Uptake of the ATP-binding cassette (ABC) transporter Ste6 into the yeast vacuole is blocked in the doa4 Mutant

Previous experiments suggested that trafficking of the a-factor transporter Ste6 of Saccharomyces cerevisiae to the yeast vacuole is regulated by ubiquitination. To define the ubiquitination-dependent step in the trafficking pathway, we examined the intracellular localization of Ste6 in the ubiquitination-deficient doa4 mutant by immunofluorescence experiments, with a Ste6-green fluorescent protein fusion protein and by sucrose density gradient fractionation. We found that Ste6 accumulated at the vacuolar membrane in the doa4 mutant and not at the cell surface. Experiments with a doa4 pep4 double mutant showed that Ste6 uptake into the lumen of the vacuole is inhibited in the doa4 mutant. The uptake defect could be suppressed by expression of additional ubiquitin, indicating that it is primarily the result of a lowered ubiquitin level (and thus of reduced ubiquitination) and not the result of a deubiquitination defect. Based on our findings, we propose that ubiquitination of Ste6 or of a trafficking factor is required for Ste6 sorting into the multivesicular bodies pathway. In addition, we obtained evidence suggesting that Ste6 recycles between an internal compartment and the plasma membrane.     

Deubiquitination Step in the Endocytic Pathway of Yeast Plasma Membrane Proteins: Crucial Role of Doa4p Ubiquitin Isopeptidase

The Fur4p uracil permease, like most yeast plasma membrane proteins, undergoes ubiquitin-dependent endocytosis and is then targeted to the vacuole (equivalent to the mammalian lysosome) for degradation. The cell surface ubiquitination of Fur4p is mediated by the essential Rsp5p ubiquitin ligase. Ubiquitination of Fur4p occurs on two target lysines, which receive two ubiquitin moieties linked through ubiquitin Lys63, a type of linkage (termed UbK63) different from that involved in proteasome recognition. We report that pep4 cells deficient for vacuolar protease activities accumulate vacuolar unubiquitinated Fur4p. In contrast, pep4 cells lacking the Doa4p ubiquitin isopeptidase accumulate ubiquitin-conjugated Fur4p. These data suggest that Fur4p undergoes Doa4p-dependent deubiquitination prior to vacuolar degradation. Compared to pep4 cells, pep4 doa4 cells have huge amounts of membrane-bound ubiquitin conjugates. This indicates that Doa4p plays a general role in the deubiquitination of membrane-bound proteins, as suggested by reports describing the suppression of some doa4 phenotypes in endocytosis and vacuolar protein sorting mutants. Some of the small ubiquitin-linked peptides that are a hallmark of Doa4 deficiency are not present in rsp5 mutant cells or after overproduction of a variant ubiquitin modified at Lys 63 (UbK63R). These data suggest that the corresponding peptides are degradation products of Rsp5p substrates and probably of ubiquitin conjugates carrying UbK63 linkages. Doa4p thus appears to be involved in the deubiquitination of endocytosed plasma membrane proteins, some of them carrying UbK63 linkages.     

Mitochondrial degradation in acetic acid‐induced yeast apoptosis: the role of Pep4 and the ADP/ATP carrier

We have previously shown that acetic acid activates a mitochondria‐dependent death process in Saccharomyces cerevisiae and that the ADP/ATP carrier (AAC) is required for mitochondrial outer membrane permeabilization and cytochrome c release. Mitochondrial fragmentation and degradation have also been shown in response to this death stimulus. Herein, we show that autophagy is not active in cells undergoing acetic acid‐induced apoptosis and is therefore not responsible for mitochondrial degradation. Furthermore, we found that the vacuolar protease Pep4p and the AAC proteins have a role in mitochondrial degradation using yeast genetic approaches. Depletion and overexpression of Pep4p, an orthologue of human cathepsin D, delays and enhances mitochondrial degradation respectively. Moreover, Pep4p is released from the vacuole into the cytosol in response to acetic acid treatment. AAC‐deleted cells also show a decrease in mitochondrial degradation in response to acetic acid and are not defective in Pep4p release. Therefore, AAC proteins seem to affect mitochondrial degradation at a step subsequent to Pep4p release, possibly triggering degradation through their involvement in mitochondrial permeabilization. The finding that both mitochondrial AAC proteins and the vacuolar Pep4p interfere with mitochondrial degradation suggests a complex regulation and interplay between mitochondria and the vacuole in yeast programmed cell death.     

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 Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast

Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Delta cells. This loss of viability and several other defects of doa4Delta cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.     

