Candida albicans VPS11 is required for vacuole biogenesis and germ tube formation

The Candida albicans vacuole has previously been observed to undergo rapid expansion during the emergence of a germ tube from a yeast cell, to occupy the majority of the parent yeast cell. Furthermore, the yeast-to-hypha switch has been implicated in the virulence of this organism. The class C vps (vacuolar protein sorting) mutants of Saccharomyces cerevisiae are defective in multiple protein delivery pathways to the vacuole and prevacuole compartment. In this study C. albicans homologues of the S. cerevisiae class C VPS genes have been identified. Deletion of a C. albicans VPS11 homologue resulted in a number of phenotypes that closely resemble those of the class C vps mutants of S. cerevisiae, including the absence of a vacuolar compartment. The C. albicans vps11Delta mutant also had much-reduced secreted lipase and aspartyl protease activities. Furthermore, vps11Delta strains were defective in yeast-hypha morphogenesis. Upon serum induction of filamentous growth, mutants showed delayed emergence of germ tubes, had a reduced apical extension rate compared to those of control strains, and were unable to form mature hyphae. These results suggest that Vps11p-mediated trafficking steps are necessary to support the rapid emergence and extension of the germ tube from the parent yeast cell.     

Molecular analysis of the yeast VPS3 gene and the role of its product in vacuolar protein sorting and vacuolar segregation during the cell cycle

vps3 mutants of the yeast Saccharomyces cerevisiae are impaired in the sorting of newly synthesized soluble vacuolar proteins and in the acidification of the vacuole (Rothman, J. H., and T. H. Stevens. Cell. 47:1041-1051; Rothman, J. H., C. T. Yamashiro, C. K. Raymond, P. M. Kane, and T. H. Stevens. 1989. J. Cell Biol. 109:93-100). The VPS3 gene, which was cloned using a novel selection procedure, encodes a low abundance, hydrophilic protein of 117 kD that most likely resides in the cytoplasm. Yeast strains bearing a deletion of the VPS3 gene (vps3-delta 1) are viable, yet their growth rate is significantly reduced relative to wild-type cells. Temperature shift experiments with strains carrying a temperature conditional vps3 allele demonstrate that cells rapidly lose the capacity to sort the vacuolar protein carboxypeptidase Y upon loss of VPS3 function. Vacuolar morphology was examined in wild-type and vps3-delta 1 yeast strains by fluorescence microscopy. The vacuoles in wild-type yeast cells are morphologically complex, and they appear to be actively partitioned between mother cells and buds during an early phase of bud growth. Vacuolar morphology in vps3-delta 1 mutants is significantly altered from the wild-type pattern, and the vacuolar segregation process seen in wild-type strains is defective in these mutants. With the exception of a vacuolar acidification defect, the phenotypes of vps3-delta 1 strains are significantly different from those of mutants lacking the vacuolar proton-translocating ATPase. These data demonstrate that the acidification defect in vps3-delta 1 cells is not the primary cause of the pleiotropic defects in vacuolar function observed in these mutants.     

Nystatin effects on vacuolar function in Saccharomyces cerevisiae.

The effects of nystatin, a polyene antibiotic, was studied in Saccharomyces cerevisiae by isolating and characterizing nystatin-sensitive mutants. We isolated a number of nystatin-sensitive mutants by ethylmethane sulfonate mutagenesis. One of these mutants, the nss1 mutant, was characterized in detail. The mutant was sensitive to stresses such as high temperature or high concentrations of monovalent and divalent cations. The nss1 mutants showed severe vacuolar protein sorting and vacuolar morphology defects. The nss1 mutant was demonstrated to have a mutational lesion in the known VPS16 gene, which is essential for vacuolar protein sorting in S. cerevisiae. All of the vacuolar deficient mutants (vps11, vps16, vps18, and vps33) were sensitive to nystatin. Nystatin was found to cause extensive enlargement of the vacuole in wild-type S. cerevisiae cells. These results are discussed with special reference to the vacuolar function of S. cerevisiae.     

