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Alibhoy AA, Chiang HL. Vacuole import and degradation pathway: Insights into a specialized autophagy pathway. World J Biol Chem 2011; 2:239-45. [PMID: 22125667 PMCID: PMC3224871 DOI: 10.4331/wjbc.v2.i11.239] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/29/2011] [Revised: 08/30/2011] [Accepted: 11/06/2011] [Indexed: 02/05/2023] Open
Abstract
Glucose deprivation induces the synthesis of pivotal gluconeogenic enzymes such as fructose-1,6-bisphosphatase, malate dehydrogenase, phosphoenolpyruvate carboxykinase and isocitrate lyase in Saccharomyces cerevisiae. However, following glucose replenishment, these gluconeogenic enzymes are inactivated and degraded. Studies have characterized the mechanisms by which these enzymes are inactivated in response to glucose. The site of degradation of these proteins has also been ascertained to be dependent on the duration of starvation. Glucose replenishment of short-term starved cells results in these proteins being degraded in the proteasome. In contrast, addition of glucose to cells starved for a prolonged period results in these proteins being degraded in the vacuole. In the vacuole dependent pathway, these proteins are sequestered in specialized vesicles termed vacuole import and degradation (Vid). These vesicles converge with the endocytic pathway and deliver their cargo to the vacuole for degradation. Recent studies have identified that internalization, as mediated by actin polymerization, is essential for delivery of cargo proteins to the vacuole for degradation. In addition, components of the target of rapamycin complex 1 interact with cargo proteins during glucose starvation. Furthermore, Tor1p dissociates from cargo proteins following glucose replenishment. Future studies will be needed to elaborate on the importance of internalization at the plasma membrane and the subsequent import of cargo proteins into Vid vesicles in the vacuole dependent degradation pathway.
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Affiliation(s)
- Abbas A Alibhoy
- Abbas A Alibhoy, Hui-Ling Chiang, Department of Cellular and Molecular Physiology, Penn State University College of Medicine, Hershey, PA 17033, United States
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Effect of PrA encoding gene-PEP4 deletion in industrial S. cerevisiae WZ65 on key enzymes in relation to the glycolytic pathway. Eur Food Res Technol 2010. [DOI: 10.1007/s00217-010-1355-y] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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Brown CR, Chiang HL. A selective autophagy pathway that degrades gluconeogenic enzymes during catabolite inactivation. Commun Integr Biol 2009; 2:177-83. [PMID: 19513275 PMCID: PMC2686377 DOI: 10.4161/cib.7711] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2008] [Accepted: 12/23/2008] [Indexed: 11/19/2022] Open
Abstract
In Saccharomyces cerevisiae, glucose starvation induces key gluconeogenic enzymes such as fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase (MDH2) and phosphoenolpyruvate carboxykinase, while glucose addition inactivates these enzymes. Significant progress has been made identifying mechanisms that mediate the "catabolite inactivation" of FBPase and MDH2. For example, the site of their degradation has been shown to change, depending on the duration of starvation. When glucose is added to short-termed starved cells, these proteins are degraded in the proteasome. However, when glucose is added to long-termed starved cells, they are degraded in the vacuole by a selective autophagy pathway. For the vacuole pathway, these proteins are first imported into novel vesicles called Vid (vacuole import and degradation) vesicles. Following import, Vid vesicles merge with the endocytic pathway. Future experiments will be directed at understanding the molecular mechanisms that regulate the switch from proteasomal to vacuolar degradation and determining the site of Vid vesicle biogenesis.
