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Raj R, Kumar A, Savithri HS, Singh P. Groundnut bud necrosis virus encoded movement protein NSm binds to GTP and ATP. 3 Biotech 2025; 15:146. [PMID: 40321847 PMCID: PMC12044118 DOI: 10.1007/s13205-025-04305-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2025] [Accepted: 04/02/2025] [Indexed: 05/08/2025] Open
Abstract
Groundnut bud necrosis virus (GBNV) is a tripartite negative sense RNA virus that belongs to tospoviridae family. The M RNA encodes non-structural protein-m (NSm), a movement protein in tospoviruses. In this communication, we demonstrate that, GBNV NSm interacts with ATP and GTP. UV crosslinking with [γ-32P] ATP indicates that GBNV NSm forms two distinct complexes with ATP one of them is Mg2+ dependent and the other is Mg2+ independent. It also binds to ATP- and GTP-coupled agarose resin and shows competition with free ATP and GTP but not with UTP and CTP. The NSm-NTP interaction was further validated by intrinsic fluorescence quenching studies. NTPs and dNTPs both could quench the intrinsic fluorescence of NSm. However, maximum quenching of fluorescence occurred in the presence of GTP, followed by ATP, suggesting that it is the preferred ligand. The extent of fluorescence quenching with different concentrations of GTP was used to calculate the binding constant, and it was found to be 3 μM, lower than that reported for other proteins that can bind NTP. This is the first report of the GTP and ATP binding property of NSm from any Tospoviruses. Further, NSm could also hydrolyze GTP. Preliminary sequence analysis suggests the presence of two putative atypical Walker A motif from amino acid sequences 51-58 and 267-274, indicating that this sequence might be involved in NTP binding. This motif is conserved in most of the tospoviruses. NSm from GBNV an Asian clade, localize to ER network and remodels it to vesicles which has been proposed to be involved in movement through plasmodesmata (PD). Therefore, GTP-NSm interaction might be involved in signaling cell to cell trafficking.
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Affiliation(s)
- Rishi Raj
- Department of Botany, School of Life Science, Mahatma Gandhi Central University, Motihari, Bihar 845401 India
| | - Abhay Kumar
- ICAR-National Research Centre on Litchi, Muzaffarpur, Bihar 842 002 India
| | - H. S. Savithri
- Department of Biochemistry, Indian Institute of Science, Bangalore, 560012 India
| | - Pratibha Singh
- Department of Botany, School of Life Science, Mahatma Gandhi Central University, Motihari, Bihar 845401 India
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A ribosome-associated chaperone enables substrate triage in a cotranslational protein targeting complex. Nat Commun 2020; 11:5840. [PMID: 33203865 PMCID: PMC7673040 DOI: 10.1038/s41467-020-19548-5] [Citation(s) in RCA: 41] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2020] [Accepted: 10/20/2020] [Indexed: 12/22/2022] Open
Abstract
Protein biogenesis is essential in all cells and initiates when a nascent polypeptide emerges from the ribosome exit tunnel, where multiple ribosome-associated protein biogenesis factors (RPBs) direct nascent proteins to distinct fates. How distinct RPBs spatiotemporally coordinate with one another to affect accurate protein biogenesis is an emerging question. Here, we address this question by studying the role of a cotranslational chaperone, nascent polypeptide-associated complex (NAC), in regulating substrate selection by signal recognition particle (SRP), a universally conserved protein targeting machine. We show that mammalian SRP and SRP receptors (SR) are insufficient to generate the biologically required specificity for protein targeting to the endoplasmic reticulum. NAC co-binds with and remodels the conformational landscape of SRP on the ribosome to regulate its interaction kinetics with SR, thereby reducing the nonspecific targeting of signalless ribosomes and pre-emptive targeting of ribosomes with short nascent chains. Mathematical modeling demonstrates that the NAC-induced regulations of SRP activity are essential for the fidelity of cotranslational protein targeting. Our work establishes a molecular model for how NAC acts as a triage factor to prevent protein mislocalization, and demonstrates how the macromolecular crowding of RPBs at the ribosome exit site enhances the fidelity of substrate selection into individual protein biogenesis pathways.
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SRP54 Negatively Regulates IFN-Beta Production and Antiviral Response by Targeting RIG-I and MDA5. Virol Sin 2020; 36:231-240. [PMID: 32767210 DOI: 10.1007/s12250-020-00267-6] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2020] [Accepted: 06/08/2020] [Indexed: 12/24/2022] Open
Abstract
During virus infection, RIG-I-like receptors (RLRs) recognize viral RNAs and recruit the adaptor protein VISA to activate downstream signaling, leading to activation of transcription factors NF-κB and IRF3, which collaborate to induce type I interferons (IFNs). IFNs further induce expression of hundreds of IFN-stimulated genes (ISGs) that suppress viral replication and facilitate the adaptive immune response. Dysregulated production of IFNs is implicated in various immune diseases. Here we identified Signal Recognition Particle 54 (SRP54) as a negative regulator of RLRs-induced antiviral signaling. Overexpression of SRP54 inhibited RNA virus-triggered induction of IFN-β and increased viral replication, whereas knockdown of SRP54 had opposite effects. Mechanistically, SRP54 interacted with both RIG-I and MDA5 and impaired their association with VISA. Our findings demonstrate that SRP54 acts as a negative regulator of RLRs-mediated innate immune response by disrupting the recruitment of VISA to RIG-I/MDA5.
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Hwang Fu YH, Chandrasekar S, Lee JH, Shan SO. A molecular recognition feature mediates ribosome-induced SRP-receptor assembly during protein targeting. J Cell Biol 2019; 218:3307-3319. [PMID: 31537711 PMCID: PMC6781444 DOI: 10.1083/jcb.201901001] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2019] [Revised: 06/28/2019] [Accepted: 08/26/2019] [Indexed: 12/21/2022] Open
Abstract
Molecular recognition features (MoRFs) provide interaction motifs in intrinsically disordered protein regions to mediate diverse cellular functions. Here we report that a MoRF element, located in the disordered linker domain of the mammalian signal recognition particle (SRP) receptor and conserved among eukaryotes, plays an essential role in sensing the ribosome during cotranslational protein targeting to the endoplasmic reticulum. Loss of the MoRF in the SRP receptor (SR) largely abolishes the ability of the ribosome to activate SRP-SR assembly and impairs cotranslational protein targeting. These results demonstrate a novel role for MoRF elements and provide a mechanism for the ribosome-induced activation of the mammalian SRP pathway. Kinetic analyses and comparison with the bacterial SRP further suggest that the SR MoRF functionally replaces the essential GNRA tetraloop in the bacterial SRP RNA, providing an example for the replacement of RNA function by proteins during the evolution of ancient ribonucleoprotein particles.
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Affiliation(s)
- Yu-Hsien Hwang Fu
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA
| | - Sowmya Chandrasekar
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA
| | - Jae Ho Lee
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA
| | - Shu-Ou Shan
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA
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Sequential activation of human signal recognition particle by the ribosome and signal sequence drives efficient protein targeting. Proc Natl Acad Sci U S A 2018; 115:E5487-E5496. [PMID: 29848629 DOI: 10.1073/pnas.1802252115] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Signal recognition particle (SRP) is a universally conserved targeting machine that mediates the targeted delivery of ∼30% of the proteome. The molecular mechanism by which eukaryotic SRP achieves efficient and selective protein targeting remains elusive. Here, we describe quantitative analyses of completely reconstituted human SRP (hSRP) and SRP receptor (SR). Enzymatic and fluorescence analyses showed that the ribosome, together with a functional signal sequence on the nascent polypeptide, are required to activate SRP for rapid recruitment of the SR, thereby delivering translating ribosomes to the endoplasmic reticulum. Single-molecule fluorescence spectroscopy combined with cross-complementation analyses reveal a sequential mechanism of activation whereby the ribosome unlocks the hSRP from an autoinhibited state and primes SRP to sample a variety of conformations. The signal sequence further preorganizes the mammalian SRP into the optimal conformation for efficient recruitment of the SR. Finally, the use of a signal sequence to activate SRP for receptor recruitment is a universally conserved feature to enable efficient and selective protein targeting, and the eukaryote-specific components confer upon the mammalian SRP the ability to sense and respond to ribosomes.
