The TOR Complex 1 Is Distributed in Endosomes and in Retrograde Vesicles That Form from the Vacuole Membrane and Plays an Important Role in the Vacuole Import and Degradation Pathway

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to cells that have been starved for 3 days, FBPase is degraded in the vacuole. FBPase is first imported to Vid (vacuole import and degradation) vesicles, and these vesicles then merge with the endocytic pathway. In this report we show that two additional gluconeogenic enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were also degraded in the vacuole via the Vid pathway. These new cargo proteins and FBPase interacted with the TORC1 complex during glucose starvation. However, Tor1p was dissociated from FBPase after the addition of glucose. FBPase degradation was inhibited in cells overexpressing TOR1, suggesting that excessive Tor1p is inhibitory. Both Tco89p and Tor1p were found in endosomes coming from the plasma membrane as well as in retrograde vesicles forming from the vacuole membrane. When TORC1 was inactivated by rapamycin, FBPase degradation was inhibited. We suggest that TORC1 interacts with multiple cargo proteins destined for the Vid pathway and plays an important role in the degradation of FBPase in the vacuole.     

Vid30 is required for the association of Vid vesicles and actin patches in the vacuole import and degradation pathway

When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase (MDH2), isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are induced. However, when glucose is added to prolonged starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. Recent evidence suggests that the Vid pathway merges with the endocytic pathway at actin patches where endocytic vesicles are formed. The convergence of the Vid pathway with the endocytic pathway allows cells to remove intracellular and extracellular proteins simultaneously. However, the genes that regulate this step of the convergence have not been identified previously. Here we show that VID30 plays a critical role for the association of Vid vesicles and actin patches. Vid30 is constitutively expressed and interacts with Vid vesicle proteins Vid24 and Sec28 but not with the cargo protein FBPase. In the absence of SEC28 or VID24, Vid30 association with actin patches was prolonged. In cells lacking the VID30 gene, FBPase and Vid24 were not localized to actin patches, suggesting that Vid30 has a role in the association of Vid vesicles and actin patches. Vid30 contains a LisH and a CTLH domain, both of which are required for FBPase degradation. When these domains were deleted, FBPase trafficking to the vacuole was impaired. We suggest that Vid30 also has a role in the Vid pathway at a later step in a process that is mediated by the LisH and CTLH domains.     

The vacuolar import and degradation pathway merges with the endocytic pathway to deliver fructose-1,6-bisphosphatase to the vacuole for degradation

The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is degraded in the vacuole when glucose is added to glucose-starved cells. Before it is delivered to the vacuole, however, FBPase is imported into intermediate carriers called Vid (vacuole import and degradation) vesicles. Here, using biochemical and genetic approaches, we identified a requirement for SEC28 in FBPase degradation. SEC28 encodes the epsilon-COP subunit of COPI (coat protein complex I) coatomer proteins. When SEC28 and other coatomer genes were mutated, FBPase degradation was defective and FBPase association with Vid vesicles was impaired. Coatomer proteins were identified as components of Vid vesicles, and they formed a protein complex with a Vid vesicle-specific protein, Vid24p. Furthermore, Vid24p association with Vid vesicles was impaired when coatomer genes were mutated. Kinetic studies indicated that Sec28p traffics to multiple locations. Sec28p was in Vid vesicles, endocytic compartments, and the vacuolar membrane in various mutants that block the FBPase degradation pathway. Sec28p was also found in vesicles adjacent to the vacuolar membrane in the ret2-1 coatomer mutant. We propose that Sec28p resides in Vid vesicles, and these vesicles converge with the endocytic pathway. After fusion, Sec28p is distributed on the vacuolar membrane, where it concentrates on vesicles that pinch off from this organelle. FBPase also utilizes the endocytic pathway for transport to the vacuole, as demonstrated by its presence in endocytic compartments in the Deltavph1 mutant. Taken together, our results indicate a strong connection between the Vid trafficking pathway and the endocytic pathway.     