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Affiliation(s)
- C Randell Brown
- Department of Cellular and Molecular Physiology; Penn State College of Medicine; Hershey, Pennsylvania USA
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Winderickx J, Holsbeeks I, Lagatie O, Giots F, Thevelein J, de Winde H. From feast to famine; adaptation to nutrient availability in yeast. ACTA ACUST UNITED AC 2002. [DOI: 10.1007/3-540-45611-2_7] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/14/2023]
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Kim J, Scott SV, Klionsky DJ. Alternative protein sorting pathways. INTERNATIONAL REVIEW OF CYTOLOGY 2000; 198:153-201. [PMID: 10804463 DOI: 10.1016/s0074-7696(00)98005-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
The term "nonclassical protein targeting" has been used to describe those pathways that have been recently discovered and differ mechanistically from the more studied "classical pathways." Because this nomenclature is rather arbitrary in terms of cellular relevance, we have chosen to group these protein sorting mechanisms under the heading "alternative protein sorting pathways" for the purpose of this review. Many of the alternative targeting pathways described are of primary importance. For example, without retrograde transport, both membrane material and targeting machinery accumulate at distal sites in the endomembrane system, preventing anterograde transport. Further, lysosome/vacuole delivery of degradative substrates by autophagic pathways is central to the role of this organelle as a primary site for intracellular degradation. Finally, targeting through the classical CPY pathway requires the ALP pathway for delivery of the vacuolar t-SNARE Vam3p. Analysis of these alternative targeting pathways provides a more complete understanding of eukaryotic cellular physiology.
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Affiliation(s)
- J Kim
- Section of Microbiology, University of California, Davis 95616, USA
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Chiang MC, Chiang HL. Vid24p, a novel protein localized to the fructose-1, 6-bisphosphatase-containing vesicles, regulates targeting of fructose-1,6-bisphosphatase from the vesicles to the vacuole for degradation. J Cell Biol 1998; 140:1347-56. [PMID: 9508768 PMCID: PMC2132677 DOI: 10.1083/jcb.140.6.1347] [Citation(s) in RCA: 71] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Glucose regulates the degradation of the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), in Saccharomyces cerevisiae. FBPase is targeted from the cytosol to a novel type of vesicle, and then to the vacuole for degradation when yeast cells are transferred from medium containing poor carbon sources to fresh glucose. To identify proteins involved in the FBPase degradation pathway, we cloned our first VID (vacuolar import and degradation) gene. The VID24 gene was identified by complementation of the FBPase degradation defect of the vid24-1 mutant. Vid24p is a novel protein of 41 kD and is synthesized in response to glucose. Vid24p is localized to the FBPase-containing vesicles as a peripheral membrane protein. In the absence of functional Vid24p, FBPase accumulates in the vesicles and fails to move to the vacuole, suggesting that Vid24p regulates FBPase targeting from the vesicles to the vacuole. FBPase sequestration into the vesicles is not affected in the vid24-1 mutant, indicating that Vid24p acts after FBPase sequestration into the vesicles has occurred. Vid24p is the first protein identified that marks the FBPase-containing vesicles and plays a critical role in delivering FBPase from the vesicles to the vacuole for degradation.
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Affiliation(s)
- M C Chiang
- Department of Cell Biology, Harvard Medical School, Boston, Massachussets 02115, USA
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Abstract
Recent characterization of the major protein-targeting systems in both yeast and mammalian cells has provided detailed descriptions of how cellular transport processes operate. Increasingly, however, novel protein-sorting mechanisms are being uncovered. These newly discovered 'alternative' mechanisms of protein sorting ensure accurate delivery of numerous cellular constituents either to their resident compartment or, in many cases, to the cellular protein-degradation machinery. Like the better characterized 'classical' protein-sorting systems, 'nonclassical' targeting mechanisms involve both membrane translocation through protein channels and vesicle-mediated transport. This review discusses our current understanding of these nonclassical protein-sorting pathways and their role in eukaryotic cells.