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Kobayashi K, Jomaa A, Lee JH, Chandrasekar S, Boehringer D, Shan SO, Ban N. Structure of a prehandover mammalian ribosomal SRP·SRP receptor targeting complex. Science 2018; 360:323-327. [PMID: 29567807 PMCID: PMC6309883 DOI: 10.1126/science.aar7924] [Citation(s) in RCA: 51] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2017] [Accepted: 03/12/2018] [Indexed: 01/13/2023]
Abstract
Signal recognition particle (SRP) targets proteins to the endoplasmic reticulum (ER). SRP recognizes the ribosome synthesizing a signal sequence and delivers it to the SRP receptor (SR) on the ER membrane followed by the transfer of the signal sequence to the translocon. Here, we present the cryo-electron microscopy structure of the mammalian translating ribosome in complex with SRP and SR in a conformation preceding signal sequence handover. The structure visualizes all eukaryotic-specific SRP and SR proteins and reveals their roles in stabilizing this conformation by forming a large protein assembly at the distal site of SRP RNA. We provide biochemical evidence that the guanosine triphosphate hydrolysis of SRP·SR is delayed at this stage, possibly to provide a time window for signal sequence handover to the translocon.
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Affiliation(s)
- Kan Kobayashi
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Otto-Stern-Weg 5, Zurich CH-8093, Switzerland
| | - Ahmad Jomaa
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Otto-Stern-Weg 5, Zurich CH-8093, Switzerland
| | - Jae Ho Lee
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
| | - Sowmya Chandrasekar
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
| | - Daniel Boehringer
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Otto-Stern-Weg 5, Zurich CH-8093, Switzerland
| | - Shu-Ou Shan
- Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
| | - Nenad Ban
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Otto-Stern-Weg 5, Zurich CH-8093, Switzerland.
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7
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Wang Y, Wei S, Chen L, Pei J, Wu H, Pei Y, Chen Y, Wang D. Transcriptomic analysis of gene expression in mice treated with troxerutin. PLoS One 2017; 12:e0188261. [PMID: 29190643 PMCID: PMC5708793 DOI: 10.1371/journal.pone.0188261] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2017] [Accepted: 11/03/2017] [Indexed: 12/13/2022] Open
Abstract
Troxerutin, a semi-synthetic derivative of the natural bioflavanoid rutin, has been reported to possess many beneficial effects in human bodies, such as vasoprotection, immune support, anti-inflammation and anti-aging. However, the effects of troxerutin on genome-wide transcription in blood cells are still unknown. In order to find out effects of troxerutin on gene transcription, a high-throughput RNA sequencing was employed to analysis differential gene expression in blood cells consisting of leucocytes, erythrocytes and platelets isolated from the mice received subcutaneous injection of troxerutin. Transcriptome analysis demonstrated that the expression of only fifteen genes was significantly changed by the treatment with troxerutin, among which 5 genes were up-regulated and 10 genes were down-regulated. Bioinformatic analysis of the fifteen differentially expressed genes was made by utilizing the Gene Ontology (GO), and the differential expression induced by troxerutin was further evaluated by real-time quantitative PCR (Q-PCR).
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Affiliation(s)
- Yuerong Wang
- Hainan Key Laboratories of Sustainable Utilization of Tropical Bioresources, Hainan University, Haikou, Hainan, China.,Laboratory of Biotechnology and Molecular Pharmacology, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan, China
| | - Shuangshuang Wei
- Hainan Key Laboratories of Sustainable Utilization of Tropical Bioresources, Hainan University, Haikou, Hainan, China.,Laboratory of Biotechnology and Molecular Pharmacology, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan, China
| | - Lintao Chen
- Hainan Key Laboratories of Sustainable Utilization of Tropical Bioresources, Hainan University, Haikou, Hainan, China.,Laboratory of Biotechnology and Molecular Pharmacology, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan, China
| | - Jinli Pei
- Hainan Key Laboratories of Sustainable Utilization of Tropical Bioresources, Hainan University, Haikou, Hainan, China.,Laboratory of Biotechnology and Molecular Pharmacology, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan, China
| | - Hao Wu
- Hainan Key Laboratories of Sustainable Utilization of Tropical Bioresources, Hainan University, Haikou, Hainan, China.,Laboratory of Biotechnology and Molecular Pharmacology, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan, China
| | - Yechun Pei
- Laboratory of Biotechnology and Molecular Pharmacology, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan, China.,Department of Animal Science, Hainan University, Haikou, Hainan, China
| | - Yibo Chen
- Laboratory of Biotechnology and Molecular Pharmacology, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan, China
| | - Dayong Wang
- Hainan Key Laboratories of Sustainable Utilization of Tropical Bioresources, Hainan University, Haikou, Hainan, China.,Laboratory of Biotechnology and Molecular Pharmacology, Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan, China
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Becker MMM, Lapouge K, Segnitz B, Wild K, Sinning I. Structures of human SRP72 complexes provide insights into SRP RNA remodeling and ribosome interaction. Nucleic Acids Res 2016; 45:470-481. [PMID: 27899666 PMCID: PMC5224484 DOI: 10.1093/nar/gkw1124] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2016] [Revised: 10/21/2016] [Accepted: 10/28/2016] [Indexed: 12/30/2022] Open
Abstract
Co-translational protein targeting and membrane protein insertion is a fundamental process and depends on the signal recognition particle (SRP). In mammals, SRP is composed of the SRP RNA crucial for SRP assembly and function and six proteins. The two largest proteins SRP68 and SRP72 form a heterodimer and bind to a regulatory site of the SRP RNA. Despite their essential roles in the SRP pathway, structural information has been available only for the SRP68 RNA-binding domain (RBD). Here we present the crystal structures of the SRP68 protein-binding domain (PBD) in complex with SRP72-PBD and of the SRP72-RBD bound to the SRP S domain (SRP RNA, SRP19 and SRP68) detailing all interactions of SRP72 within SRP. The SRP72-PBD is a tetratricopeptide repeat, which binds an extended linear motif of SRP68 with high affinity. The SRP72-RBD is a flexible peptide crawling along the 5e- and 5f-loops of SRP RNA. A conserved tryptophan inserts into the 5e-loop forming a novel type of RNA kink-turn stabilized by a potassium ion, which we define as K+-turn. In addition, SRP72-RBD remodels the 5f-loop involved in ribosome binding and visualizes SRP RNA plasticity. Docking of the S domain structure into cryo-electron microscopy density maps reveals multiple contact sites between SRP68/72 and the ribosome, and explains the role of SRP72 in the SRP pathway.
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Affiliation(s)
- Matthias M M Becker
- Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany
| | - Karine Lapouge
- Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany
| | - Bernd Segnitz
- Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany
| | - Klemens Wild
- Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany
| | - Irmgard Sinning
- Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany
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9
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Comparison of inter- and intraspecies variation in humans and fruit flies. GENOMICS DATA 2014; 3:49-54. [PMID: 26484147 PMCID: PMC4536057 DOI: 10.1016/j.gdata.2014.11.010] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/19/2014] [Revised: 11/12/2014] [Accepted: 11/12/2014] [Indexed: 12/17/2022]
Abstract
Variation is essential to species survival and adaptation during evolution. This variation is conferred by the imperfection of biochemical processes, such as mutations and alterations in DNA sequences, and can also be seen within genomes through processes such as the generation of antibodies. Recent sequencing projects have produced multiple versions of the genomes of humans and fruit flies (Drosophila melanogaster). These give us a chance to study how individual gene sequences vary within and between species. Here we arranged human and fly genes in orthologous pairs and compared such within-species variability with their degree of conservation between flies and humans. We observed that a significant number of proteins associated with mRNA translation are highly conserved between species and yet are highly variable within each species. The fact that we observe this in two species whose lineages separated more than 700 million years ago suggests that this is the result of a very ancient process. We hypothesize that this effect might be attributed to a positive selection for variability of virus-interacting proteins that confers a general resistance to viral hijacking of the mRNA translation machinery within populations. Our analysis points to this and to other processes resulting in positive selection for gene variation.