A functional analysis of the Candida albicans homolog of Saccharomyces cerevisiae VPS4

To investigate the role of the prevacuolar secretion pathway in the trafficking of vacuolar proteins in Candida albicans, the C. albicans homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene VPS4 was cloned and analyzed. Candida albicans VPS4 encodes a deduced AAA-type ATPase that is 75.6% similar to S. cerevisiae Vps4p, and plasmids bearing C. albicans VPS4 complemented the abnormal vacuolar morphology and carboxypeptidase missorting in S. cerevisiae vps4 null mutants. Candida albicans vps4Delta null mutants displayed a characteristic class E vacuolar morphology and multilamellar structures consistent with an aberrant prevacuolar compartment. The C. albicans vps4Delta mutant degraded more extracellular bovine serum albumin than did wild-type strains, which implied that this mutant secreted more extracellular protease activity. These phenotypes were complemented when a wild-type copy of VPS4 was reintroduced into its proper locus. Using a series of protease inhibitors, the origin of this extracellular protease activity was identified as a serine protease, and genetic analyses using a C. albicans vps4Deltaprc1Delta mutant identified this missorted vacuolar protease as carboxypeptidase Y. Unexpectedly, C. albicans Sap2p was not detected in culture supernatants of the vps4Delta mutants. These results indicate that C. albicans VPS4 is required for vacuolar biogenesis and proper sorting of vacuolar proteins.     

Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants.

The collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunofluorescence microscopy. Most of the vps mutants were found to possess vacuolar morphologies that differed significantly from wild-type vacuoles. Furthermore, mutants representing independent vps complementation groups were found to share aberrant morphological features. Six distinct classes of vacuolar morphology were observed. Mutants from eight vps complementation groups were defective both for vacuolar segregation from mother cells into developing buds and for acidification of the vacuole. Another group of mutants, represented by 13 complementation groups, accumulated a novel organelle distinct from the vacuole that contained a late-Golgi protein, active vacuolar H(+)-ATPase complex, and soluble vacuolar hydrolases. We suggest that this organelle may represent an exaggerated endosome-like compartment. None of the vps mutants appeared to mislocalize significant amounts of the vacuolar membrane protein alkaline phosphatase. Quantitative immunoprecipitations of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) were performed to determine the extent of the sorting defect in each vps mutant. A good correlation between morphological phenotype and the extent of the CPY sorting defect was observed.     

A putative zinc finger protein, Saccharomyces cerevisiae Vps18p, affects late Golgi functions required for vacuolar protein sorting and efficient alpha-factor prohormone maturation.

Saccharomyces cerevisiae strains carrying vps18 mutations are defective in the sorting and transport of vacuolar enzymes. The precursor forms of these proteins are missorted and secreted from the mutant cells. Most vps18 mutants are temperature sensitive for growth and are defective in vacuole biogenesis; no structure resembling a normal vacuole is seen. A plasmid complementing the temperature-sensitive growth defect of strains carrying the vps18-4 allele was isolated from a centromere-based yeast genomic library. Integrative mapping experiments indicated that the 26-kb insert in this plasmid was derived from the VPS18 locus. A 4-kb minimal complementing fragment contains a single long open reading frame predicted to encode a 918-amino-acid hydrophilic protein. Comparison of the VPS18 sequence with the PEP3 sequence reported in the accompanying paper (R. A. Preston, H. F. Manolson, K. Becherer, E. Weidenhammer, D. Kirkpatrick, R. Wright, and E. W. Jones, Mol. Cell. Biol. 11:5801-5812, 1991) shows that the two genes are identical. Disruption of the VPS18/PEP3 gene (vps18 delta 1::TRP1) is not lethal but results in the same vacuolar protein sorting and growth defects exhibited by the original temperature-sensitive vps18 alleles. In addition, vps18 delta 1::TRP1 MAT alpha strains exhibit a defect in the Kex2p-dependent processing of the secreted pheromone alpha-factor. This finding suggests that vps18 mutations alter the function of a late Golgi compartment which contains Kex2p and in which vacuolar proteins are thought to be sorted from proteins destined for the cell surface. The Vps18p sequence contains a cysteine-rich, zinc finger-like motif at the COOH terminus. A mutant in which the first cysteine of this motif was changed to serine results in a temperature-conditional carboxypeptidase Y sorting defect shortly after a shift to nonpermissive conditions. We identified a similar cysteine-rich motif near the COOH terminus of another Vps protein, the Vps11/Pep5/End1 protein. Preston et al. (Mol. Cell. Biol. 11:5801-5812, 1991) present evidence that the Vps18/Pep3 protein colocalizes with the Vps11/Pep5 protein to the cytosolic face of the vacuolar membrane. Together with the similar phenotypes exhibited by both vps11 and vps18 mutants, this finding suggests that they may function at a common step during vacuolar protein sorting and that the integrity of their zinc finger motifs may be required for this function.     