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8
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Huang PH, Chiang HL. Identification of novel vesicles in the cytosol to vacuole protein degradation pathway. J Cell Biol 1997; 136:803-10. [PMID: 9049246 PMCID: PMC2132494 DOI: 10.1083/jcb.136.4.803] [Citation(s) in RCA: 84] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/1996] [Revised: 12/10/1996] [Indexed: 02/03/2023] Open
Abstract
The key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae are starved of glucose. FBPase is targeted from the cytosol to the yeast vacuole for degradation when glucose-starved cells are replenished with fresh glucose. Several vid mutants defective in the glucose-induced degradation of FBPase in the vacuole have been isolated. In some vid mutants, FBPase is found in punctate structures in the cytoplasm. When extracts from these cells are fractionated, a substantial amount of FBPase is sedimentable in the high speed pellet, suggesting that FBPase is associated with intracellular structures in these vid mutants. In this paper we investigated whether FBPase association with intracellular structures also existed in wild-type cells. We report the purification of novel FBPase-associated vesicles from wild-type cells to near homogeneity. Kinetic studies indicate that FBPase association with these vesicles is stimulated by glucose and occurs only transiently, suggesting that these vesicles are intermediate in the FBPase degradation pathway. Fractionation analysis demonstrates that these vesicles are distinct from known organelles such as the vacuole, ER, Golgi, mitochondria, peroxisomes, endosomes, COPI, or COPII vesicles. Under EM, these vesicles are 30-40 nm in diam. Proteinase K experiments indicate that the majority of FBPase is sequestered inside the vesicles. We propose that FBPase is imported into these vesicles before entering the vacuole.
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Affiliation(s)
- P H Huang
- Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA
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Betting J, Seufert W. A yeast Ubc9 mutant protein with temperature-sensitive in vivo function is subject to conditional proteolysis by a ubiquitin- and proteasome-dependent pathway. J Biol Chem 1996; 271:25790-6. [PMID: 8824207 DOI: 10.1074/jbc.271.42.25790] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
The UBC9 gene of the yeast Saccharomyces cerevisiae is essential for cell viability and encodes a soluble protein of the nucleus that is metabolically stable. Products of mutant alleles selected to confer temperature-sensitive in vivo function were found to be extremely short-lived at the restrictive but long-lived at the permissive condition. An extragenic suppressor mutation was isolated which increased thermoresistance of a ubc9-1 strain. This suppressor turned out to stabilize the mutated gene product, indicating that the physiological activity of ubc9-1 protein is primarily controlled by conditional proteolysis. The labile ubc9-1 protein appears to be a substrate for ubiquitination, and its turnover was substantially reduced by expression of a ubiquitin derivative that interferes with formation of multi-ubiquitin chains. Stabilization resulted also from competitive inhibition of Ubc4-related ubiquitin-conjugating enzymes. Activity of the proteasome complex was crucial to rapid breakdown, whereas vacuolar proteases were dispensable. Thus, the heat-denatured ubc9-1 protein is targeted for proteolysis by the ubiquitin-proteasome pathway and may serve as a useful tool to further define the process by which a misfolded polypeptide is recognized.
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Affiliation(s)
- J Betting
- Institut für Genetik und Mikrobiologie, Universität München, Maria-Ward-Strasse 1a, D-80638 München, Germany
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Chiang HL, Schekman R, Hamamoto S. Selective uptake of cytosolic, peroxisomal, and plasma membrane proteins into the yeast lysosome for degradation. J Biol Chem 1996; 271:9934-41. [PMID: 8626630 DOI: 10.1074/jbc.271.17.9934] [Citation(s) in RCA: 88] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
When glucose-starved cells are replenished with glucose, the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is selectively targeted from the cytosol to the yeast lysosome (vacuole) for degradation. The glucose-induced targeting of FBPase to the vacuole for degradation occurs in cells grown under a variety of metabolic conditions. Immunoelectron microscopic studies demonstrate that the uptake of FBPase by the vacuole is mediated in part by an autophagic process. FBPase can be found on the vacuolar membrane and also at the sites of membrane invaginations. Furthermore, FBPase is associated with different forms of vesicles, which are induced to accumulate inside the vacuole. We have identified peroxisomes as the organelles that are delivered to the vacuole for degradation when cells are replenished with glucose. Ultrastructural studies indicate that peroxisomes are engulfed by the vacuole by an autophagic process, leading to the destruction of whole organelles in the vacuole. Furthermore, the galactose transporter (Gal2p) is also delivered from the plasma membrane to the vacuole for degradation in response to glucose. Gal2p is delivered to the vacuole through the endocytic pathway, as mutants defective in receptor-mediated endocytosis fail to degrade Gal2p in response to glucose.