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Denks K, Vogt A, Sachelaru I, Petriman NA, Kudva R, Koch HG. The Sec translocon mediated protein transport in prokaryotes and eukaryotes. Mol Membr Biol 2014; 31:58-84. [DOI: 10.3109/09687688.2014.907455] [Citation(s) in RCA: 123] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
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11
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SIMIBI twins in protein targeting and localization. Nat Struct Mol Biol 2013; 20:776-80. [DOI: 10.1038/nsmb.2605] [Citation(s) in RCA: 61] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2013] [Accepted: 05/07/2013] [Indexed: 12/31/2022]
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12
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Veith C, Schmitt S, Veit F, Dahal BK, Wilhelm J, Klepetko W, Marta G, Seeger W, Schermuly RT, Grimminger F, Ghofrani HA, Fink L, Weissmann N, Kwapiszewska G. Cofilin, a hypoxia-regulated protein in murine lungs identified by 2DE: Role of the cytoskeletal protein cofilin in pulmonary hypertension. Proteomics 2013; 13:75-88. [DOI: 10.1002/pmic.201200206] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2012] [Revised: 10/08/2012] [Accepted: 10/29/2012] [Indexed: 01/18/2023]
Affiliation(s)
- Christine Veith
- Universities of Giessen and Marburg Lung Center (UGMLC); Giessen Germany
| | - Sigrid Schmitt
- Department of Biochemistry; University of Giessen; Giessen Germany
| | - Florian Veit
- Universities of Giessen and Marburg Lung Center (UGMLC); Giessen Germany
| | - Bhola Kumar Dahal
- Universities of Giessen and Marburg Lung Center (UGMLC); Giessen Germany
| | - Jochen Wilhelm
- Universities of Giessen and Marburg Lung Center (UGMLC); Giessen Germany
| | - Walter Klepetko
- Department of Cardiac Surgery; University of Vienna; Vienna Austria
| | - Gabriel Marta
- Department of Cardiac Surgery; University of Vienna; Vienna Austria
| | - Werner Seeger
- Universities of Giessen and Marburg Lung Center (UGMLC); Giessen Germany
| | | | | | | | - Ludger Fink
- Universities of Giessen and Marburg Lung Center (UGMLC); Giessen Germany
| | - Norbert Weissmann
- Universities of Giessen and Marburg Lung Center (UGMLC); Giessen Germany
| | - Grazyna Kwapiszewska
- Universities of Giessen and Marburg Lung Center (UGMLC); Giessen Germany
- Ludwig Boltzmann Institute for Lung Vascular Research; Graz Austria
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13
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Luirink J, Yu Z, Wagner S, de Gier JW. Biogenesis of inner membrane proteins in Escherichia coli. BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 2011; 1817:965-76. [PMID: 22201544 DOI: 10.1016/j.bbabio.2011.12.006] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/18/2011] [Revised: 12/05/2011] [Accepted: 12/12/2011] [Indexed: 11/26/2022]
Abstract
The inner membrane proteome of the model organism Escherichia coli is composed of inner membrane proteins, lipoproteins and peripherally attached soluble proteins. Our knowledge of the biogenesis of inner membrane proteins is rapidly increasing. This is in particular true for the early steps of biogenesis - protein targeting to and insertion into the membrane. However, our knowledge of inner membrane protein folding and quality control is still fragmentary. Furthering our knowledge in these areas will bring us closer to understand the biogenesis of individual inner membrane proteins in the context of the biogenesis of the inner membrane proteome of Escherichia coli as a whole. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.
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Affiliation(s)
- Joen Luirink
- Section of Molecular Microbiology, Department of Molecular Cell Biology, VU University, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
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14
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Yang MJ, Zhang X. Molecular dynamics simulations reveal structural coordination of Ffh-FtsY heterodimer toward GTPase activation. Proteins 2011; 79:1774-85. [DOI: 10.1002/prot.23000] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2010] [Revised: 01/03/2011] [Accepted: 01/17/2011] [Indexed: 11/08/2022]
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15
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Lewis NE, Marty NJ, Kathir KM, Rajalingam D, Kight AD, Daily A, Kumar TKS, Henry RL, Goforth RL. A dynamic cpSRP43-Albino3 interaction mediates translocase regulation of chloroplast signal recognition particle (cpSRP)-targeting components. J Biol Chem 2010; 285:34220-30. [PMID: 20729200 PMCID: PMC2962520 DOI: 10.1074/jbc.m110.160093] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2010] [Revised: 08/16/2010] [Indexed: 12/31/2022] Open
Abstract
The chloroplast signal recognition particle (cpSRP) and its receptor, chloroplast FtsY (cpFtsY), form an essential complex with the translocase Albino3 (Alb3) during post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCPs). Here, we describe a combination of studies that explore the binding interface and functional role of a previously identified cpSRP43-Alb3 interaction. Using recombinant proteins corresponding to the C terminus of Alb3 (Alb3-Cterm) and various domains of cpSRP43, we identify the ankyrin repeat region of cpSRP43 as the domain primarily responsible for the interaction with Alb3-Cterm. Furthermore, we show Alb3-Cterm dissociates a cpSRP·LHCP targeting complex in vitro and stimulates GTP hydrolysis by cpSRP54 and cpFtsY in a strictly cpSRP43-dependent manner. These results support a model in which interactions between the ankyrin region of cpSRP43 and the C terminus of Alb3 promote distinct membrane-localized events, including LHCP release from cpSRP and release of targeting components from Alb3.
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Affiliation(s)
| | | | | | | | | | - Anna Daily
- Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701
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16
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Yang M, Zhang X, Han K. Molecular dynamics simulation of SRP GTPases: Towards an understanding of the complex formation from equilibrium fluctuations. Proteins 2010; 78:2222-37. [DOI: 10.1002/prot.22734] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
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17
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Zhang Y, Li Q, Zhu F, Cui J, Li K, Li Q, Wang R, Wang W, Wang W, Yan W. Subcellular localization of APMCF1 and its biological significance of expression pattern in normal and malignant human tissues. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2009; 28:111. [PMID: 19664239 PMCID: PMC2731735 DOI: 10.1186/1756-9966-28-111] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/27/2009] [Accepted: 08/09/2009] [Indexed: 12/01/2022]
Abstract
Background APMCF1 is a novel human gene first cloned from apoptotic MCF-7 cells. Our previous study found ectogenic APMCF1 could induce G1 arrest in hepatocarcinoma cell line HHCC. In order to search its broad expression profile for further understanding of its mechanism in tumor, we investigated a subcellular location of APMCF1 and performed an immunohistochemistry study including various tumor and normal tissues. Discovery from the expression characterization of AMPCF1 may have applicability in the analysis of its biological function in tumor. Methods We investigated subcellular localization of APMCF1 by transient transfection in green monkey kidney epithelial cells (COS-7) with a fusion protein vector pEGFP-APMCF1 and detected expression profile in a broad range of normal and malignant human tissues via tissue microarray (TMA) by immunohistochemistry with polyclonal antibody first produced in our laboratory. Results EGFP-APMCF1 was generally localized in the cytoplasm of COS-7 cell. Positive staining of APMCF1 was found in liver, lung, breast, colon, stomach, esophagus and testis, exhibited a ubiquitous expression pattern while its expression was up-regulated in tumor tissues compared with corresponding normal tissues. Normal brain neuron cells also showed expression of APMCF1, but negative in gliocyte cells and glioma. Both the normal and tumor tissues of ovary were absent of APMCF1 expression. Positive immunostaining for APMCF1 with large samples in liver, colon, esophagus, lung and breast carcinomas were 96% (51/53), 80% (44/55), 57% (30/53), 58% (33/57) and 34% (16/47) respectively. Conclusion These results revealed a cytoplastic expression pattern of APMCF1 and up-regulated in tumour tissues suggesting APMCF1 may have potential relationship with oncogenesis. The data presented should serve as a useful reference for further studies of APMCF1 functions in tumorigenesis and might provide a potential anti-tumor target.
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Affiliation(s)
- Yaqing Zhang
- Department of Pathology, State Key Laboratory of GI Cancer Biology, Xijing Hospital, Fourth Military Medical University, Shaanxi Province, PR China.
