RESUMO
The vacuole/lysosome is a primary catabolic site in the eukaryotic cell. One implication of its cellular role is that delivery systems must exist to target both hydrolytic enzymes and substrates destined for degradation to this organelle. A number of nonclassical vacuolar targeting pathways that deliver degradative substrates and at least one resident enzyme from the cytosol to the vacuole have recently been described. The pathways identified so far include cytoplasm to vacuole targeting, macroautophagy, pexophagy and vacuolar import and degradation. Cytological, genetic and molecular genetic approaches have begun to provide insight into the molecular basis of these processes.
Assuntos
Citoplasma/metabolismo , Organelas/metabolismo , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Vacúolos/metabolismo , Aminopeptidases/metabolismo , Transporte Biológico , Meios de Cultura , Endopeptidases/metabolismo , Microcorpos/metabolismo , Mitocôndrias/metabolismo , FagossomosRESUMO
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.
RESUMO
Several protein-targeting fields have recently converged in their observations of what once was thought to be a rare phenomenon: the transport of folded and oligomerized proteins across membranes. Three of the newly characterized pathways that are known to accommodate folded substrates are the peroxisomal targeting machinery for matrix proteins, the twin-arginine translocation (Tat) of bacteria and the related DeltapH-dependent pathway of plant plastids, and the cytoplasm-to-vacuole targeting (Cvt) pathway in Saccharomyces cerevisiae. Current work strives to understand the molecular mechanisms that accomplish transport of folded substrates. The aim of this commentary is to highlight our knowledge of transport mechanisms, point out areas for future research and address how paradigms of classical protein translocation have shaped current views.
Assuntos
Membrana Celular/metabolismo , Dobramento de Proteína , Proteínas/metabolismo , Transporte Biológico , Humanos , Proteínas/químicaRESUMO
The Saccharomyces cerevisiae APE1 gene product, aminopeptidase I (API), is a soluble hydrolase that has been shown to be localized to the vacuole. API lacks a standard signal sequence and contains an unusual amino-terminal propeptide. We have examined the biosynthesis of API in order to elucidate the mechanism of its delivery to the vacuole. API is synthesized as an inactive precursor that is matured in a PEP4-dependent manner. The half-time for processing is approximately 45 min. The API precursor remains in the cytoplasm after synthesis and does not enter the secretory pathway. The precursor does not receive glycosyl modifications, and removal of its propeptide occurs in a sec-independent manner. Neither the precursor nor mature form of API are secreted into the extracellular fraction in vps mutants or upon overproduction, two additional characteristics of soluble vacuolar proteins that transit through the secretory pathway. Overproduction of API results in both an increase in the half-time of processing and the stable accumulation of precursor protein. These results suggest that API enters the vacuole by a posttranslational process not used by most previously studied resident vacuolar proteins and will be a useful model protein to analyze this alternative mechanism of vacuolar localization.
Assuntos
Aminopeptidases/farmacocinética , Compartimento Celular , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Aminopeptidases/análise , Aminopeptidases/biossíntese , Transporte Biológico , Concanavalina A/farmacologia , Genes Fúngicos , Glicosilação/efeitos dos fármacos , Glicosiltransferases/metabolismo , Mutagênese Insercional , Precursores de Proteínas/análise , Precursores de Proteínas/biossíntese , Precursores de Proteínas/farmacocinética , Tunicamicina/farmacologia , Vacúolos/químicaRESUMO
Autophagy is a degradative pathway by which cells sequester nonessential, bulk cytosol into double-membrane vesicles (autophagosomes) and deliver them to the vacuole for recycling. Using this strategy, eukaryotic cells survive periods of nutritional starvation. Under nutrient-rich conditions, autophagy machinery is required for the delivery of a resident vacuolar hydrolase, aminopeptidase I, by the cytoplasm to vacuole targeting (Cvt) pathway. In both pathways, the vesicle formation process requires the function of the starvation-induced Aut7 protein, which is recruited from the cytosol to the forming Cvt vesicles and autophagosomes. The membrane binding of Aut7p represents an early step in vesicle formation. In this study, we identify several requirements for Aut7p membrane association. After synthesis in the cytosol, Aut7p is proteolytically cleaved in an Aut2p-dependent manner. While this novel processing event is essential for Aut7p membrane binding, Aut7p must undergo additional physical interactions with Aut1p and the autophagy (Apg) conjugation complex before recruitment to the membrane. Lack of these interactions results in a cytosolic distribution of Aut7p rather than localization to forming Cvt vesicles and autophagosomes. This study assigns a functional role for the Apg conjugation system as a mediator of Aut7p membrane recruitment. Further, we demonstrate that Aut1p, which physically interacts with components of the Apg conjugation complex and Aut7p, constitutes an additional factor required for Aut7p membrane recruitment. These findings define a series of steps that results in the modification of Aut7p and its subsequent binding to the sequestering transport vesicles of the autophagy and cytoplasm to vacuole targeting pathways.