Vid30 is required for the association of Vid vesicles and actin patches in the vacuole import and degradation pathway

When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase (MDH2), isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are induced. However, when glucose is added to prolonged starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. Recent evidence suggests that the Vid pathway merges with the endocytic pathway at actin patches where endocytic vesicles are formed. The convergence of the Vid pathway with the endocytic pathway allows cells to remove intracellular and extracellular proteins simultaneously. However, the genes that regulate this step of the convergence have not been identified previously. Here we show that VID30 plays a critical role for the association of Vid vesicles and actin patches. Vid30 is constitutively expressed and interacts with Vid vesicle proteins Vid24 and Sec28 but not with the cargo protein FBPase. In the absence of SEC28 or VID24, Vid30 association with actin patches was prolonged. In cells lacking the VID30 gene, FBPase and Vid24 were not localized to actin patches, suggesting that Vid30 has a role in the association of Vid vesicles and actin patches. Vid30 contains a LisH and a CTLH domain, both of which are required for FBPase degradation. When these domains were deleted, FBPase trafficking to the vacuole was impaired. We suggest that Vid30 also has a role in the Vid pathway at a later step in a process that is mediated by the LisH and CTLH domains.     

The Vacuole Import and Degradation Pathway Utilizes Early Steps of Endocytosis and Actin Polymerization to Deliver Cargo Proteins to the Vacuole for Degradation

When glucose is added to yeast cells that are starved for 3 days, fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase 2 are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. In this study, we examined the distribution of FBPase at the ultrastructural level. FBPase was observed in areas close to the plasma membrane and in cytoplasmic structures that are heterogeneous in size and density. We have isolated these intracellular structures that contain FBPase, the Vid vesicle marker Vid24p, and the endosomal marker Pep12p. They appeared irregular in size and shape. In yeast, actin polymerization plays an important role in early steps of endocytosis. Mutants that affect actin polymerization inhibited FBPase degradation, suggesting that actin polymerization is important for FBPase degradation. Both FBPase and malate dehydrogenase 2 were associated with actin patches. Vid vesicle proteins such as Vid24p or Sec28p were also at actin patches, although they dissociated from these structures at later time points. We propose that Vid24p and Sec28p are present at actin patches during glucose starvation. Cargo proteins arrive at these sites following the addition of glucose, and the endocytic vesicles then pinch off from the plasma membrane. Following the fusion of endosomes with the vacuole, cargo proteins are then degraded in the vacuole.     

The Vid vesicle to vacuole trafficking event requires components of the SNARE membrane fusion machinery

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is targeted to Vid vesicles when glucose-starved cells are replenished with glucose. Vid vesicles then deliver FBPase to the vacuole for degradation. A modified alkaline phosphatase assay was developed to study the trafficking of Vid vesicles to the vacuole. For this assay, FBPase was fused with a truncated form of alkaline phosphatase. Under in vivo conditions, FBPase-delta60Pho8p was targeted to the vacuole via Vid vesicles, and it exhibited Pep4p- and Vid24p-dependent alkaline phosphatase activation. Vid vesicle-vacuole targeting was reconstituted using Vid vesicles that contained FBPase-delta60Pho8p. These vesicles were incubated with vacuoles in the presence of cytosol and an ATP-regenerating system. Under in vitro conditions, alkaline phosphatase was also activated in a Pep4p- and Vid24p-dependent manner. The GTPase Ypt7p was identified as an essential component in Vid vesicle-vacuole trafficking. Likewise, a number of v-SNAREs (Ykt6p, Nyv1p, Vti1p) and homotypic fusion vacuole protein sorting complex family members (Vps39p and Vps41p) were required for the proper function of Vid vesicles. In contrast, the t-SNARE Vam3p was a necessary vacuolar component. Vid vesicle-vacuole trafficking exhibits characteristics similar to heterotypic membrane fusion events.     

The type 1 phosphatase Reg1p-Glc7p is required for the glucose-induced degradation of fructose-1,6-bisphosphatase in the vacuole

Protein phosphatases play an important role in vesicular trafficking and membrane fusion processes. The type 1 phosphatase Glc7p and its regulatory subunit Reg1p were identified as required components in the glucose-induced targeting of the key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) to the vacuole for degradation. The interaction of Reg1p with Glc7p was important for the transport of FBPase from intermediate vacuole import and degradation (Vid) vesicles to vacuoles. The glc7-T152K mutant strain exhibited a reduced Reg1p binding along with defects in FBPase degradation and Vid vesicle trafficking to the vacuole. In this mutant, Vid vesicles were the most defective components, whereas the vacuole was also defective. Shp1p and Glc8p regulate Glc7p phosphatase activity and are required for FBPase degradation. In the Deltashp1 and Deltaglc8 strains, Reg1p-Glc7p interaction was not affected, suggesting that phosphatase activity is also necessary for FBPase degradation. Similar to those seen in the glc7-T152K mutant, the Deltashp1 and Deltaglc8 mutants exhibited severely defective Vid vesicles, but partially defective vacuoles. Taken together, our results suggest that Reg1p-Glc7p interaction and Glc7p phosphatase activity play a required role in the Vid vesicle to vacuole-trafficking step along the FBPase degradation pathway.     