The Candida albicans vacuole is required for differentiation and efficient macrophage killing

Yeast-hypha differentiation is believed to be necessary for the normal progression of Candida albicans infections. The emergence and extension of a germ tube from a parental yeast cell are accompanied by dynamic changes in vacuole size and morphology. Although vacuolar function is required during this process, it is unclear if it is vacuolar expansion or some other vacuolar function that is important. We previously described a C. albicans vps11Delta mutant which lacked a recognizable vacuole compartment and with defects in multiple vacuolar functions. These include sensitivities to stress, reduced proteolytic activities, and severe defects in filamentation. Herein we utilize a partially functional VPS11 allele (vps11hr) to help define which vacuolar functions are required for differentiation and which influence interaction with macrophages. Mutant strains harboring this allele are not osmotically or temperature sensitive and have normal levels of secreted aspartyl protease and carboxypeptidase Y activity but have a fragmented vacuole morphology. Moreover, this mutant is defective in filamentation, suggesting that the major role the vacuole plays in yeast-hypha differentiation may relate directly to its morphology. The results of this study support the hypothesis that vacuole expansion is required during germ tube emergence. Both vps11 mutants were severely attenuated in their ability to kill a macrophage cell line. The viability of the vps11delta mutant was significantly reduced during macrophage interaction compared to that in the control strains, while the vps11hr mutant was unaffected. This implies some vacuolar functions are required for Candida survival within the macrophage, while additional vacuolar functions are required to inflict injury on the macrophage.     

The yeast vps class E mutants: the beginning of the molecular genetic analysis of multivesicular body biogenesis

In 1992, Raymond et al. published a compilation of the 41 yeast vacuolar protein sorting (vps) mutant groups and described a large class of mutants (class E vps mutants) that accumulated an exaggerated prevacuolar endosome-like compartment. Further analysis revealed that this "class E compartment" contained soluble vacuolar hydrolases, vacuolar membrane proteins, and Golgi membrane proteins unable to recycle back to the Golgi complex, yet these class E vps mutants had what seemed to be normal vacuoles. The 13 class E VPS genes were later shown to encode the proteins that make up the complexes required for formation of intralumenal vesicles in late endosomal compartments called multivesicular bodies, and for the sorting of ubiquitinated cargo proteins into these internal vesicles for eventual delivery to the vacuole or lysosome.     

Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis.

vps33 mutants missort and secrete multiple vacuolar hydrolases and exhibit extreme defects in vacuolar morphology. Toward a molecular understanding of the role of the VPS33 gene in vacuole biogenesis, we have cloned this gene from a yeast genomic library by complementation of a temperature-sensitive vps33 mutation. Gene disruption demonstrated that VPS33 was not essential but was required for growth at high temperatures. At the permissive temperature, vps33 null mutants exhibited defects in vacuolar protein localization and vacuole morphology similar to those seen in most of the original mutant alleles. Sequence analysis revealed a putative open reading frame sufficient to encode a protein of 691 amino acids. Hydropathy analysis indicated that the deduced product of the VPS33 gene is generally hydrophilic, contains no obvious signal sequence or transmembrane domains, and is therefore unlikely to enter the secretory pathway. Polyclonal antisera raised against TrpE-Vps33 fusion proteins recognized a protein in yeast cells of the expected molecular weight, approximately 75,000. In cell fractionation studies, Vps33p behaved as a cytosolic protein. The predicted VPS33 gene product possessed sequence similarity with a number of ATPases and ATP-binding proteins specifically in their ATP-binding domains. One vps33 temperature-sensitive mutant contained a missense mutation near this region of sequence similarity; the mutation resulted in a Leu-646----Pro substitution in Vps33p. This temperature-sensitive mutant strain contained normal vacuoles at the permissive temperature but lacked vacuoles specifically in the bud at the nonpermissive temperature. Our data suggest that Vps33p acts in the cytoplasm to facilitate Golgi-to-vacuole protein delivery. We propose that as a consequence of the vps33 protein-sorting defects, abnormalities in vacuolar morphology and vacuole assembly result.     

A yeast protein related to a mammalian Ras-binding protein, Vps9p, is required for localization of vacuolar proteins.