Assuntos
Autofagia/fisiologia , Cisteína Endopeptidases/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases , Proteínas de Saccharomyces cerevisiae , Transdução de Sinais/fisiologia , Animais , Proteína 5 Relacionada à Autofagia , Proteína 7 Relacionada à Autofagia , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Biomarcadores , Membrana Celular/metabolismo , Cisteína Endopeptidases/genética , Citoplasma/metabolismo , Proteínas Fúngicas/genética , Proteínas de Fluorescência Verde , Proteínas de Choque Térmico/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Mutagênese , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Coelhos , Saccharomyces cerevisiae , Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína Ligases , Vacúolos/metabolismoRESUMO
Although the majority of known vacuolar proteins transit through the secretory pathway, two vacuole-resident proteins have been identified that reach this organelle by an alternate pathway. These polypeptides are targeted to the vacuole directly from the cytoplasm by a novel import mechanism. The best characterized protein that uses this pathway is aminopeptidase I (API). API is synthesized as a cytoplasmic precursor containing an amino-terminal propeptide that is cleaved off when the protein reaches the vacuole. To dissect the biochemistry of this pathway, we have reconstituted the targeting of API in vitro in a permeabilized cell system. Based on several criteria, the in vitro import assay faithfully reconstitutes the in vivo reaction. After incubation under import conditions, API is processed by a vacuolar-resident protease, copurifies with a vacuole-enriched fraction, and becomes inaccessible to the cytoplasm. These observations demonstrate that API has passed from the cytoplasm to the vacuole. The reconstituted import process is dependent on time, temperature, and energy. ATP gamma S inhibits this reaction, indicating that API transport is ATP driven. API import is also inhibited by GTP gamma S, suggesting that this process may be mediated by a GTP-binding protein. In addition, in vitro import requires a functional vacuolar ATPase; import is inhibited both in the presence of the specific V-ATPase inhibitor bafilomycin A1, and in a yeast strain in which one of the genes encoding a V-ATPase subunit has been disrupted.