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18
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Grudnik P, Bange G, Sinning I. Protein targeting by the signal recognition particle. Biol Chem 2009; 390:775-82. [DOI: 10.1515/bc.2009.102] [Citation(s) in RCA: 128] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Abstract
Protein targeting by the signal recognition particle (SRP) is universally conserved and starts with the recognition of a signal sequence in the context of a translating ribosome. SRP54 and FtsY, two multidomain proteins with guanosine triphosphatase (GTPase) activity, are the central elements of the SRP system. They have to coordinate the presence of a signal sequence with the presence of a vacant translocation channel in the membrane. For coordination the two GTPases form a unique, nearly symmetric heterodimeric complex in which the activation of GTP hydrolysis plays a key role for membrane insertion of substrate proteins. Recent results are integrated in an updated perception of the order of events in SRP-mediated protein targeting.
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Abstract
The Signal Recognition Particle (SRP) plays a critical role in the sorting of nascent secretory and membrane proteins. Remarkably, this function has been conserved from bacteria, where SRP delivers proteins to the inner membrane, through to eukaryotes, where SRP is required for targeting of proteins to the endoplasmic reticulum. This review focuses on present understanding of SRP structure and function and the relationship between the two. Furthermore, the similarities and differences in the structure, function and cellular role of SRP in bacteria, chloroplasts, fungi and mammals will be stressed.
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Affiliation(s)
- Martin R Pool
- Faculty of Life Sciences, University of Manchester, Manchester, UK.
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20
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Cross BCS, Sinning I, Luirink J, High S. Delivering proteins for export from the cytosol. Nat Rev Mol Cell Biol 2009; 10:255-64. [PMID: 19305415 DOI: 10.1038/nrm2657] [Citation(s) in RCA: 160] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Correct protein function depends on delivery to the appropriate cellular or subcellular compartment. Following the initiation of protein synthesis in the cytosol, many bacterial and eukaryotic proteins must be integrated into or transported across a membrane to reach their site of function. Whereas in the post-translational delivery pathway ATP-dependent factors bind to completed polypeptides and chaperone them until membrane translocation is initiated, a GTP-dependent co-translational pathway operates to couple ongoing protein synthesis to membrane transport. These distinct pathways provide different solutions for the maintenance of proteins in a state that is competent for membrane translocation and their delivery for export from the cytosol.
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Affiliation(s)
- Benedict C S Cross
- Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK
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21
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Multiple conformational switches in a GTPase complex control co-translational protein targeting. Proc Natl Acad Sci U S A 2009; 106:1754-9. [PMID: 19174514 DOI: 10.1073/pnas.0808573106] [Citation(s) in RCA: 90] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The "GTPase switch" paradigm, in which a GTPase switches between an active, GTP-bound state and an inactive, GDP-bound state through the recruitment of nucleotide exchange factors (GEFs) or GTPase activating proteins (GAPs), has been used to interpret the regulatory mechanism of many GTPases. A notable exception to this paradigm is provided by two GTPases in the signal recognition particle (SRP) and the SRP receptor (SR) that control the co-translational targeting of proteins to cellular membranes. Instead of the classical "GTPase switch," both the SRP and SR undergo a series of discrete conformational rearrangements during their interaction with one another, culminating in their reciprocal GTPase activation. Here, we show that this series of rearrangements during SRP-SR binding and activation provide important control points to drive and regulate protein targeting. Using real-time fluorescence, we showed that the cargo for SRP--ribosomes translating nascent polypeptides with signal sequences--accelerates SRP.SR complex assembly over 100-fold, thereby driving rapid delivery of cargo to the membrane. A series of subsequent rearrangements in the SRP x SR GTPase complex provide important driving forces to unload the cargo during late stages of protein targeting. Further, the cargo delays GTPase activation in the SRP.SR complex by 8-12 fold, creating an important time window that could further improve the efficiency and fidelity of protein targeting. Thus, the SRP and SR GTPases, without recruiting external regulatory factors, constitute a self-sufficient system that provides exquisite spatial and temporal control of a complex cellular process.
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22
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Field MC, Lumb JH, Adung'a VO, Jones NG, Engstler M. Chapter 1 Macromolecular Trafficking and Immune Evasion in African Trypanosomes. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2009; 278:1-67. [DOI: 10.1016/s1937-6448(09)78001-3] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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Dalley JA, Selkirk A, Pool MR. Access to ribosomal protein Rpl25p by the signal recognition particle is required for efficient cotranslational translocation. Mol Biol Cell 2008; 19:2876-84. [PMID: 18448667 PMCID: PMC2441686 DOI: 10.1091/mbc.e07-10-1074] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2007] [Revised: 04/10/2008] [Accepted: 04/18/2008] [Indexed: 11/11/2022] Open
Abstract
Targeting of proteins to the endoplasmic reticulum (ER) occurs cotranslationally necessitating the interaction of the signal recognition particle (SRP) and the translocon with the ribosome. Biochemical and structural studies implicate ribosomal protein Rpl25p as a major ribosome interaction site for both these factors. Here we characterize an RPL25GFP fusion, which behaves as a dominant mutant leading to defects in co- but not posttranslational translocation in vivo. In these cells, ribosomes still interact with ER membrane and the translocon, but are defective in binding SRP. Overexpression of SRP can restore ribosome binding of SRP, but only partially rescues growth and translocation defects. Our results indicate that Rpl25p plays a critical role in the recruitment of SRP to the ribosome.
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Affiliation(s)
- Jane A. Dalley
- Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom
| | - Alexander Selkirk
- Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom
| | - Martin R. Pool
- Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom
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Origins and evolution of cotranslational transport to the ER. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2007; 607:52-60. [PMID: 17977458 DOI: 10.1007/978-0-387-74021-8_4] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/08/2023]
Abstract
All living organisms possess the ability to translocate proteins across biological membranes. This is a fundamental necessity since proteins function in different locations yet are synthesized in one compartment only, the cytosol. Even though different transport systems exist, the pathway that is dominantly used to translocate secretory and membrane proteins is known as the cotranslational transport pathway. It evolved only once and is in its core conserved throughout all kingdoms of life. The process is characterized by a well understood sequence of events: first, an N-terminal signal sequence of a nascent polypeptide is recognized on the ribosome by the signal recognition particle (SRP), then the SRP-ribosome complex is targeted to the membrane via the SRP receptor. Next, the nascent chain is transferred from SRP to the protein conducting channel, through which it is cotranslationally threaded. All the essential components of the system have been identified. Recent structural and biochemical studies have unveiled some of the intricate regulatory circuitry of the process. These studies also shed light on the accessory components unique to eukaryotes, pointing to early events in eukaryotic evolution.
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Hainzl T, Huang S, Sauer-Eriksson AE. Interaction of signal-recognition particle 54 GTPase domain and signal-recognition particle RNA in the free signal-recognition particle. Proc Natl Acad Sci U S A 2007; 104:14911-6. [PMID: 17846429 PMCID: PMC1986587 DOI: 10.1073/pnas.0702467104] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2007] [Indexed: 11/18/2022] Open
Abstract
The signal-recognition particle (SRP) is a ubiquitous protein-RNA complex that targets proteins to cellular membranes for insertion or secretion. A key player in SRP-mediated protein targeting is the evolutionarily conserved core consisting of the SRP RNA and the multidomain protein SRP54. Communication between the SRP54 domains is critical for SRP function, where signal sequence binding at the M domain directs receptor binding at the GTPase domain (NG domain). These SRP activities are linked to domain rearrangements, for which the role of SRP RNA is not clear. In free SRP, a direct interaction of the GTPase domain with SRP RNA has been proposed but has never been structurally verified. In this study, we present the crystal structure at 2.5-A resolution of the SRP54-SRP19-SRP RNA complex of Methanococcus jannaschii SRP. The structure reveals an RNA-bound conformation of the SRP54 GTPase domain, in which the domain is spatially well separated from the signal peptide binding site. The association of both the N and G domains with SRP RNA in free SRP provides further structural evidence for the pivotal role of SRP RNA in the regulation of the SRP54 activity.
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Affiliation(s)
- Tobias Hainzl
- Umeå Center for Molecular Pathogenesis, Umeå University, SE-901 87 Umeå, Sweden.