Identification of Novel Vesicles in the Cytosol to Vacuole Protein Degradation Pathway

The key gluconeogenic enzyme, fructose1,6-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.     

The heat shock protein Ssa2p is required for import of fructose-1, 6-bisphosphatase into Vid vesicles

Fructose-1,6-bisphosphatase (FBPase) is targeted to the vacuole for degradation when Saccharomyces cerevisiae are shifted from low to high glucose. Before vacuolar import, however, FBPase is sequestered inside a novel type of vesicle, the vacuole import and degradation (Vid) vesicles. Here, we reconstitute import of FBPase into isolated Vid vesicles. FBPase sequestration into Vid vesicles required ATP and cytosol, but was inhibited if ATP binding proteins were depleted from the cytosol. The heat shock protein Ssa2p was identified as one of the ATP binding proteins involved in FBPase import. A Deltassa2 strain exhibited a significant decrease in the rate of FBPase degradation in vivo as compared with Deltassa1, Deltassa3, or Deltassa4 strains. Likewise, in vitro import was impaired for the Deltassa2 strain, but not for the other Deltassa strains. The cytosol was identified as the site of the Deltassa2 defect; Deltassa2 cytosol did not stimulate FBPase import into import competent Vid vesicles, but wild-type cytosol supported FBPase import into competent Deltassa2 vesicles. The addition of purified recombinant Ssa2p stimulated FBPase import into Deltassa2 Vid vesicles, providing Deltassa2 cytosol was present. Thus, Ssa2p, as well as other undefined cytosolic proteins are required for the import of FBPase into vesicles.     

Biochemical analysis of fructose-1,6-bisphosphatase import into vacuole import and degradation vesicles reveals a role for UBC1 in vesicle biogenesis

When Saccharomyces cerevisiae are shifted from medium containing poor carbon sources to medium containing fresh glucose, the key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is imported into Vid (vacuole import and degradation) vesicles and then to the vacuole for degradation. Here, we show that FBPase import is independent of vacuole functions and proteasome degradation. However, FBPase import required the ubiquitin-conjugating enzyme Ubc1p. A strain containing a deletion of the UBC1 gene exhibited defective FBPase import. Furthermore, FBPase import was inhibited when cells overexpressed the K48R/K63R ubiquitin mutant that fails to form multiubiquitin chains. The defects in FBPase import seen for the Deltaubc1 and the K48R/K63R mutants were attributed to the Vid vesicle fraction. In the Deltaubc1 mutant, the level of the Vid vesicle-specific marker Vid24p was reduced in the vesicle fraction, suggesting that UBC1 is required for either Vid vesicle production or Vid24p binding to Vid vesicles. However, the K48R/K63R mutant did not prevent Vid24p binding to Vid vesicles, indicating that ubiquitin chain formation is dispensable for Vid24p binding to these structures. Our results support the findings that ubiquitin conjugation and ubiquitin chain formation play important roles in a number of cellular processes including organelle biogenesis.     

Degradation of the Gluconeogenic Enzyme Fructose-1,6-Bisphosphatase is Dependent on the Vacuolar ATPase

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced during glucose starvation. After the addition of glucose, inactivated FBPase is selectively targeted to a novel type of Vid (vacuolar import and degradation) vesicle and then to the vacuole for degradation. To identify proteins involved in this pathway, we screened various libraries for mutants that failed to degrade FBPase. Via these approaches, subunits of the vacuolar H+ ATPase (V-ATPase) have been identified repeatedly. The VATPase has established roles in endocytosis, sorting of carboxypeptidase Y and homotypic vacuole fusion. Here, we show that Stv1p, Vph1p, and other subunits of the VATPase are required for FBPase degradation. VPH1 and V0 domain subunits such as Vma3p were required for both Vid vesicle and vacuole function, as determined by an in vitro fusion assay. However, STV1 was only required for the proper function of the Vid vesicles. We also show that the V1 domain participates in the Vid vesicle to vacuoletrafficking step, since most of the V1 subunits are necessary for Vid vesicle-vacuole fusionto occur. The V0 and V1 domains are assembled following a glucose shift and theassembly is independent of protein kinase A and RAV genes. Assembly of the V0 complexis necessary for FBPase trafficking, since mutants that block the assembly and transport ofV0 out of the ER were defective in FBPase degradation.     