Processing and maturation of carboxypeptidase Y and alkaline phosphatase in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Schizosaccharomyces pombe carboxypeptidase Y (CPY) is synthesized as a zymogen and transported into the vacuole where maturation and activation occurs. The 110-kDa S. pombe CPY precursor is processed twice and finally converted to a mature form consisting of polypeptides of approximately 19 and 32 kDa linked by a single disulfide bond. In Saccharomyces cerevisiae, maturation of CPY occurs mostly through the activity of vacuolar aspartyl protease Pep4p, whereas a Pep4p homolog has not been found in the S. pombe genome database. Based on analysis of protease-deficient mutants, we found that S. pombe CPY was not able to be processed or activated in isp6Δpsp3Δ double disruptants. Both Isp6p and Psp3p are subtilase-type serine proteases with related sequences. Moreover, alkaline phosphatase of S. pombe was found to be localized at the vacuolar membrane and was also unprocessed in isp6Δpsp3Δ double disruptants. Vacuolar localization of GFP-fused Isp6p and Psp3p was determined by fluorescence microscopy. These results suggest that the two serine proteases Isp6p and Psp3p are functional in the vacuole and are involved in proteolytic processing of vacuolar proteins.     

A family of small coiled-coil-forming proteins functioning at the late endosome in yeast

The multispanning membrane protein Ste6, a member of the ABC-transporter family, is transported to the yeast vacuole for degradation. To identify functions involved in the intracellular trafficking of polytopic membrane proteins, we looked for functions that block Ste6 transport to the vacuole upon overproduction. In our screen, we identified several known vacuolar protein sorting (VPS) genes (SNF7/VPS32, VPS4, and VPS35) and a previously uncharacterized open reading frame, which we named MOS10 (more of Ste6). Sequence analysis showed that Mos10 is a member of a small family of coiled-coil-forming proteins, which includes Snf7 and Vps20. Deletion mutants of all three genes stabilize Ste6 and show a "class E vps phenotype." Maturation of the vacuolar hydrolase carboxypeptidase Y was affected in the mutants and the endocytic tracer FM4-64 and Ste6 accumulated in a dot or ring-like structure next to the vacuole. Differential centrifugation experiments demonstrated that about half of the hydrophilic proteins Mos10 and Vps20 was membrane associated. The intracellular distribution was further analyzed for Mos10. On sucrose gradients, membrane-associated Mos10 cofractionated with the endosomal t-SNARE Pep12, pointing to an endosomal localization of Mos10. The growth phenotypes of the mutants suggest that the "Snf7-family" members are involved in a cargo-specific event.     

Ubiquitin-independent entry into the yeast recycling pathway

The yeast a-factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway links to vacuolar degradation of the receptor, while a ligand-dependent uptake pathway links primarily to recycling and thus, receptor reutilization. Ste3p ubiquitination triggers its uptake into the constitutive pathway. The present work considers the role of the receptor ubiquitination associated with the Ste3p ligand-dependent endocytosis mechanism. The doa4Δ mutation which reduces the cellular availability of ubiquitin blocks the Ste3p constitutive uptake. Uptake into the Ste3p ligand-dependent recycling pathway, however, continues unimpaired. The ubiquitin independence of Ste3p ligand-dependent uptake was further indicated by analysis of receptor mutants having Lys-to-Arg substitutions at all possible ubiquitin acceptor sites. Again, the ligand-induced internalization was unimpaired. Furthermore, no discernible effect was seen on either recycling or on the slow PEP4 -dependent turnover of the receptor (for receptor internalized via the ligand-dependent mechanism, trafficking to the vacuole/lysosome is the minor, alternate fate to recycling). However, one striking effect of the Lys-to-Arg mutations was noted. Following a prolonged exposure of the cells to the a-factor ligand, rather than being delivered to the vacuolar lumen, the Lys-to-Arg receptor was found to localize instead to the limiting membrane of the vacuole. Thus, while receptor ubiquitination clearly is not required for either the a-factor-dependent uptake into recycling pathway or for the recycling itself, it does affect the routing of receptor to the vacuole, likely by affecting the routing through the late endosomal, multivesicular body: ubiquitinated receptor may be selected into the internal, lumenal vesicles, while unmodified receptor may be left to reside at the limiting external membrane.     