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26
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Sahdev S, Khattar SK, Saini KS. Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem 2007. [PMID: 17874175 DOI: 10.1007/s11010‐007‐9603‐6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Among the various expression systems employed for the over-production of proteins, bacteria still remains the favorite choice of a Protein Biochemist. However, even today, due to the lack of post-translational modification machinery in bacteria, recombinant eukaryotic protein production poses an immense challenge, which invariably leads to the production of biologically in-active protein in this host. A number of techniques are cited in the literature, which describe the conversion of inactive protein, expressed as an insoluble fraction, into a soluble and active form. Overall, we have divided these methods into three major groups: Group-I, where the factors influencing the formation of insoluble fraction are modified through a stringent control of the cellular milieu, thereby leading to the expression of recombinant protein as soluble moiety; Group-II, where protein is refolded from the inclusion bodies and thereby target protein modification is avoided; Group-III, where the target protein is engineered to achieve soluble expression through fusion protein technology. Even within the same family of proteins (e.g., tyrosine kinases), optimization of standard operating protocol (SOP) may still be required for each protein's over-production at a pilot-scale in Escherichia coli. However, once standardized, this procedure can be made amenable to the industrial production for that particular protein with minimum alterations.
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Affiliation(s)
- Sudhir Sahdev
- Department of Biotechnology & Bioinformatics, New Drug Discovery Research, Ranbaxy Research Laboratories-R&D-3, 20-Sector 18 Udyog Vihar, Gurgaon, India.
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27
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Sahdev S, Khattar SK, Saini KS. Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem 2007; 307:249-64. [PMID: 17874175 DOI: 10.1007/s11010-007-9603-6] [Citation(s) in RCA: 265] [Impact Index Per Article: 14.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2007] [Accepted: 08/27/2007] [Indexed: 12/13/2022]
Abstract
Among the various expression systems employed for the over-production of proteins, bacteria still remains the favorite choice of a Protein Biochemist. However, even today, due to the lack of post-translational modification machinery in bacteria, recombinant eukaryotic protein production poses an immense challenge, which invariably leads to the production of biologically in-active protein in this host. A number of techniques are cited in the literature, which describe the conversion of inactive protein, expressed as an insoluble fraction, into a soluble and active form. Overall, we have divided these methods into three major groups: Group-I, where the factors influencing the formation of insoluble fraction are modified through a stringent control of the cellular milieu, thereby leading to the expression of recombinant protein as soluble moiety; Group-II, where protein is refolded from the inclusion bodies and thereby target protein modification is avoided; Group-III, where the target protein is engineered to achieve soluble expression through fusion protein technology. Even within the same family of proteins (e.g., tyrosine kinases), optimization of standard operating protocol (SOP) may still be required for each protein's over-production at a pilot-scale in Escherichia coli. However, once standardized, this procedure can be made amenable to the industrial production for that particular protein with minimum alterations.
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Affiliation(s)
- Sudhir Sahdev
- Department of Biotechnology & Bioinformatics, New Drug Discovery Research, Ranbaxy Research Laboratories-R&D-3, 20-Sector 18 Udyog Vihar, Gurgaon, India.
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28
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Bange G, Petzold G, Wild K, Parlitz RO, Sinning I. The crystal structure of the third signal-recognition particle GTPase FlhF reveals a homodimer with bound GTP. Proc Natl Acad Sci U S A 2007; 104:13621-5. [PMID: 17699634 PMCID: PMC1959431 DOI: 10.1073/pnas.0702570104] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2007] [Indexed: 11/18/2022] Open
Abstract
Flagella are well characterized as the organelles of locomotion and allow bacteria to react to environmental changes. The assembly of flagella is a multistep process and relies on a complex type III export machinery located in the cytoplasmic membrane. The FlhF protein is essential for the placement and assembly of polar flagella and has been classified as a signal-recognition particle (SRP)-type GTPase. SRP GTPases appeared early in evolution and form a unique subfamily within the guanine nucleotide binding proteins with only three members: the signal sequence-binding protein SRP54, the SRP receptor FtsY, and FlhF. We report the crystal structures of FlhF from Bacillus subtilis in complex with GTP and GMPPNP. FlhF shares SRP GTPase-specific features such as the presence of an N-terminal alpha-helical domain and the I-box insertion. It forms a symmetric homodimer sequestering a composite active site that contains two head-to-tail arranged nucleotides similar to the heterodimeric SRP-targeting complex. However, significant differences to the GTPases of SRP and the SRP receptor include the formation of a stable homodimer with GTP as well as severe modifications and even the absence of motifs involved in regulation of the other two SRP GTPases. Our results provide insights into SRP GTPases and their roles in two fundamentally different protein-targeting routes that both rely on efficient protein delivery to a secretion channel.
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Affiliation(s)
- Gert Bange
- Heidelberg University Biochemistry Center (BZH), INF 328, 69120 Heidelberg, Germany
| | - Georg Petzold
- Heidelberg University Biochemistry Center (BZH), INF 328, 69120 Heidelberg, Germany
| | - Klemens Wild
- Heidelberg University Biochemistry Center (BZH), INF 328, 69120 Heidelberg, Germany
| | - Richard O. Parlitz
- Heidelberg University Biochemistry Center (BZH), INF 328, 69120 Heidelberg, Germany
| | - Irmgard Sinning
- Heidelberg University Biochemistry Center (BZH), INF 328, 69120 Heidelberg, Germany
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29
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Mitra K, Frank J, Driessen A. Co- and post-translational translocation through the protein-conducting channel: analogous mechanisms at work? Nat Struct Mol Biol 2007; 13:957-64. [PMID: 17082791 DOI: 10.1038/nsmb1166] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Many proteins are translocated across, or integrated into, membranes. Both functions are fulfilled by the 'translocon/translocase', which contains a membrane-embedded protein-conducting channel (PCC) and associated soluble factors that drive translocation and insertion reactions using nucleotide triphosphates as fuel. This perspective focuses on reinterpreting existing experimental data in light of a recently proposed PCC model comprising a front-to-front dimer of SecY or Sec61 heterotrimeric complexes. In this new framework, we propose (i) a revised model for SRP-SR-mediated docking of the ribosome-nascent polypeptide to the PCC; (ii) that the dynamic interplay between protein substrate, soluble factors and PCC controls the opening and closing of a transmembrane channel across, and/or a lateral gate into, the membrane; and (iii) that co- and post-translational translocation, involving the ribosome and SecA, respectively, not only converge at the PCC but also use analogous mechanisms for coordinating protein translocation.
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30
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Schaffitzel C, Oswald M, Berger I, Ishikawa T, Abrahams JP, Koerten HK, Koning RI, Ban N. Structure of the E. coli signal recognition particle bound to a translating ribosome. Nature 2006; 444:503-6. [PMID: 17086205 DOI: 10.1038/nature05182] [Citation(s) in RCA: 107] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2006] [Accepted: 08/18/2006] [Indexed: 11/09/2022]
Abstract
The prokaryotic signal recognition particle (SRP) targets membrane proteins into the inner membrane. It binds translating ribosomes and screens the emerging nascent chain for a hydrophobic signal sequence, such as the transmembrane helix of inner membrane proteins. If such a sequence emerges, the SRP binds tightly, allowing the SRP receptor to lock on. This assembly delivers the ribosome-nascent chain complex to the protein translocation machinery in the membrane. Using cryo-electron microscopy and single-particle reconstruction, we obtained a 16 A structure of the Escherichia coli SRP in complex with a translating E. coli ribosome containing a nascent chain with a transmembrane helix anchor. We also obtained structural information on the SRP bound to an empty E. coli ribosome. The latter might share characteristics with a scanning SRP complex, whereas the former represents the next step: the targeting complex ready for receptor binding. High-resolution structures of the bacterial ribosome and of the bacterial SRP components are available, and their fitting explains our electron microscopic density. The structures reveal the regions that are involved in complex formation, provide insight into the conformation of the SRP on the ribosome and indicate the conformational changes that accompany high-affinity SRP binding to ribosome nascent chain complexes upon recognition of the signal sequence.