Degradation of the gluconeogenic enzyme fructose-1, 6-bisphosphatase is dependent on the vacuolar ATPase

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced during glucose starvation. After the addition of glucose, inactivated FBPase is selectively targeted to Vid (vacuolar import and degradation) vesicles and then to the vacuole for degradation. To identify proteins involved in this pathway, we screened various libraries for mutants that failed to degrade FBPase. Via these approaches, subunits of the vacuolar- H+ -ATPase (V-ATPase) have been identified repeatedly. The V-ATPase has established roles in endocytosis, sorting of carboxypeptidase Y and homotypic vacuole fusion. Here, we show that mutants lacking Stv1p, Vph1p, and other subunits of the V-ATPase are defective for FBPase degradation. FBPase was detected in Vid vesicles. However, most FBPase was resistant to proteinase K digestion in the Deltavph1 or vma mutants, whereas the majority of FBPase was sensitive to proteinase K digestion in the Deltastv1 mutant. Therefore, STV1 and VPH1 have distinct functions in FBPase degradation. In cells lacking V0 genes, Vma2p and Vma5p were still detected on Vid vesicles and vacuoles, suggesting that the distribution of V1 proteins is independent of V0 genes. The V0 and V1 domains are assembled following a glucose shift and the assembly is not regulated by protein kinase A and RAV genes. Assembly of the V0 complex is necessary for FBPase trafficking, since mutants that block the assembly and transport of V0 out of the ER were defective in FBPase degradation.     