Assuntos
Citoplasma/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Vacúolos/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/fisiologia , Aminopeptidases/genética , Aminopeptidases/metabolismo , Transporte Biológico/fisiologia , Compartimento Celular/fisiologia , Membrana Celular/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Hidrólise , Mutação/fisiologia , Saccharomyces cerevisiae/citologia , Temperatura , Fatores de TempoRESUMO
Rapamycin, an antifungal macrolide antibiotic, mimics starvation conditions in Saccharomyces cerevisiae through activation of a general G(0) program that includes widespread effects on translation and transcription. Macroautophagy, a catabolic membrane trafficking phenomenon, is a prominent part of this response. Two views of the induction of autophagy may be considered. In one, up-regulation of proteins involved in autophagy causes its induction, implying that autophagy is the result of a signal transduction mechanism leading from Tor to the transcriptional and translational machinery. An alternative hypothesis postulates the existence of a dedicated signal transduction mechanism that induces autophagy directly. We tested these possibilities by assaying the effects of cycloheximide and specific mutations on the induction of autophagy. We find that induction of autophagy takes place in the absence of de novo protein synthesis, including that of specific autophagy-related proteins that are up-regulated in response to rapamycin. We also find that dephosphorylation of Apg13p, a signal transduction event that correlates with the onset of autophagy, is also independent of new protein synthesis. Finally, our data indicate that autophagosomes that form in the absence of protein synthesis are significantly smaller than normal, indicating a role for de novo protein synthesis in the regulation of autophagosome expansion. Our results define the existence of a signal transduction-dependent nucleation step and a separate autophagosome expansion step that together coordinate autophagosome biogenesis.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Autofagia/fisiologia , Fosfatidilinositol 3-Quinases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/fisiologia , Vacúolos/enzimologia , Proteínas Adaptadoras de Transdução de Sinal , Aminopeptidases/metabolismo , Antifúngicos/farmacologia , Autofagia/efeitos dos fármacos , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteínas de Ciclo Celular , Cicloeximida/farmacologia , Citoplasma/enzimologia , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/fisiologia , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/fisiologia , Nitrogênio/farmacologia , Fenótipo , Fosfoproteínas/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Sirolimo/farmacologia , Proteínas de Transporte VesicularRESUMO
The yeast vacuolar protein aminopeptidase I (API) is synthesized as a cytosolic precursor that is transported to the vacuole by a nonclassical targeting mechanism. Recent genetic studies indicate that the biosynthetic pathway that transports API uses many of the same molecular components as the degradative autophagy pathway. This overlap coupled with both in vitro and in vivo analysis of API import suggested that, like autophagy, API transport is vesicular. Subcellular fractionation experiments demonstrate that API precursor (prAPI) initially enters a nonvacuolar cytosolic compartment. In addition, subvacuolar vesicles containing prAPI were purified from a mutant strain defective in breakdown of autophagosomes, further indicating that prAPI enters the vacuole inside a vesicle. The purified subvacuolar vesicles do not appear to contain vacuolar marker proteins. Immunogold EM confirms that prAPI is localized in cytosolic and in subvacuolar vesicles in a mutant strain defective in autophagic body degradation. These data suggest that cytosolic vesicles containing prAPI fuse with the vacuole to release a membrane-bounded intermediate compartment that is subsequently broken down, allowing API maturation.
Assuntos
Aminopeptidases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Vacúolos/metabolismo , Aminopeptidases/genética , Animais , Transporte Biológico , Microscopia Imunoeletrônica , Mutação , Precursores de Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , TemperaturaRESUMO
Yeast vacuole protein targeting (vpt) mutants exhibit defects in the sorting and processing of multiple vacuolar hydrolases. To evaluate the impact these vpt mutations have on the biogenesis and functioning of the lysosome-like vacuole, we have used light and electron microscopic techniques to analyze the vacuolar morphology in the mutants. These observations have permitted us to assign the vpt mutants to three distinct classes. The class A vpt mutants (26 complementation groups) contain 1-3 large vacuoles that are morphologically indistinguishable from those in the parental strain, suggesting that only a subset of the proteins destined for delivery to this compartment is mislocalized. One class A mutant (vpt13) is very sensitive to low pH and exhibits a defect in vacuole acidification. Consistent with a potential role for vacuolar pH in protein sorting, we found that bafilomycin A1, a specific inhibitor of the vacuolar ATPase, as well as the weak base ammonium acetate and the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, collapse the pH gradient across the vacuolar membrane and cause the missorting and secretion of two vacuolar hydrolases in wild-type cells. Mutants in the three class B vpt complementation groups exhibit a fragmented vacuole morphology. In these mutants, no large normal vacuoles are observed. Instead, many (20-40) smaller vacuole-like organelles accumulate. The class C vpt mutants, which constitute four complementation groups, exhibit extreme defects in vacuole biogenesis. The mutants lack any organelle resembling a normal vacuole but accumulate other organelles including vesicles, multilamellar membrane structures, and Golgi-related structures. Heterozygous class C zygotes reassemble normal vacuoles rapidly, indicating that some of the accumulated aberrant structures may be intermediates in vacuole formation. These class C mutants also exhibit sensitivity to osmotic stress, suggesting an osmoregulatory role for the vacuole. The vpt mutants should provide insights into the normal physiological role of the vacuole, as well as allowing identification of components required for vacuole protein sorting and/or vacuole assembly.