The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways

The Saccharomyces cerevisiae DOA4 gene encodes a deubiquitinating enzyme that is required for rapid degradation of ubiquitin-proteasome pathway substrates. Both genetic and biochemical data suggest that Doa4 acts in this pathway by facilitating ubiquitin recycling from ubiquitinated intermediates targeted to the proteasome. Here we describe the isolation of 12 spontaneous extragenic suppressors of the doa4-1 mutation; these involve seven different genes, six of which were cloned. Surprisingly, all of the cloned DID (Doa4-independent degradation) genes encode components of the vacuolar protein-sorting (Vps) pathway. In particular, all are class E Vps factors, which function in the maturation of a late endosome/prevacuolar compartment into multivesicular bodies that then fuse with the vacuole. Four of the six Did proteins are structurally related, suggesting an overlap in function. In wild-type and several vps strains, Doa4-green fluorescent protein displays a cytoplasmic/nuclear distribution. However, in cells lacking the Vps4/Did6 ATPase, a large fraction of Doa4-green fluorescent protein, like several other Vps factors, concentrates at the late endosome-like class E compartment adjacent to the vacuole. These results suggest an unanticipated connection between protein deubiquitination and endomembrane protein trafficking in which Doa4 acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole.     

Pep12p is a multifunctional yeast syntaxin that controls entry of biosynthetic, endocytic and retrograde traffic into the prevacuolar compartment

Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of the endosomal syntaxin, Pep12p. Many vacuolar proteins, such as the soluble vacuolar hydrolase, carboxypeptidase Y (CPY), traverse the prevacuolar compartment (PVC) en route to the vacuole. Here we show that deletion of the carboxy-terminal transmembrane domain of Pep12p results in a temperature-conditional block in transport of CPY to the PVC. The PVC also receives traffic from the early endosome and the vacuole, and mutation in PEP12 also blocks these other trafficking pathways into the PVC. Therefore, Pep12p is a multifunctional syntaxin that is required for all known trafficking pathways into the yeast PVC. Finally, we found that the internalized pheromone receptor, Ste3p, can cycle out of the PVC in a VPS27 -independent fashion.     

Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein

The folding and assembly of proteins in the endoplasmic reticulum (ER) lumen and membrane are monitored by ER quality control. Misfolded or unassembled proteins are retained in the ER and, if they cannot fold or assemble correctly, ultimately undergo ER-associated degradation (ERAD) mediated by the ubiquitin-proteasome system. Whereas luminal and integral membrane ERAD substrates both require the proteasome for their degradation, the ER quality control machinery for these two classes of proteins likely differs because of their distinct topologies. Here we establish the requirements for the ERAD of Ste6p*, a multispanning membrane protein with a cytosolic mutation, and compare them with those for mutant form of carboxypeptidase Y (CPY*), a soluble luminal protein. We show that turnover of Ste6p* is dependent on the ubiquitin-protein isopeptide ligase Doa10p and is largely independent of the ubiquitin-protein isopeptide ligase Hrd1p/Der3p, whereas the opposite is true for CPY*. Furthermore, the cytosolic Hsp70 chaperone Ssa1p and the Hsp40 co-chaperones Ydj1p and Hlj1p are important in ERAD of Ste6p*, whereas the ER luminal chaperone Kar2p is dispensable, again opposite their roles in CPY* turnover. Finally, degradation of Ste6p*, unlike CPY*, does not appear to require the Sec61p translocon pore but, like CPY*, could depend on the Sec61p homologue Ssh1p. The ERAD pathways for Ste6p* and CPY* converge at a post-ubiquitination, pre-proteasome step, as both require the ATPase Cdc48p. Our results demonstrate that ERAD of Ste6p* employs distinct machinery from that of the soluble luminal substrate CPY* and that Ste6p* is a valuable model substrate to dissect the cellular machinery required for the ERAD of multispanning membrane proteins with a cytosolic mutation.     

Vac1p coordinates Rab and phosphatidylinositol 3-kinase signaling in Vps45p-dependent vesicle docking/fusion at the endosome

The vacuolar protein sorting (VPS) pathway of Saccharomyces cerevisiae mediates transport of vacuolar protein precursors from the late Golgi to the lysosome-like vacuole. Sorting of some vacuolar proteins occurs via a prevacuolar endosomal compartment and mutations in a subset of VPS genes (the class D VPS genes) interfere with the Golgi-to-endosome transport step. Several of the encoded proteins, including Pep12p/Vps6p (an endosomal target (t) SNARE) and Vps45p (a Sec1p homologue), bind each other directly [1]. Another of these proteins, Vac1p/Pep7p/Vps19p, associates with Pep12p and binds phosphatidylinositol 3-phosphate (PI(3)P), the product of the Vps34 phosphatidylinositol 3-kinase (PI 3-kinase) [1] [2]. Here, we demonstrate that Vac1p genetically and physically interacts with the activated, GTP-bound form of Vps21p, a Rab GTPase that functions in Golgi-to-endosome transport, and with Vps45p. These results implicate Vac1p as an effector of Vps21p and as a novel Sec1p-family-binding protein. We suggest that Vac1p functions as a multivalent adaptor protein that ensures the high fidelity of vesicle docking and fusion by integrating both phosphoinositide (Vps34p) and GTPase (Vps21p) signals, which are essential for Pep12p- and Vps45p-dependent targeting of Golgi-derived vesicles to the prevacuolar endosome.     