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

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.Protein degradation is an important process that is tightly regulated. In mammalian cells, serum starvation induces protein degradation by lysosomes (Dice, 1990; Hayes and Dice, 1996). Cytosolic proteins containing a pentapeptide sequence are targeted to the lysosome for degradation in a process mediated by a heat shock protein (Chiang and Dice, 1988; Chiang et al., 1989; Terlecky et al., 1992; Terlecky and Dice, 1993; Cuervo et al., 1994). The receptor protein for this selective proteolysis pathway has been identified recently to be LGP96 (Cuervo and Dice, 1996). Overexpression of the receptor protein increases the degradation of cytosolic proteins in lysosomes both in vivo and in vitro (Cuervo and Dice, 1996).In Saccharomyces cerevisiae, the vacuole is functionally homologous to the lysosome and takes up proteins by several mechanisms. Most vacuole resident proteinases such as carboxypeptidase Y (CPY)¹ enter the vacuole through the secretory pathway (Hasilik and Tanner, 1978; Hemmings et al., 1981; Rothman and Stevens, 1986; Banta et al., 1988; Jones, 1991). CPY is synthesized and processed sequentially in the ER and the Golgi. Sorting occurs in the late Golgi by the CPY receptor encoded by the PEP1/ VPS10 gene (Marcusson et al., 1994; Cooper and Stevens, 1996). CPY is delivered to the vacuole from the prevacuolar or endosomal compartment and the receptor protein recycles back to the Golgi (Marcusson et al., 1994; Cooper and Stevens, 1996). Other vacuolar proteins such as α-mannosidase or aminopeptidase I are imported from the cytosol to the vacuole, independent of the secretory pathway (Yoshihisa and Anraku, 1990; Klionsky et al., 1992; Harding et al., 1995, 1996; Scott et al., 1996). Plasma membrane proteins can be internalized by endocytosis and transported through early endosomes to late endosomes, from which they are directed to the vacuole for degradation (Davis et al., 1993; Raths et al., 1993; Kolling and Hollenberg, 1994; Schandel and Jennes, 1994; Lai et al., 1995; Riballo et al., 1995). Organelles such as peroxisomes or mitochondria can be engulfed by the vacuoles by autophagy (Takeshige et al., 1992; Tuttle and Dunn, 1995; Chiang et al., 1996). The key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae cells are grown in medium containing poor carbon sources. When cells are transferred to medium containing fresh glucose, FBPase is rapidly inactivated (Gancedo, 1971). Using isogenic strains differing only at the PEP4 gene, we have demonstrated that FBPase is targeted from the cytosol to the vacuole for degradation when cells are transferred from poor carbon sources to fresh glucose (Chiang and Schekman, 1991). The PEP4 gene encodes proteinase A, whose activity is required for the maturation of proteinase B and proteinase C (Zubenko and Jones, 1981; Jones, 1991). As a result, the pep4 strain reduces the vacuolar proteolytic activity to 30% of the wild-type level (Zubenko and Jones, 1981; Jones, 1991; Chiang et al., 1996). The glucose-induced distribution of FBPase from the cytosol to the vacuole has been observed in the pep4 cell by cell fractionation techniques, immunofluorescence microscopy, and immunoelectron microscopy (Chiang and Schekman, 1991; Chiang et al., 1996). FBPase targeting into the vacuole always occurs, regardless of whether cells are transferred to glucose from acetate, ethanol, galactose, or oleate (Chiang and Schekman, 1994; Chiang et al., 1996).To dissect the FBPase degradation pathway, we have taken a genetic approach. Several vid (vacuolar import and degradation) mutants that fail to degrade FBPase in response to glucose have been isolated (Hoffman and Chiang, 1996). Most vid mutants block FBPase in the cytosol. However, in the vid14-1, vid15-1, and vid16-1 mutants, FBPase is found in punctate structures in the cytoplasm. When cell extracts from one of these mutants are fractionated, a substantial amount of FBPase is found in the high speed pellet, suggesting that FBPase is associated with intracellular structures in these mutants (Hoffman and Chiang, 1996). This association is also observed in wild-type cells (Huang and Chiang, 1997).The FBPase-containing vesicles have been purified from wild-type cells to near homogeneity using a combination of differential centrifugation, gel filtration, and equilibrium centrifugation in sucrose gradients (Huang and Chiang, 1997). The purified fractions contain 30–40-nm-diam vesicles and are essentially free of other organelles. Kinetic studies indicate that FBPase association with these vesicles is induced by glucose, occurs only transiently, and precedes the association with the vacuole. The FBPase-containing vesicles are distinct from mitochondria, peroxisomes, endosomes, vacuoles, ER, Golgi, or transport vesicles such as the coat protein (COPI or COPII)-containing vesicles as analyzed by protein markers and electron microscopy (Huang and Chiang, 1997).The vesicles were predicted to contain proteins involved in FBPase targeting and sequestration into the vesicles, as well as proteins participating in carrying FBPase from the vesicles to the vacuole for degradation. To identify such factors, we cloned our first VID gene. The VID24 gene was identified by complementation of the degradation defect of the vid24-1 mutant. Vid24p is a novel 41-kD protein and is synthesized in response to glucose. A significant portion of the Vid24p is localized to the FBPase-containing vesicles as a peripheral protein. The deletion of Vid24p abolishes the degradation of FBPase, but does not cause significant change in growth, sporulation, germination, osmolarity sensitivity, or processing of CPY. In the absence of functional Vid24p, FBPase accumulates in the vesicles and fails to move to the vacuole. FBPase is sequestered inside the vesicles in the vid24-1 mutant, suggesting that Vid24p acts after FBPase sequestration into the vesicles has occurred. Vid24p is the first protein identified that is localized to the FBPase-containing vesicles and plays a critical role in delivering FBPase from the vesicles to the vacuole for degradation.     

Cyclophilin A mediates Vid22p function in the import of fructose-1,6-bisphosphatase into Vid vesicles

Fructose-1,6-bisphosphatase (FBPase) is synthesized in yeast during glucose starvation but is rapidly degraded in the vacuole following the addition of glucose. FBPase trafficking to the vacuole involves two distinct steps, import into intermediate transport vesicles (Vid vesicles) and Vid vesicle trafficking to the vacuole. FBPase import into Vid vesicles requires the VID22 gene. However, VID22 affects FBPase import indirectly through a cytosolic factor. To identify the required cytosolic component, wild type cytosol was fractionated and screened for proteins that complement Deltavid22 mutant cytosol using an in vitro assay that reproduces FBPase import into Vid vesicles. Cyclophilin A (Cpr1p) was identified as a cytosolic protein that mediates Vid22p function in FBPase import. Mutants lacking Cpr1p were defective in FBPase import. Furthermore, the addition of purified Cpr1p restored FBPase import in both the Deltacpr1 and the Deltavid22 mutants. The cyclosporin A binding pocket is important for Cpr1p function, since cyclosporin A binding-deficient mutants failed to complement FBPase import in Deltacpr1 and Deltavid22 mutants. The levels of Cpr1p were reduced in the Deltavid22 mutants, implying that the expression of Cpr1p is regulated by Vid22p. Our results suggest that Cpr1p mediates Vid22p function and is directly involved in the import of FBPase into Vid vesicles.     