Assuntos
Compartimento Celular , Saccharomyces cerevisiae/genética , Vacúolos/ultraestrutura , Proteínas Fúngicas/fisiologia , Teste de Complementação Genética , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Peso Molecular , Morfogênese , Mutação , Fenótipo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/ultraestrutura , Vacúolos/fisiologiaRESUMO
Aminopeptidase I (API) is transported into the yeast vacuole by the cytoplasm to vacuole targeting (Cvt) pathway. Genetic evidence suggests that autophagy, a major degradative pathway in eukaryotes, and the Cvt pathway share largely the same cellular machinery. To understand the mechanism of the Cvt import process, we examined the native state of API. Dodecameric assembly of precursor API in the cytoplasm and membrane binding were rapid events, whereas subsequent vacuolar import appeared to be rate limiting. A unique temperature-sensitive API-targeting mutant allowed us to kinetically monitor its oligomeric state during translocation. Our findings indicate that API is maintained as a dodecamer throughout its import and will be useful to study the posttranslational movement of folded proteins across biological membranes.
Assuntos
Aminopeptidases/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Autofagia , Transporte Biológico , Citoplasma/metabolismo , Proteínas Fúngicas/metabolismo , Membranas Intracelulares/metabolismo , Substâncias Macromoleculares , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/enzimologia , Deleção de Sequência , Vacúolos/enzimologia , Vacúolos/metabolismoRESUMO
Stress conditions lead to a variety of physiological responses at the cellular level. Autophagy is an essential process used by animal, plant, and fungal cells that allows for both recycling of macromolecular constituents under conditions of nutrient limitation and remodeling the intracellular structure for cell differentiation. To elucidate the molecular basis of autophagic protein transport to the vacuole/lysosome, we have undertaken a morphological and biochemical analysis of this pathway in yeast. Using the vacuolar hydrolase aminopeptidase I (API) as a marker, we provide evidence that the autophagic pathway overlaps with the biosynthetic pathway, cytoplasm to vacuole targeting (Cvt), used for API import. Before targeting, the precursor form of API is localized mostly in restricted regions of the cytosol as a complex with spherical particles (termed Cvt complex). During vegetative growth, the Cvt complex is selectively wrapped by a membrane sac forming a double membrane-bound structure of approximately 150 nm diam, which then fuses with the vacuolar membrane. This process is topologically the same as macroautophagy induced under starvation conditions in yeast (Baba, M., K. Takeshige, N. Baba, and Y. Ohsumi. 1994. J. Cell Biol. 124:903-913). However, in contrast with autophagy, API import proceeds constitutively in growing conditions. This is the first demonstration of the use of an autophagy-like mechanism for biosynthetic delivery of a vacuolar hydrolase. Another important finding is that when cells are subjected to starvation conditions, the Cvt complex is now taken up by an autophagosome that is much larger and contains other cytosolic components; depending on environmental conditions, the cell uses an alternate pathway to sequester the Cvt complex and selectively deliver API to the vacuole. Together these results indicate that two related but distinct autophagy-like processes are involved in both biogenesis of vacuolar resident proteins and sequestration of substrates to be degraded.