In the yeast Saccharomyces cerevisiae, mutations in vacuolar protein sorting (VPS) genes result in secretion of proteins normally localized to the vacuole. Characterization of the VPS pathway has provided considerable insight into mechanisms of protein sorting and vesicle-mediated intracellular transport. We have cloned VPS9 by complementation of the vacuolar protein sorting defect of vps9 cells, characterized its gene product, and investigated its role in vacuolar protein sorting. Cells with a vps9 disruption exhibit severe vacuolar protein sorting defects and a temperature-sensitive growth defect at 38 degrees C. Electron microscopic examination of delta vps9 cells revealed the appearance of novel reticular membrane structures as well as an accumulation of 40- to 50-nm-diameter vesicles, suggesting that Vps9p may be required for the consumption of transport vesicles containing vacuolar protein precursors. A temperature-conditional allele of vps9 was constructed and used to investigate the function of Vps9p. Immediately upon shifting of temperature-conditional vps9 cells to the nonpermissive temperature, newly synthesized carboxypeptidase Y was secreted, indicating that Vps9p function is directly required in the VPS pathway. Antibodies raised against Vps9p immunoprecipitate a rare 52-kDa protein that fractionates with cytosolic proteins following cell lysis and centrifugation. Analysis of the VPS9 DNA sequence predicts that Vps9p is related to human proteins that bind Ras and negatively regulate Ras-mediated signaling. We term the related regions of Vps9p and these Ras-binding proteins a GTPase binding homology domain and suggest that it defines a family of proteins that bind monomeric GTPases. Vps9p may bind and serve as an effector of a rab GTPase, like Vps2lp, required for vacuolar protein sorting.     

Saccharomyces cerevisiae Mutants Affected in Vacuole Assembly or Vacuolar H+-ATPase are Hypersensitive to Lead (Pb) Toxicity

Lead is an important environmental pollutant. The role of vacuole, in Pb detoxification, was studied using a vacuolar protein sorting mutant strain (vps16Δ), belonging to class C mutants. Cells disrupted in VPS16 gene, did not display a detectable vacuolar-like structure. Based on the loss of cell proliferation capacity, it was found that cells from vps16Δ mutant exhibited a hypersensitivity to Pb-induced toxicity, compared to wild type (WT) strain. The function of vacuolar H⁺-ATPase (V-ATPase), in Pb detoxification, was evaluated using mutants with structurally normal vacuoles but defective in subunits of catalytic (vma1Δ or vma2Δ) or membrane domain (vph1Δ or vma3Δ) of V-ATPase. All mutants tested, lacking a functional V-ATPase, displayed an increased susceptibility to Pb, comparatively to cells from WT strain. Modification of vacuolar morphology, in Pb-exposed cells, was visualized using a Vma2p-GFP strain. The treatment of yeast cells with Pb originated the fusion of the medium size vacuolar lobes into one enlarged vacuole. In conclusion, it was found that vacuole plays an important role in the detoxification of Pb in Saccharomyces cerevisiae; in addition, a functional V-ATPase was required for Pb compartmentalization.     

Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases.

vps35 mutants of Saccharomyces cerevisiae exhibit severe defects in the localization of carboxypeptidase Y, a soluble vacuolar hydrolase. We have cloned the wild-type VPS35 gene by complementation of the vacuolar protein sorting defect exhibited by the vps35-17 mutant. Sequence analysis revealed an open reading frame predicted to encode a protein of 937 amino acids that lacks any obvious hydrophobic domains. Subcellular fractionation studies indicated that 80% of Vps35p peripherally associates with a membranous particulate cell fraction. The association of Vps35p with this fraction appears to be saturable; when overproduced, the vast majority of Vps35p remains in a soluble fraction. Disruption of the VPS35 gene demonstrated that it is not essential for yeast cell growth. However, the null allele of VPS35 results in a differential defect in the sorting of vacuolar carboxypeptidase Y (CPY), proteinase A (PrA), proteinase B (PrB), and alkaline phosphatase (ALP). proCPY was quantitatively missorted and secreted by delta vps35 cells, whereas almost all of proPrA, proPrB, and proALP were retained within the cell and converted to their mature forms, indicating delivery to the vacuole. Based on these observations, we propose that alternative pathways exist for the sorting and/or delivery of proteins to the vacuole.     