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Affiliation(s)
- H L Chiang
- Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA
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Medintz I, Jiang H, Han EK, Cui W, Michels CA. Characterization of the glucose-induced inactivation of maltose permease in Saccharomyces cerevisiae. J Bacteriol 1996; 178:2245-54. [PMID: 8636025 PMCID: PMC177932 DOI: 10.1128/jb.178.8.2245-2254.1996] [Citation(s) in RCA: 71] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
The addition of glucose to maltose-fermenting Saccharomyces cerevisiae cells causes a rapid and irreversible loss of the ability to transport maltose, resulting both from the repression of transcription of the maltose permease gene and from the inactivation of maltose permease. The latter is referred to as glucose-induced inactivation or catabolite inactivation. We describe an analysis of this process in a maltose-fermenting strain expressing a hemagglutinin (HA)-tagged allele of MAL61, encoding maltose permease. The transfer of maltose-induced cells expressing the Mal61/HA protein to rich medium containing glucose produces a decrease in maltose transport rates which is paralleled by a decrease in Mal61/HA maltose permease protein levels. In nitrogen starvation medium, glucose produces a biphasic inactivation, i.e., an initial, rapid loss in transport activity (inhibition) followed by a slower decrease in transport activity, which correlates with a decrease in the amount of maltose permease protein (proteolysis). The inactivation in both rich and nitrogen-starved media results from a decrease in Vmax with no apparent change in Km. Using strains carrying mutations in END3, REN1(VPS2), PEP4, and PRE1 PRE2, we demonstrate that the proteolysis of Mal61/HAp is dependent on endocytosis and vacuolar proteolysis and is independent of the proteosome. Moreover, we show that the Mal61/HA maltose permease is present in differentially phosphorylated forms.
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Affiliation(s)
- I Medintz
- Biology Department, Queens College, City University of New York, Flushing 11367, USA
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Abstract
The yeast vacuole, which is equivalent to the lysosome of higher eukaryotes, is one of the best characterized degradative organelles. This review describes the biosynthesis and function of yeast vacuolar proteases. Most of these enzymes are delivered to the vacuole via the early compartments of the secretory pathway and the endosome, while one of them is directly imported from the cytoplasm. The proteases are synthesized as precursors which undergo many post-translational modifications before the final active form is generated. Proteolytic activation by developments in the analysis of the functions of vacuolar proteolysis are described. Substrates of the vacuolar proteases are mostly imported via endocytosis or autophagocytosis, and vacuolar proteolysis appears to be mainly important under nutritional stress conditions and sporulation.
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Affiliation(s)
- H B Van Den Hazel
- Department of Yeast Genetics, Carlsberg Laboratory, Copenhagen-Valby, Denmark
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Abstract
Protein degradation is an essential process in cells. Degradation of intracellular proteins increases when cells are starved of nutrients. Lysosomes are responsible for the enhanced protein degradation during starvation. To understand the degradation process that occurs in lysosomes, we studied the catabolite inactivation of fructose-1,6-bisphosphatase (FBPase) in Saccharomyces cerevisiae. Fructose-1,6-bisphosphatase, a key enzyme in the gluconeogenesis pathway, is induced when cells are starved of glucose and is degraded when cells are replenished with glucose. We have shown that catabolite inactivation of FBPase is mediated by a selective import of the enzyme into the vacuole (yeast lysosome) for degradation. Glucose-induced degradation of FBPase serves to regulate metabolism to prevent the energy futile cycle. In addition to FBPase, we have also demonstrated that peroxisomes, which are important in the oxidation of fatty acids, are delivered to the vacuole for degradation in response to glucose. Furthermore, the galactose transporter, which is induced when cells are grown in galactose, is internalized and delivered to the vacuole for degradation when cells are transferred to glucose. Key words: protein degradation, yeast vacuole, catabolite inactivation, fructose-1,6-bisphosphatase, galactose permease, autophagic vacuole.
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