Assuntos
Aminopeptidases/metabolismo , Autofagia , Citoplasma/metabolismo , Lisossomos/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Vacúolos/metabolismo , Transporte Biológico , Meios de Cultura , Citoplasma/ultraestrutura , Membranas Intracelulares/metabolismo , Lisossomos/ultraestrutura , Microscopia Imunoeletrônica , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Vacúolos/ultraestruturaRESUMO
In Saccharomyces cerevisiae the vacuolar protein aminopeptidase I (API) is localized to the vacuole independent of the secretory pathway. The alternate targeting mechanism used by this protein has not been characterized. API is synthesized as a 61-kD soluble cytosolic precursor. Upon delivery to the vacuole, the amino-terminal propeptide is removed by proteinase B (PrB) to yield the mature 50-kD hydrolase. We exploited this delivery-dependent maturation event in a mutant screen to identify genes whose products are involved in API targeting. Using antiserum to the API propeptide, we isolated mutants that accumulate precursor API. These mutants, designated cvt, fall into eight complementation groups, five of which define novel genes. These five complementation groups exhibit a specific defect in maturation of API, but do not have a significant effect on vacuolar protein targeting through the secretory pathway. Localization studies show that precursor API accumulates outside of the vacuole in all five groups, indicating that they are blocked in API targeting and/or translocation. Future analysis of these gene products will provide information about the subcellular components involved in this alternate mechanism of vacuolar protein localization.
Assuntos
Citoplasma/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Vacúolos/fisiologia , Alelos , Aminopeptidases/metabolismo , Testes Genéticos , Concentração de Íons de Hidrogênio , Cinética , Mutação/fisiologia , Fenótipo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle. When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters bulk cytosol for delivery, breakdown, and recycling in the vacuole by the autophagy pathway. Each of these overlapping alternative transport pathways is specifically mobilized depending on environmental cues. The basic mechanism of cargo packaging and delivery involves the formation of a double-membrane transport vesicle around prAPI and/or bulk cytosol. Upon completion, these Cvt and autophagic vesicles are targeted to the vacuole to allow delivery of their lumenal contents. Key questions remain regarding the origin and formation of the transport vesicle. In this study, we have cloned the APG9/CVT7 gene and characterized the gene product. Apg9p/Cvt7p is the first characterized integral membrane protein required for Cvt and autophagy transport. Biochemical and morphological analyses indicate that Apg9p/Cvt7p is localized to large perivacuolar punctate structures, but does not colocalize with typical endomembrane marker proteins. Finally, we have isolated a temperature conditional allele of APG9/CVT7 and demonstrate the direct role of Apg9p/Cvt7p in the formation of the Cvt and autophagic vesicles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specialized compartment essential for these vesicle-mediated alternative targeting pathways.
Assuntos
Autofagia/fisiologia , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Animais , Transporte Biológico , Centrifugação com Gradiente de Concentração , Proteínas Fúngicas/genética , Proteínas de Membrana/genética , Coelhos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Frações Subcelulares , Vacúolos/metabolismoRESUMO
Aminopeptidase I (API) is a soluble leucine aminopeptidase resident in the yeast vacuole (Frey, J., and K.H. Rohm. 1978. Biochim. Biophys. Acta. 527:31-41). The precursor form of API contains an amino-terminal 45-amino acid propeptide, which is removed by proteinase B (PrB) upon entry into the vacuole. The propeptide of API lacks a consensus signal sequence and it has been demonstrated that vacuolar localization of API is independent of the secretory pathway (Klionsky, D.J., R. Cueva, and D.S. Yaver. 1992. J. Cell Biol. 119:287-299). The predicted secondary structure for the API propeptide is composed of an amphipathic alpha-helix followed by a beta-turn and another alpha-helix, forming a helix-turn-helix structure. With the use of mutational analysis, we determined that the API propeptide is essential for proper transport into the vacuole. Deletion of the entire propeptide from the API molecule resulted in accumulation of a mature-sized protein in the cytosol. A more detailed examination using random mutagenesis and a series of smaller deletions throughout the propeptide revealed that API localization is severely affected by alterations within the predicted first alpha-helix. In vitro studies indicate that mutations in this predicted helix prevent productive binding interactions from taking place. In contrast, vacuolar import is relatively insensitive to alterations in the second predicted helix of the propeptide. Examination of API folding revealed that mutations that affect entry into the vacuole did not affect the structure of API. These data indicate that the API propeptide serves as a vacuolar targeting determinant at a critical step along the cytoplasm to vacuole targeting pathway.