Vacuole import and degradation pathway: Insights into a specialized autophagy pathway

Glucose deprivation induces the synthesis of pivotagluconeogenic enzymes such as fructose-1,6-bisphos-phatase, malate dehydrogenase, phosphoenolpyruvatecarboxykinase and isocitrate lyase in Saccharomycescerevisiae. However, following glucose replenishment,these gluconeogenic enzymes are inactivated and de-graded. Studies have characterized the mechanismsby which these enzymes are inactivated in response toglucose. The site of degradation of these proteins hasalso been ascertained to be dependent on the dura-tion of starvation. Glucose replenishment of short-termstarved cells results in these proteins being degradedin the proteasome. In contrast, addition of glucose tocells starved for a prolonged period results in theseproteins being degraded in the vacuole. In the vacuoledependent pathway, these proteins are sequestered inspecialized vesicles termed vacuole import and degra-dation (Vid). These vesicles converge with the endo-cytic pathway and deliver their cargo to the vacuolefor degradation. Recent studies have identified thatinternalization, as mediated by actin polymerization, isessential for delivery of cargo proteins to the vacuolefor degradation. In addition, components of the targetof rapamycin complex 1 interact with cargo proteins during glucose starvation. Furthermore, Tor1p dissoci-ates from cargo proteins following glucose replenish-ment. 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.     

Isolation of Degradation-Deficient Mutants Defective in the Targeting of Fructose-1,6-Bisphosphatase into the Vacuole for Degradation in Saccharomyces Cerevisiae

The key regulatory enzyme in the gluconeogenesis pathway, fructose-1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae are grown in medium containing a poor carbon source. FBPase is targeted to the yeast vacuole for degradation when glucose-starved cells are replenished with fresh glucose. To identify genes involved in the FBPase degradation pathway, mutants that failed to degrade FBPase in response to glucose were isolated using a colony-blotting procedure. These vacuolar import and degradation-deficient (vid) mutants were placed into 20 complementation groups. They are distinct from the known sec, vps or pep mutants affecting protein secretion, vacuolar sorting and vacuolar proteolysis in that they sort CpY correctly and regulate osmotic pressure normally. Despite the presence of FBPase antigen in these mutants, FBPase is completely inactivated in all vid mutants, indicating that the c-AMP-dependent signal transduction pathway and inactivation must function properly in vid mutants. vid mutants block FBPase degradation by accumulating FBPase in the cytosol and also in small vesicles in the cytoplasm. FBPase may be targeted to small vesicles before uptake by the vacuole.     

Regulation of Hxt3 and Hxt7 Turnover Converges on the Vid30 Complex and Requires Inactivation of the Ras/cAMP/PKA Pathway in Saccharomyces cerevisiae

Eukaryotic cells adjust their intracellular protein complement as a mechanism to adapt to changing environmental signals. In Saccharomyces cerevisiae the hexose transporters Hxt3 and Hxt7 are expressed and function on the plasma membrane in high and low glucose abundance, respectively. By contrast, Hxt3 is endocytosed and degraded in the vacuole when cells are starved of glucose and Hxt7 in response to rapamycin treatment or when nitrogen is limiting. Yeast uses several signaling pathways, including the TORC1 and Ras/cAMP/Protein Kinase A (PKA) pathways, to adapt to nutrient changes in the environment. The multi-protein Vid30 complex (Vid30c), an E3 ubiquitin ligase required for the degradation of FBPase, assists in this adaptation process in a mechanism that is poorly understood. Here we show the endocytosis and the subsequent degradation of both Hxt3 and Hxt7, in response to different nutrient signals, is dependent on components of the Vid30c. Additionally, we define the signaling events required for the turnover of Hxt3 and Hxt7 by showing that Hxt3 turnover requires Ras2 and PKA inactivation, whereas Hxt7 turnover requires TORC1 and Ras2 inactivation. Further investigation led us to identify Rim15, a kinase that is inhibited by both the TORC1 and Ras/cAMP/PKA pathways, as a key downstream effector in signaling both turnover events. Finally, we show that the turnover of both Hxt3 and Hxt7 is dependent on the essential E3 ubiquitin ligase, Rsp5, indicating that the role of the Vid30c might be indirect of Hxt ubiquitylation.     