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Affiliation(s)
- Christiane Schaffitzel
- ETH Zurich, Institute for Molecular Biology and Biophysics, HPK Building, Schafmattstrasse 20, 8093 Zurich, Switzerland
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31
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Schlenker O, Hendricks A, Sinning I, Wild K. The structure of the mammalian signal recognition particle (SRP) receptor as prototype for the interaction of small GTPases with Longin domains. J Biol Chem 2006; 281:8898-906. [PMID: 16439358 DOI: 10.1074/jbc.m512415200] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
The eukaryotic signal recognition particle (SRP) and its receptor (SR) play a central role in co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum. The SR is a heterodimeric complex assembled by the two GTPases SRalpha and SRbeta, which is membrane-anchored. Here we present the 2.45-A structure of mammalian SRbeta in its Mg2+ GTP-bound state in complex with the minimal binding domain of SRalpha termed SRX. SRbeta is a member of the Ras-GTPase superfamily closely related to Arf and Sar1, while SRX belongs to the SNARE-like superfamily with a fold also known as longin domain. SRX binds to the P loop and the switch regions of SRbeta-GTP. The binding mode and structural similarity with other GTPase-effector complexes suggests a co-GAP (GTPase-activating protein) function for SRX. Comparison with the homologous yeast structure and other longin domains reveals a conserved adjustable hydrophobic surface within SRX which is of central importance for the SRbeta-GTP:SRX interface. A helix swap in SRX results in the formation of a dimer in the crystal structure. Based on structural conservation we present the SRbeta-GTP:SRX structure as a prototype for conserved interactions in a variety of GTPase regulated targeting events occurring at endomembranes.
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Affiliation(s)
- Oliver Schlenker
- Biochemie-Zentrum der Universität Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany
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32
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Abstract
Gram-negative bacteria such as Escherichia coli are surrounded by two membranes, the inner membrane and the outer membrane. The biogenesis of most inner membrane proteins (IMPs), typical alpha-helical proteins, appears to follow a partly conserved cotranslational pathway. Targeting involves a relatively simple signal recognition particle (SRP) and SRP-receptor. Insertion of most IMPs into the membrane occurs via the Sec-translocon, which is also used for the vectorial transport of secretory proteins. Similar to eukaryotic systems, little is known about the later stages of biogenesis of IMPs, the folding and assembly in the lipid bilayer. Recently, YidC has been identified as a factor that assists in the integration, folding, and assembly of IMPs both in association with the Sec-translocon and separately. This review deals mainly with recent structural and biochemical data from various experimental systems that offer new insight into the different stages of biogenesis of E. coli IMPs.
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Affiliation(s)
- Joen Luirink
- Department of Microbiology, Institute of Molecular Cell Biology, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands.
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33
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Mukhopadhyay A, Ni L, Weiner H. A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria. Biochem J 2005; 382:385-92. [PMID: 15153070 PMCID: PMC1133951 DOI: 10.1042/bj20040065] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2004] [Revised: 05/18/2004] [Accepted: 05/21/2004] [Indexed: 11/17/2022]
Abstract
The dual signal approach, i.e. a mitochondrial signal at the N-terminus and an ER (endoplasmic reticulum) or a peroxisomal signal at the C-terminus of EGFP (enhanced green fluorescent protein), was employed in transfected HeLa cells to test for a co-translational import model. The signal peptide from OTC (ornithine transcarbamylase) or arginase II was fused to the N-terminus of EGFP, and an ER or peroxisomal signal was fused to its C-terminus. The rationale was that if the free preprotein remained in the cytosol, it could be distributed between the two organelles by using a post-translational pathway. The resulting fusion proteins were imported exclusively into mitochondria, suggesting that co-translational import occurred. Native preALDH (precursor of rat liver mitochondrial aldehyde dehydrogenase), preOTC and rhodanese, each with the addition of a C-terminal ER or peroxisomal signal, were also translocated only to the mitochondria, again showing that a co-translational import pathway exists for these native proteins. Import of preALDH(sp)-DHFR, a fusion protein consisting of the leader sequence (signal peptide) of preALDH fused to DHFR (dihydrofolate reductase), was studied in the presence of methotrexate, a substrate analogue for DHFR. It was found that 70% of the preALDH(sp)-DHFR was imported into mitochondria in the presence of methotrexate, implying that 70% of the protein utilized the co-translational import pathway and 30% used the post-translational import pathway. Thus it appears that co-translational import is a major pathway for mitochondrial protein import. A model is proposed to explain how competition between binding factors could influence whether or not a cytosolic carrier protein, such as DHFR, uses the co- or post-translational import pathway.
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Affiliation(s)
- Abhijit Mukhopadhyay
- Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, IN 47907-2063, U.S.A
| | - Li Ni
- Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, IN 47907-2063, U.S.A
| | - Henry Weiner
- To whom correspondence should be addressed (email )
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34
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Abstract
Co-translational targeting of secretory and membrane proteins to the translocation machinery is mediated by the signal recognition particle (SRP) and its membrane-bound receptor (SR) in all three domains of life. Although the overall composition of the SRP system differs, the central ribonucleoprotein core and the general mechanism of GTP-dependent targeting are highly conserved. Recently, structural studies have contributed significantly to our understanding of the molecular organization of SRP. SRP appears as a structurally flexible particle modulated and regulated by its interactions with the ribosome-nascent chain complex, the translocon and the SR. The SRP core (SRP54 with its cognate RNA binding site) plays a central role in these interactions and communicates the different binding states by long-range interdomain communication. Based on recent structures of SRP54, a model for signal peptide binding stimulating the GTP affinity during the first step of the SRP cycle is presented. The model is placed in the context of the recent structures of mammalian SRP bound to a ribosome-nascent chain complex and of a subcomplex of SRP-SR.
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Affiliation(s)
- Klemens Wild
- Biochemie-Zentrum der Universität Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany.
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35
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Wild K, Halic M, Sinning I, Beckmann R. SRP meets the ribosome. Nat Struct Mol Biol 2004; 11:1049-53. [PMID: 15523481 DOI: 10.1038/nsmb853] [Citation(s) in RCA: 82] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2004] [Accepted: 10/04/2004] [Indexed: 11/09/2022]
Abstract
Cotranslational targeting directly couples synthesis of proteins to their translocation across or insertion into membranes. The signal recognition particle (SRP) and its membrane-bound receptor facilitate the targeting of the translation machinery, the ribosome, via recognition of a signal sequence in the nascent peptide chain. By combining structures of free and ribosome-bound SRP we derive a structural model describing the dynamic nature of SRP when it meets the ribosome.
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Affiliation(s)
- Klemens Wild
- Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany
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36
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Abstract
The signal recognition particle (SRP) directs integral membrane and secretory proteins to the cellular protein translocation machinery during translation. The SRP is an evolutionarily conserved RNA-protein complex whose activities are regulated by GTP hydrolysis. Recent structural investigations of SRP functional domains and interactions provide new insights into the mechanisms of SRP activity in all cells, leading toward a comprehensive understanding of protein trafficking by this elegant pathway.
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Affiliation(s)
- Jennifer A Doudna
- Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California at Berkeley, Berkeley, California 94705, USA.
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37
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Rosendal KR, Wild K, Montoya G, Sinning I. Crystal structure of the complete core of archaeal signal recognition particle and implications for interdomain communication. Proc Natl Acad Sci U S A 2003; 100:14701-6. [PMID: 14657338 PMCID: PMC299766 DOI: 10.1073/pnas.2436132100] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2003] [Indexed: 11/18/2022] Open
Abstract
Targeting of secretory and membrane proteins by the signal recognition particle (SRP) is evolutionarily conserved, and the multidomain protein SRP54 acts as the key player in SRP-mediated protein transport. Binding of a signal peptide to SRP54 at the ribosome is coordinated with GTP binding and subsequent complex formation with the SRP receptor. Because these functions are localized to distinct domains of SRP54, communication between them is essential. We report the crystal structures of SRP54 from the Archaeon Sulfolobus solfataricus with and without its cognate SRP RNA binding site (helix 8) at 4-A resolution. The two structures show the flexibility of the SRP core and the position of SRP54 relative to the RNA. A long linker helix connects the GTPase (G domain) with the signal peptide binding (M) domain, and a hydrophobic contact between the N and M domains relates the signal peptide binding site to the G domain. Hinge regions are identified in the linker between the G and M domains (292-LGMGD) and in the N-terminal part of the M domain, which allow for structural rearrangements within SRP54 upon signal peptide binding at the ribosome.