Assuntos
Aminopeptidases/metabolismo , Citoplasma/metabolismo , Proteínas Fúngicas/metabolismo , Sinais Direcionadores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Vacúolos/metabolismo , Sequência de Aminoácidos , Aminopeptidases/química , Sequência de Bases , Transporte Biológico , Compartimento Celular , Proteínas Fúngicas/química , Sequências Hélice-Volta-Hélice , Membranas Intracelulares/metabolismo , Dados de Sequência Molecular , Mutagênese , Sinais Direcionadores de Proteínas/química , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/ultraestruturaRESUMO
Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.
Assuntos
Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais/fisiologia , Vesículas Transportadoras/metabolismo , Vacúolos/metabolismo , Aminopeptidases/genética , Aminopeptidases/metabolismo , Western Blotting , Proteínas de Transporte/genética , Fracionamento Celular , Membrana Celular/metabolismo , Citosol/metabolismo , Glucose/metabolismo , Humanos , Microscopia de Fluorescência , Peroxissomos/metabolismo , Pichia/genética , Pichia/metabolismo , Pichia/ultraestrutura , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestruturaRESUMO
Macroautophagy is a dynamic process involving the rearrangement of subcellular membranes to sequester cytoplasm and organelles for delivery to the lysosome or vacuole where the sequestered cargo is degraded and recycled. This process takes place in all eukaryotic cells. It is highly regulated through the action of various kinases, phosphatases, and guanosine triphosphatases (GTPases). The core protein machinery that is necessary to drive formation and consumption of intermediates in the macroautophagy pathway includes a ubiquitin-like protein conjugation system and a protein complex that directs membrane docking and fusion at the lysosome or vacuole. Macroautophagy plays an important role in developmental processes, human disease, and cellular response to nutrient deprivation.
Assuntos
Autofagia , Citoplasma/metabolismo , Organelas/metabolismo , Fagossomos/metabolismo , Animais , Humanos , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Fusão de Membrana , Organelas/ultraestrutura , Fagossomos/ultraestrutura , Proteínas/metabolismo , Leveduras/genética , Leveduras/metabolismo , Leveduras/ultraestruturaRESUMO
Unicellular eukaryotic organisms must be capable of rapid adaptation to changing environments. While such changes do not normally occur in the tissues of multicellular organisms, developmental and pathological changes in the environment of cells often require adaptation mechanisms not dissimilar from those found in simpler cells. Autophagy is a catabolic membrane-trafficking phenomenon that occurs in response to dramatic changes in the nutrients available to yeast cells, for example during starvation or after challenge with rapamycin, a macrolide antibiotic whose effects mimic starvation. Autophagy also occurs in animal cells that are serum starved or challenged with specific hormonal stimuli. In macroautophagy, the form of autophagy commonly observed, cytoplasmic material is sequestered in double-membrane vesicles called autophagosomes and is then delivered to a lytic compartment such as the yeast vacuole or mammalian lysosome. In this fashion, autophagy allows the degradation and recycling of a wide spectrum of biological macromolecules. While autophagy is induced only under specific conditions, salient mechanistic aspects of autophagy are functional in a constitutive fashion. In Saccharomyces cerevisiae, induction of autophagy subverts a constitutive membrane-trafficking mechanism called the cytoplasm-to-vacuole targeting pathway from a specific mode, in which it carries the resident vacuolar hydrolase, aminopeptidase I, to a nonspecific bulk mode in which significant amounts of cytoplasmic material are also sequestered and recycled in the vacuole. The general aim of this review is to focus on insights gained into the mechanism of autophagy in yeast and also to review our understanding of the physiological significance of autophagy in both yeast and higher organisms.