Arabidopsis VPS35, a retromer component, is required for vacuolar protein sorting and involved in plant growth and leaf senescence

The retromer complex is responsible for retrograde transport,which is coordinated with anterograde transport in the secretorypathway including vacuolar protein sorting. Yeast VPS35 is acomponent of the retromer complex that is essential for recognitionof specific cargo molecules. The physiological function of VPS35has not been determined in vacuolar protein sorting in higherorganisms. Arabidopsis thaliana has three VPS35 homologs designatedVPS35a, VPS35b and VPS35c. We isolated four vps35 mutants (vps35a-1,vps35b-1, vps35b-2 and vps35c-1) and then generated four doublemutants and one triple mutant. vps35a-1 vps35c-1 exhibited nounusual phenotypes. On the other hand, vps35b-1 vps35c-1 andthe triple mutant (vps35a-1 vps35b-2 vps35c-1) exhibited severephenotypes: dwarfism, early leaf senescence and fragmentationof protein storage vacuoles (PSVs). In addition, these mutantsmis-sorted storage proteins by secreting them out of the cellsand accumulated a higher level of vacuolar sorting receptor(VSR) than the wild type. VPS35 was localized in pre-vacuolarcompartments (PVCs), some of which contained VSR. VPS35 wasimmunoprecipitated with VPS29/MAG1, another component of theretromer complex. Our findings suggest that VPS35, mainly VPS35b,is involved in sorting proteins to PSVs in seeds, possibly byrecycling VSR from PVCs to the Golgi complex, and is also involvedin plant growth and senescence in vegetative organs.     

VPS21 encodes a rab5-like GTP binding protein that is required for the sorting of yeast vacuolar proteins.

Many of the vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae exhibit severe defects in the sorting of vacuolar proteins but still retain near-normal vacuole morphology. The gene affected in one such mutant, vps21, has been cloned and found to encode a member of the ras-like GTP binding protein family. Sequence comparisons with other known GTP binding proteins indicate that Vps21p is unique but shares striking similarity with mammalian rab5 proteins (> 50% identity and > 70% similarity). Regions with highest similarity are clustered within the putative GTP binding motifs and the proposed effector domains of the Vps21/rab5 proteins. Point mutations constructed within these conserved regions inactivate Vps21p function; the mutant cells missort and secrete the soluble vacuolar hydrolase carboxypeptidase Y (CPY). Cells carrying a complete deletion of the VPS21 coding sequence (i) are viable but exhibit a growth defect at 38 degrees C, (ii) missort multiple vacuolar proteins, (iii) accumulate 40-50 nm vesicles and (iv) contain a large vacuole. VPS21 encodes a 22 kDa protein that binds GTP and fractionates with subcellular membranes. Mutant analysis indicates that the association with a membrane(s) is dependent on geranylgeranylation of the C-terminal cysteine residue(s) of Vps21p. We propose that Vps21p functions in the targeting and/or fusion of transport vesicles that mediate the delivery of proteins to the vacuole.     

Endosome to Golgi Retrieval of the Vacuolar Protein Sorting Receptor, Vps10p, Requires the Function of the VPS29, VPS30, and VPS35 Gene Products

Mutations in the S. cerevisiae VPS29 and VPS30 genes lead to a selective protein sorting defect in which the vacuolar protein carboxypeptidase Y (CPY) is missorted and secreted from the cell, while other soluble vacuolar hydrolases like proteinase A (PrA) are delivered to the vacuole. This phenotype is similar to that seen in cells with mutations in the previously characterized VPS10 and VPS35 genes. Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for soluble vacuolar hydrolases like CPY and PrA, while Vps35p is a peripheral membrane protein which cofractionates with membranes enriched in Vps10p. The sequences of the VPS29, VPS30, and VPS35 genes do not yet give any clues to the functions of their products. Each is predicted to encode a hydrophilic protein with homologues in the human and C. elegans genomes. Interestingly, mutations in the VPS29, VPS30, or VPS35 genes change the subcellular distribution of the Vps10 protein, resulting in a shift of Vps10p from the Golgi to the vacuolar membrane. The route that Vps10p takes to reach the vacuole in a vps35 mutant does not depend upon Sec1p mediated arrival at the plasma membrane but does require the activity of the pre-vacuolar endosomal t-SNARE, Pep12p. A temperature conditional allele of the VPS35 gene was generated and has been found to cause missorting/secretion of CPY and also Vps10p to mislocalize to a vacuolar membrane fraction at the nonpermissive temperature. Vps35p continues to cofractionate with Vps10p in vps29 mutants, suggesting that Vps10p and Vps35p may directly interact. Together, the data indicate that the VPS29, VPS30, and VPS35 gene products are required for the normal recycling of Vps10p from the prevacuolar endosome back to the Golgi where it can initiate additional rounds of vacuolar hydrolase sorting.     