Characterization of Vta1p, a class E Vps protein in Saccharomyces cerevisiae

We identified VTA1 in a screen for mutations that result in altered vacuole morphology. Deletion of VTA1 resulted in delayed trafficking of the lipophilic dye FM4-64 to the vacuole and altered vacuolar morphology when cells were exposed to the dye 5-(and 6)-carboxy-2',7'-dichlorofluorescein diacetate (CDCFDA). Deletion of class E vacuolar protein sorting (VPS) genes, which encode proteins that affect multivesicular body formation, also showed altered vacuolar morphology upon exposure to high concentrations of CDCFDA. These results suggest a VPS defect for Deltavta1 cells. Deletion of VTA1 did not affect growth on raffinose and only mildly affected carboxypeptidase S sorting. Turnover of the surface protein Ste3p, the a-factor receptor, was affected in Deltavta1 cells with the protein accumulating on the vacuolar membrane. Likewise the alpha-factor receptor Ste2p accumulated on the vacuolar membrane in Deltavta1 cells. We demonstrated that many class E VPS deletion strains are hyper-resistant to the cell wall disruption agent calcofluor white. Deletion of VTA1 or VPS60, another putative class E gene, resulted in calcofluor white hypersensitivity. A Vta1p-green fluorescent protein fusion protein transiently associated with a Pep12p-positive compartment. This localization was altered by deletion of many of the class E VPS genes, indicating that Vta1p binds to endosomes in a manner dependent on the assembly of the endosomal sorting complexes required for transport. Membrane-associated Vta1p co-purified with Vps60p, suggesting that Vta1p is a class E Vps protein that interacts with Vps60p on a prevacuolar compartment.     

The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants.

We are investigating the transport and turnover of the multispanning membrane protein Ste6. The Ste6 protein is a member of the ABC-transporter family and is required for the secretion of the yeast mating pheromone a-factor. In contrast to the prevailing view that Ste6 is a plasma membrane protein, we found that Ste6 is mainly associated with internal membranes and not with the cell surface. Fractionation and immunofluorescence data are compatible with a Golgi localization of Ste6. Despite its mostly intracellular localization, the Ste6 protein is in contact with the cell surface, as demonstrated by the finding that Ste6 accumulates in the plasma membrane in endocytosis mutants. The Ste6 protein which accumulates in the plasma membrane in endocytosis mutants is ubiquitinated. Ste6 is thus the second protein in yeast besides MAT alpha 2 for which ubiquitination has been demonstrated. Ste6 is a very unstable protein (half-life 13 min) which is stabilized approximately 3-fold in a ubc4 ubc5 mutant, implicating the ubiquitin system in the degradation of Ste6. The strongest stabilizing effect on Ste6 is, however, observed in the vacuolar pep4 mutant (half-life > 2 h), suggesting that most of Ste6 is degraded in the vacuole. Secretory functions are required for efficient degradation of Ste6, indicating that Ste6 enters the secretory pathway and is transported to the vacuole by vesicular carriers.     

Proteolytic Function of GPI-Anchored Plasma Membrane Protease Yps1p in the Yeast Vacuole and Golgi

Yps1p is a member of the GPI-anchored aspartic proteases which reside at the plasma membrane of Saccharomyces cerevisiae. Here we show that in Δerg6 cells, where a late biosynthetic step of the membrane lipid ergosterol is blocked, part of Yps1p was targeted to the vacuole. There it overtook proteolytic functions of the Pep4p protease, resulting in processing of pro-CPY to CPY in cells lacking the PEP4 gene. Yps1p was enriched in membrane microdomains, as it could be isolated in detergent-insoluble complexes from both normal and Δerg6 cells. Vacuolar Yps1 caused degradation of a mammalian sialyltransferase ectodomain fusion protein (ST6Ne), which was directed from the Golgi to the vacuole in both normal and Δerg6 cells. Unexpectedly, ST6Ne was degraded also when arrested in the Golgi in a temperature-sensitive sec7–1 mutant. Newly synthesized Yps1p, in transit to the plasma membrane, was also involved in the Golgi-associated degradation. These data show that GPI-anchored proteases, whose biological roles are unknown, may reside and function in different subcellular locations.     