Assuntos
Leveduras/fisiologia , Autofagia/genética , Leveduras/genéticaRESUMO
Nutrient starvation in the yeast Saccharomyces cerevisiae leads to a number of physiological changes that accompany entry into stationary phase. The expression of genes whose products play a role in stress adaptation is regulated in a manner that allows the cell to sense and respond to changing environmental conditions. We have identified a novel yeast gene, YGP1, that displays homology to the sporulation-specific SPS100 gene. The expression of YGP1 is regulated by nutrient availability. The gene is expressed at a basal level during "respiro-fermentative" (logarithmic) growth. When the glucose concentration in the medium falls below 1%, the YGP1 gene is derepressed and the gene product, gp37, is synthesized at levels up to 50-fold above the basal level. The glucose-sensing mechanism is independent of the SNF1 pathway and does not operate when cells are directly shifted to a low glucose concentration. The expression of YGP1 also responds to the depletion of nitrogen and phosphate, indicating a general response to nutrient deprivation. These results suggest that the YGP1 gene product may be involved in cellular adaptations prior to stationary phase and may be a useful marker protein for monitoring early events associated with the stress response.
Assuntos
Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Genes Fúngicos , Glicoproteínas/biossíntese , Glicoproteínas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Clonagem Molecular , Meios de Cultura , DNA Fúngico/genética , DNA Fúngico/metabolismo , Escherichia coli , Fermentação , Proteínas Fúngicas/isolamento & purificação , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Glicoproteínas/isolamento & purificação , Glicosídeo Hidrolases/biossíntese , Glicosídeo Hidrolases/genética , Glicosilação , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Esporos Fúngicos/fisiologia , Trealase/genética , beta-FrutofuranosidaseRESUMO
An inactive precursor form of proteinase A (PrA) transits through the early secretory pathway before final vacuolar delivery. We used gene fusions between the gene coding for PrA (PEP4) and the gene coding for the secretory enzyme invertase (SUC2) to identify vacuolar protein-sorting information in the PrA precursor. We found that the 76-amino-acid preprosegment of PrA contains at least two sorting signals: an amino-terminal signal peptide that is cleaved from the protein at the level of the endoplasmic reticulum followed by the prosegment which functions as a vacuolar protein-sorting signal. PrA-invertase hybrid proteins that carried this sequence information were accurately sorted to the yeast vacuole as determined by cell fractionation and immunolocalization studies. Hybrid proteins lacking all or a portion of the PrA prosegment were secreted from the cell. Our gene fusion data together with an analysis of the wild-type PrA protein indicated that N-linked carbohydrate modifications are not required for vacuolar sorting of this protein. Furthermore, results obtained with a set of deletion mutations constructed in the PrA prosegment indicated that this sequence also contributes to proper folding of this polypeptide into a stable transit-competent molecule.
Assuntos
Ácido Aspártico Endopeptidases , Endopeptidases/metabolismo , Proteínas Fúngicas/metabolismo , Precursores de Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Endopeptidases/genética , Proteínas Fúngicas/genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Glicosilação , Conformação Proteica , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Vacúolos/metabolismo , beta-FrutofuranosidaseRESUMO
The NH2 terminus of the yeast F1-ATPase beta subunit precursor directs the import of this protein into mitochondria. To define the functionally important components of this import signal, oligonucleotide-directed mutagenesis was used to introduce a series of deletion and missense mutations into the gene encoding the F1-beta subunit precursor. Among these mutations were three nonoverlapping deletions, two within the 19-amino-acid presequence (delta 5-12 and delta 16-19) and one within the mature protein (delta 28-34). Characterization of the mitochondrial import properties of various mutant F1-beta subunit proteins containing different combinations of these deletions showed that import was blocked only when all three deletions were combined. Mutant proteins containing all possible single and pairwise combinations of these deletions were found to retain the ability to direct mitochondrial import of the F1-beta subunit. These data suggest that the F1-beta subunit contains redundant import information at its NH2 terminus. In fact, we found that deletion of the entire F1-beta subunit presequence did not prevent import, indicating that a functional mitochondrial import signal is present near the NH2 terminus of the mature protein. Furthermore, by analyzing mitochondrial import of the various mutant proteins in [rho-] yeast, we obtained evidence that different segments of the F1-beta subunit import signal may act in an additive or cooperative manner to optimize the import properties of this protein.