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Affiliation(s)
- Ken R Rosendal
- Biochemie-Zentrum Heidelberg, INF 328, D-69120 Heidelberg, Germany
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38
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MacKenzie JA, Payne RM. Ribosomes specifically bind to mammalian mitochondria via protease-sensitive proteins on the outer membrane. J Biol Chem 2003; 279:9803-10. [PMID: 14668341 DOI: 10.1074/jbc.m307167200] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The interaction of ribosomes with specific components of membranes is one of the central themes to the co-translational targeting and import of proteins. To examine ribosome binding to mammalian mitochondria, we used ribosome-nascent chain complexes (RNCs) to follow the in vitro binding of ribosomes that correspond to the initial targeting stage of proteins. Mitochondria were found to contain a limited number of RNC binding sites on the outer membrane. It required more than twice the amount of non-translating ribosomes to inhibit RNC binding by one-half, indicating that RNCs have a competitive binding advantage. In addition, we found that RNCs bind mainly through the ribosomal component and not the nascent chain. RNCs bind via protease-sensitive proteins on the outer membrane, as well as by protease-insensitive components suggesting that two classes of receptors exist. We also show that binding is sensitive to cation conditions. Nearly all of the binding was inhibited in 0.5 m KCl, indicating that they interact with the membrane primarily through electrostatic interactions. In addition, disruption of RNC structure by removing magnesium causes the complete inhibition of binding under normal binding conditions indicating that it is the intact ribosome that is crucial for binding and not the nascent chain. These findings support the hypothesis that the outer mitochondrial membrane contains receptors specific for ribosomes, which would support the conditions necessary for co-translational import.
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Affiliation(s)
- James A MacKenzie
- Section on Cardiology, Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1081, USA
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39
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Mandon EC, Jiang Y, Gilmore R. Dual recognition of the ribosome and the signal recognition particle by the SRP receptor during protein targeting to the endoplasmic reticulum. J Cell Biol 2003; 162:575-85. [PMID: 12913112 PMCID: PMC2173783 DOI: 10.1083/jcb.200303143] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
We have analyzed the interactions between the signal recognition particle (SRP), the SRP receptor (SR), and the ribosome using GTPase assays, biosensor experiments, and ribosome binding assays. Possible mechanisms that could contribute to an enhanced affinity between the SR and the SRP-ribosome nascent chain complex to promote protein translocation under physiological ionic strength conditions have been explored. Ribosomes or 60S large ribosomal subunits activate the GTPase cycle of SRP54 and SRalpha by providing a platform for assembly of the SRP-SR complex. Biosensor experiments revealed high-affinity, saturable binding of ribosomes or large ribosomal subunits to the SR. Remarkably, the SR has a 100-fold higher affinity for the ribosome than for SRP. Proteoliposomes that contain the SR bind nontranslating ribosomes with an affinity comparable to that shown by the Sec61 complex. An NH2-terminal 319-residue segment of SRalpha is necessary and sufficient for binding of SR to the ribosome. We propose that the ribosome-SR interaction accelerates targeting of the ribosome nascent chain complex to the RER, while the SRP-SR interaction is crucial for maintaining the fidelity of the targeting reaction.
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Affiliation(s)
- Elisabet C Mandon
- Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605-2324, USA
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40
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Yan W, Wang WL, Zhu F, Chen SQ, Li QL, Wang L. Isolation of a novel member of small G protein superfamily and its expression in colon cancer. World J Gastroenterol 2003; 9:1719-24. [PMID: 12918107 PMCID: PMC4611530 DOI: 10.3748/wjg.v9.i8.1719] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: APMCF1 is a novel human gene whose transcripts are up-regulated in apoptotic MCF-7 cells. In order to learn more about this gene’s function in other tumors, we cloned its full length cDNA and prepared its polyclonal antibody to investigate its expression in colon cancers with immunohistochemistry.
METHODS: With the method of 5’ rapid amplification of cDNA end (RACE) and EST assembled in GenBank, we extended the length of APMCF1 at 5’ end. Then the sequence encoding the APMCF1 protein was amplified by RT-PCR from the total RNA of apoptotic MCF-7 cells and cloned into the prokaryotic expression vector pGEX-KG to construct recombinant expression vector pGEX-APMCF1. The GST-APMCF1 fusion protein was expressed in E. coli and used to immunize rabbits to get the rabbit anti-APMCF1 serum. The specificity of polyclonal anti-APMCF1 antibody was determined by Western blot. Then we investigated the expression of Apmcf1 in colon cancers and normal colonic mucosa with immunohistochemistry.
RESULTS: A cDNA fragment with a length of 1745 bp was obtained. APMCF1 was mapped to chromosome 3q22.2 and spanned at least 14.8 kb of genomic DNA with seven exons and six introns contained. Bioinformatic analysis showed the protein encoded by APMCF1 contained a small GTP-binding protein (G proteins) domain and was homologous to mouse signal recognition particle receptor β(SRβ). A coding region covering 816 bp was cloned and polyclonal anti-APMCF1 antibody was prepared successfully. The immunohistochemistry study showed that APMCF1 had a strong expression in colon cancer.
CONCLUSION: APMCF1 may be the gene coding human signal recognition particle receptor β and belongs to the small-G protein superfamily. Its strong expression pattern in colon cancer suggests it may play a role in colon cancer development.
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Affiliation(s)
- Wei Yan
- Department of Patholology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China
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41
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Legate KR, Andrews DW. The beta-subunit of the signal recognition particle receptor is a novel GTP-binding protein without intrinsic GTPase activity. J Biol Chem 2003; 278:27712-20. [PMID: 12759365 DOI: 10.1074/jbc.m302158200] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The beta-subunit of the signal recognition particle receptor (SRbeta), a member of the Ras family of small molecular weight GTPases, is involved in the targeting of nascent polypeptide chains to the protein translocation machinery in the endoplasmic reticulum membrane. We purified SRbeta from an expressing strain of Escherichia coli and investigated the properties of the isolated GTPase. We find that, unlike other Ras family GTPases, most SRbeta purifies bound to GTP, and SRbeta-bound GTP is not easily exchanged with solution GTP. SRbeta possesses no detectable GTPase activity. Although a stable interaction between SRbeta and ribosomes is observed, SRbeta is not stimulated to hydrolyze GTP when incubated with ribosomes or ribosome-nascent chains. A GTPase mutant harboring a mutation in a region predicted to be functionally important, based on observations made in related GTPases, binds GTP with faster kinetics and appears to be a less stable protein but otherwise displays similar properties to the wild-type SRbeta GTPase. Our results demonstrate that as an isolated GTPase, SRbeta functions differently from the Arf- and Ras-type GTPases that it is most closely related to by sequence.
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MESH Headings
- Animals
- Chromatography, High Pressure Liquid
- Cross-Linking Reagents/pharmacology
- DNA, Complementary/metabolism
- Dose-Response Relationship, Drug
- Endoplasmic Reticulum/metabolism
- Escherichia coli/metabolism
- GTP Phosphohydrolases/metabolism
- GTP-Binding Proteins/metabolism
- Guanosine Triphosphate/metabolism
- Humans
- Hydrolysis
- Intracellular Membranes/metabolism
- Kinetics
- Mutagenesis, Site-Directed
- Mutation
- Plasmids/metabolism
- Precipitin Tests
- Protein Binding
- Protein Structure, Tertiary
- Receptors, Cytoplasmic and Nuclear/chemistry
- Receptors, Cytoplasmic and Nuclear/physiology
- Receptors, Peptide/chemistry
- Receptors, Peptide/physiology
- Ribosomes/metabolism
- Saccharomyces cerevisiae/metabolism
- Spectrometry, Fluorescence
- Time Factors
- Ultraviolet Rays
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Affiliation(s)
- Kyle R Legate
- Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada
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42
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Nagai K, Oubridge C, Kuglstatter A, Menichelli E, Isel C, Jovine L. Structure, function and evolution of the signal recognition particle. EMBO J 2003; 22:3479-85. [PMID: 12853463 PMCID: PMC165607 DOI: 10.1093/emboj/cdg337] [Citation(s) in RCA: 117] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
The signal recognition particle (SRP) is a ribonucleoprotein particle essential for the targeting of signal peptide-bearing proteins to the prokaryotic plasma membrane or the eukaryotic endoplasmic reticulum membrane for secretion or membrane insertion. SRP binds to the signal peptide emerging from the exit site of the ribosome and forms a ribosome nascent chain (RNC)-SRP complex. The RNC-SRP complex then docks in a GTP-dependent manner with a membrane-anchored SRP receptor and the protein is translocated across or integrated into the membrane through a channel called the translocon. Recently considerable progress has been made in understanding the architecture and function of SRP.