The protective role of yeast Cathepsin D in acetic acid-induced apoptosis depends on ANT (Aac2p) but not on the voltage-dependent channel (Por1p)

We have previously shown that the yeast Cathepsin D (CatD) Pep4p translocates from the vacuole to the cytosol during acetic acid-induced apoptosis and is required for efficient mitochondrial degradation, though its specific role in this process is still elusive. Here, we show that the protective role of Pep4p in acetic acid-induced apoptosis depends on its catalytic activity and is independent of the yeast voltage-dependent anion channel Por1p (which has no role on mitochondrial degradation) but dependent on AAC proteins, the yeast adenine nucleotide translocator. Our results demonstrate a differential interplay between yeast vacuolar CatD and mitochondrial proteins involved in apoptosis regulation.     

Ceramide signaling targets the PP2A-like protein phosphatase Sit4p to impair vacuolar function,vesicular trafficking and autophagy in Isc1p deficient cells

The vacuoles play important roles in cellular homeostasis and their functions include the digestion of cytoplasmic material and organelles derived from autophagy. Conserved nutrient signaling pathways regulate vacuolar function and autophagy, ensuring normal cell and organismal development and aging. Recent evidence implicates sphingolipids in the modulation of these processes, but the impact of ceramide signaling on vacuolar dynamics and autophagy remains largely unknown. Here, we show that yeast cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase type 2, exhibit vacuolar fragmentation and dysfunctions, namely decreased Pep4p-mediated proteolysis and V-ATPase activity, which impairs vacuolar acidification. Moreover, these phenotypes are suppressed by downregulation of the ceramide-activated protein phosphatase Sit4p. The isc1Δ cells also exhibit defective Cvt and vesicular trafficking in a Sit4p-dependent manner, ultimately contributing to a reduced autophagic flux. Importantly, these phenotypes are also suppressed by downregulation of the nutrient signaling kinase TORC1, which is known to inhibit Sit4p and autophagy, or Sch9p. These results support a model in which Sit4p functions downstream of Isc1p in a TORC1-independent, ceramide-dependent signaling branch that impairs vacuolar function and vesicular trafficking, leading to autophagic defects in yeast.     

Regulation of copper-dependent endocytosis and vacuolar degradation of the yeast copper transporter, Ctr1p, by the Rsp5 ubiquitin ligase

The Saccharomyces cerevisiae high-affinity copper transporter, Ctr1p, mediates cellular uptake of Cu(I). We report that when copper (50 microm CuSO(4)) is added to the growth medium of copper-starved cells, Ctr1p is rapidly internalized by endocytosis, delivered to the lumen of the lysosome-like vacuole and slowly degraded by vacuolar proteases. Through analysis of the trafficking and degradation of Ctr1p mutants, two lysine residues in the C-terminal cytoplasmic tail of Ctr1p, Lys340 and Lys345, were found to be critical for copper-dependent endocytosis and degradation. In response to copper addition, Ctr1p was found to be ubiquitylated and a mutation in the Rsp5 ubiquitin ligase largely abolished ubiquitylation, endocytosis and degradation. In a strain lacking the Rsp5p accessory factors Bul1p and Bul2p, endocytosis and degradation of Ctr1p-green fluorescent protein were substantially diminished. Surprisingly, a Ctr1p mutant that lacks Lys340 and Lys345 was still ubiquitylated in a copper-dependent manner, indicating that ubiquitylation of Ctr1p on other sites is insufficient to drive copper-dependent endocytosis and degradation. This study demonstrates that copper regulates turnover of Ctr1p by stimulating Rsp5p-dependent endocytosis and degradation of Ctr1p in the vacuole.     

The proteolytic systems and heterologous proteins degradation in the methylotrophic yeastPichia pastoris

ThePichia pastoris expression system has been successfully used for production of various recombinant heterogeneous proteins. The productivity ofP. pastoris can be improved substantially by bioreactor cultivations. However, heterologous proteins degradation increases as well in high-cell density culture. Proteolytic degradation is a serious problem since the yeast has been employed to express recombinant proteins. In this review, some of the recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels on the cytosolic proteasome, vacuolar proteases, and proteases located within the secretory pathway inP. pastoris, are reviewed.