Degradation of the gluconeogenic enzymes fructose-1,6-bisphosphatase and malate dehydrogenase is mediated by distinct proteolytic pathways and signaling events

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is subjected to catabolite inactivation and degradation when glucose-starved cells are replenished with fresh glucose. In various studies, the proteasome and the vacuole have each been reported to be the major site of FBPase degradation. Because different growth conditions were used in these studies, we examined whether variations in growth conditions could alter the site of FBPase degradation. Here, we demonstrated that FBPase was degraded outside the vacuole (most likely in the proteasome), when glucose was added to cells that were grown in low glucose media for a short period of time. By contrast, cells that were grown in the same low glucose media for longer periods of time degraded FBPase in the vacuole in response to glucose. Another gluconeogenic enzyme malate dehydrogenase (MDH2) showed the same degradation characteristics as FBPase in that the short term starvation of cells led to a non-vacuolar degradation, whereas long term starvation resulted in the vacuolar degradation of this protein. The N-terminal proline is required for the degradation of FBPase and MDH2 for both the vacuolar and non-vacuolar proteolytic pathways. The cAMP signaling pathway and the phosphorylation of glucose were needed for the vacuolar-dependent degradation of FBPase and MDH2. By contrast, the cAMP-dependent signaling pathway was not involved in the non-vacuolar degradation of these proteins, although the phosphorylation of glucose was required.     

Two distinct proteolytic systems responsible for glucose-induced degradation of fructose-1,6-bisphosphatase and the Gal2p transporter in the yeast Saccharomyces cerevisiae share the same protein components of the glucose signaling pathway.

Addition of glucose to Saccharomyces cerevisiae inactivates the galactose transporter Gal2p and fructose-1,6-bisphosphatase (FBPase) by a mechanism called glucose- or catabolite-induced inactivation, which ultimately results in a degradation of both proteins. It is well established, however, that glucose induces internalization of Gal2p into the endocytotic pathway and its subsequent proteolysis in the vacuole, whereas FBPase is targeted to the 26 S proteasome for proteolysis under similar inactivation conditions. Here we report that two distinct proteolytic systems responsible for specific degradation of two conditionally short-lived protein targets, Gal2p and FBPase, utilize most (if not all) protein components of the same glucose sensing (signaling) pathway. Indeed, initiation of Gal2p and FBPase proteolysis appears to require rapid transport of those substrates of the Hxt transporters that are at least partially metabolized by hexokinase Hxk2p. Also, maltose transported via the maltose-specific transporter(s) generates an appropriate signal that culminates in the degradation of both proteins. In addition, Grr1p and Reg1p were found to play a role in transduction of the glucose signal for glucose-induced proteolysis of Gal2p and FBPase. Thus, one signaling pathway initiates two different proteolytic mechanisms of catabolite degradation, proteasomal proteolysis and endocytosis followed by lysosomal proteolysis.     

The Hsp70 chaperone Ssa1 is essential for catabolite induced degradation of the gluconeogenic enzyme fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphatase (FBPase) is a key regulatory enzyme of gluconeogenesis. In the yeast Saccharomyces cerevisiae, it is only expressed when cells are grown in medium with nonfermentable carbon sources. Addition of glucose to cells leads to inactivation of FBPase and degradation via the ubiquitin-proteasome system. Polyubiquitination of FBPase is carried out by the Gid complex, a multi-subunit ubiquitin ligase. Using tandem affinity purification and subsequent mass spectrometry we identified the Hsp70 chaperone Ssa1 as a novel interaction partner of FBPase. Studies with the temperature-sensitive mutant ssa1-45^ts showed that Ssa1 is essential for polyubiquitination of FBPase by the Gid complex. Moreover, we show that degradation of an additional gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, is also affected in ssa1-45^ts cells demonstrating that Ssa1 plays a general role in elimination of gluconeogenic enzymes.