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Affiliation(s)
- Kiyoshi Nagai
- MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
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43
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Helmers J, Schmidt D, Glavy JS, Blobel G, Schwartz T. The beta-subunit of the protein-conducting channel of the endoplasmic reticulum functions as the guanine nucleotide exchange factor for the beta-subunit of the signal recognition particle receptor. J Biol Chem 2003; 278:23686-90. [PMID: 12750387 DOI: 10.1074/jbc.c300180200] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Cotranslational protein transport to the endoplasmic reticulum is controlled by the concerted interaction of three GTPases: the SRP54 subunit of the signal recognition particle (SRP) and the alpha- and beta-subunits of the SRP receptor (SR). SRbeta is related to ADP-ribosylation factor (ARF)-type GTPases, and the recently published crystal structure of SRbeta-GTP in complex with the binding domain of SRalpha suggested that SRbeta, like all ARF-type GT-Pases, requires a guanine nucleotide exchange factor (GEF) for function. Searching the sequence data base, we identified significant sequence similarity between the Sec7 domain of ARF-GEFs and the cytosolic domains of the beta-subunits of the two homologous heterotrimeric protein-conducting channels in yeast. Using a fluorescence nucleotide exchange assay, we show that the beta-subunits of the heterotrimeric protein-conducting channels function as the GEFs for SRbeta. Both the cytosolic domain of Sec61beta as well as the holo-Sec61beta, when part of the isolated trimeric Sec61p complex, function as the GEF for SRbeta, whereas the same Sec61beta, when part of the heptameric complex that facilitates posttranslational protein transport, is inactive as the GEF for SRbeta
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Affiliation(s)
- Jurgen Helmers
- Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, USA.
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44
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Johnson AE, Chen JC, Flanagan JJ, Miao Y, Shao Y, Lin J, Bock PE. Structure, function, and regulation of free and membrane-bound ribosomes: the view from their substrates and products. COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY 2003; 66:531-41. [PMID: 12762055 DOI: 10.1101/sqb.2001.66.531] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Affiliation(s)
- A E Johnson
- Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, Departments of Chemistry and of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA
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45
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Koch HG, Moser M, Müller M. Signal recognition particle-dependent protein targeting, universal to all kingdoms of life. Rev Physiol Biochem Pharmacol 2003; 146:55-94. [PMID: 12605305 DOI: 10.1007/s10254-002-0002-9] [Citation(s) in RCA: 119] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/05/2022]
Abstract
The signal recognition particle (SRP) and its membrane-bound receptor represent a ubiquitous protein-targeting device utilized by organisms as different as bacteria and humans, archaea and plants. The unifying concept of SRP-dependent protein targeting is that SRP binds to signal sequences of newly synthesized proteins as they emerge from the ribosome. In eukaryotes this interaction arrests or retards translation elongation until SRP targets the ribosome-nascent chain complexes via the SRP receptor to the translocation channel. Such channels are present in the endoplasmic reticulum of eukaryotic cells, the thylakoids of chloroplasts, or the plasma membrane of prokaryotes. The minimal functional unit of SRP consists of a signal sequence-recognizing protein and a small RNA. The as yet most complex version is the mammalian SRP whose RNA, together with six proteinaceous subunits, undergo an intricate assembly process. The preferential substrates of SRP possess especially hydrophobic signal sequences. Interactions between SRP and its receptor, the ribosome, the signal sequence, and the target membrane are regulated by GTP hydrolysis. SRP-dependent protein targeting in bacteria and chloroplasts slightly deviate from the canonical mechanism found in eukaryotes. Pro- and eukaryotic cells harbour regulatory mechanisms to prevent a malfunction of the SRP pathway.
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Affiliation(s)
- H-G Koch
- Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Strasse 7, 79104, Freiburg, Germany.
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46
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Schwartz T, Blobel G. Structural basis for the function of the beta subunit of the eukaryotic signal recognition particle receptor. Cell 2003; 112:793-803. [PMID: 12654246 DOI: 10.1016/s0092-8674(03)00161-2] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
Protein translocation across and insertion into membranes is a process essential to all life forms. In higher eukaryotes, this process is initiated by targeting the translating ribosome to the endoplasmic reticulum via the signal recognition particle (SRP) and its membrane-associated heterodimeric receptor (SR). This targeting step is regulated by three G proteins, SRP54, SR alpha, and SR beta, which act in concert. Little is known about the regulatory role of SR beta. Here, we present the 1.7 A crystal structure of the SR beta-GTP subunit in complex with the interaction domain of SR alpha. Strikingly, the binding interface overlaps largely with the switch 1 region of SR beta. This finding, together with additional biochemical data, shows that the eukaryotic SR is a conditional and not an obligate heterodimer. The results suggest that the GTP/GDP switch cycle of SR beta functions as a regulatory switch for the receptor dimerization. We discuss the implications for the translocation pathway.
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Affiliation(s)
- Thomas Schwartz
- Howard Hughes Medical Institute, Laboratory of Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
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47
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Schnell DJ, Hebert DN. Protein translocons: multifunctional mediators of protein translocation across membranes. Cell 2003; 112:491-505. [PMID: 12600313 DOI: 10.1016/s0092-8674(03)00110-7] [Citation(s) in RCA: 165] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
Protein translocation systems consist of complex molecular machines whose activities are not limited to unidirectional protein targeting. Protein translocons and their associated receptor systems can be viewed as dynamic modular units whose interactions, and therefore functions, are regulated in response to specific signals. This flexibility allows translocons to interact with multiple signal receptor systems to manage the targeting of topologically distinct classes of proteins, to mediate targeting to different suborganellar compartments, and to respond to stress and developmental cues. Furthermore, the activities of translocons are tightly coordinated with downstream events, thereby providing a direct link between targeting and protein maturation.
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Affiliation(s)
- Danny J Schnell
- Program in Plant Biology, University of Massachusetts, Amherst, MA 01003, USA.
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48
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Abstract
The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein that associates with ribosomes to mediate the targeting of membrane and secretory proteins to biological membranes. In higher eukaryotes, SRP biogenesis involves the sequential binding of SRP19 and SRP54 proteins to the S domain of 7S RNA. The recently determined crystal structures of SRP19 in complex with the S domain, and that of the ternary complex of SRP19, the S domain and the M domain of SRP54, provide insight into the molecular basis of S-domain assembly and SRP function.
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49
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Pool MR, Stumm J, Fulga TA, Sinning I, Dobberstein B. Distinct modes of signal recognition particle interaction with the ribosome. Science 2002; 297:1345-8. [PMID: 12193787 DOI: 10.1126/science.1072366] [Citation(s) in RCA: 145] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
Signal recognition particle (SRP), together with its receptor (SR), mediates the targeting of ribosome-nascent chain complexes to the endoplasmic reticulum. Using protein cross-linking, we detected distinct modes in the binding of SRP to the ribosome. During signal peptide recognition, SRP54 is positioned at the exit site close to ribosomal proteins L23a and L35. When SRP54 contacts SR, SRP54 is rearranged such that it is no longer close to L23a. This repositioning may allow the translocon to dock with the ribosome, leading to insertion of the signal peptide into the translocation channel.
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Affiliation(s)
- Martin R Pool
- Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), D-69120 Heidelberg, Germany.
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50
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Abstract
The signal recognition particle (SRP) and its membrane-associated receptor (SR) catalyze targeting of nascent secretory and membrane proteins to the protein translocation apparatus of the cell. Components of the SRP pathway and salient features of the molecular mechanism of SRP-dependent protein targeting are conserved in all three kingdoms of life. Recent advances in the structure determination of a number of key components in the eukaryotic and prokaryotic SRP pathway provide new insight into the molecular basis of SRP function, and they set the stage for future work toward an integrated picture that takes into account the dynamic and contextual properties of this remarkable cellular machine.
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Affiliation(s)
- R J Keenan
- Maxygen, 515 Galveston Drive, Redwood City, California 94063, USA.
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