Novel Golgi to vacuole delivery pathway in yeast: identification of a sorting determinant and required transport component.

More than 40 vacuolar protein sorting (vps) mutants have been identified which secrete proenzyme forms of soluble vacuolar hydrolases to the cell surface. A subset of these mutants has been found to show selective defects in the sorting of two vacuolar membrane proteins. Under non-permissive conditions, vps45tsf (SEC1 homolog) and pep12/vps6tsf (endosomal t-SNARE) mutants efficiently sort alkaline phosphatase (ALP) to the vacuole while multiple soluble vacuolar proteins and the membrane protein carboxypeptidase yscS (CPS) are no longer delivered to the vacuole. Vacuolar localization of ALP in these mutants does not require transport to the plasma membrane followed by endocytic uptake, as double mutants of pep12tsf and vps45tsf with sec1 and end3 sort and mature ALP at the non-permissive temperature. Given the demonstrated role of t-SNAREs such as Pep12p in transport vesicle recognition, our results indicate that ALP and CPS are packaged into distinct transport intermediates. Consistent with ALP following an alternative route to the vacuole, isolation of a vps41tsf mutant revealed that at non-permissive temperature ALP is mislocalized while vacuolar delivery of CPS and CPY is maintained. A series of domain-swapping experiments was used to define the sorting signal that directs selective packaging and transport of ALP. Our data demonstrate that the amino-terminal 16 amino acid portion of the ALP cytoplasmic tail domain contains a vacuolar sorting signal which is responsible for the active recognition, packaging and transport of ALP from the Golgi to the vacuole via a novel delivery pathway.     

Defect of vacuolar protein sorting stimulates proteolytic processing of human urokinase-type plasminogen activator in the yeast Hansenula polymorpha

Human urokinase-type plasminogen activator (uPA) is poorly secreted by yeast cells. Here, we have selected Hansenula polymorpha mutants with increased productivity of active extracellular uPA. Several of the obtained mutants also demonstrated a defect of sorting of carboxypeptidase Y to the vacuole and the mutant loci have been identified in six of them. All these mutations damaged genes involved in protein traffic between the Golgi apparatus and the vacuole, namely PEP3, VPS8, VPS10, VPS17, and VPS35. We have shown that inactivation of the VPS10 gene encoding the vacuolar protein sorting receptor does not increase uPA secretion but stimulates its proteolytic processing.     

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.     

Arabidopsis vacuolar sorting mutants (green fluorescent seed) can be identified efficiently by secretion of vacuole-targeted green fluorescent protein in their seeds

Two Arabidopsis thaliana genes have been shown to function in vacuolar sorting of seed storage proteins: a vacuolar sorting receptor, VSR1/ATELP1, and a retromer component, MAIGO1 (MAG1)/VPS29. Here, we show an efficient and simple method for isolating vacuolar sorting mutants of Arabidopsis. The method was based on two findings in this study. First, VSR1 functioned as a sorting receptor for beta-conglycinin by recognizing the vacuolar targeting signal. Second, when green fluorescent protein (GFP) fusion with the signal (GFP-CT24) was expressed in vsr1, mag1/vps29, and wild-type seeds, both vsr1and mag1/vps29 gave strongly fluorescent seeds but the wild type did not, suggesting that a defect in vacuolar sorting provided fluorescent seeds by the secretion of GFP-CT24 out of the cells. We mutagenized transformant seeds expressing GFP-CT24. From approximately 3,000,000 lines of M2 seeds, we obtained >100 fluorescent seeds and designated them green fluorescent seed (gfs) mutants. We report 10 gfs mutants, all of which caused missorting of storage proteins. We mapped gfs1 to VSR1, gfs2 to KAM2/GRV2, gfs10 to the At4g35870 gene encoding a novel membrane protein, and the others to different loci. This method should provide valuable insights into the complex molecular mechanisms underlying vacuolar sorting of storage proteins.     

Yeast Vps55p,a functional homolog of human obesity receptor gene-related protein,is involved in late endosome to vacuole trafficking

The Saccharomyces cerevisiae VPS55 (YJR044c) gene encodes a small protein of 140 amino acids with four potential transmembrane domains. VPS55 belongs to a family of genes of unknown function, including the human gene encoding the obesity receptor gene-related protein (OB-RGRP). Yeast cells with a disrupted VPS55 present normal vacuolar morphology, but exhibit an abnormal secretion of the Golgi form of the soluble vacuolar carboxypeptidase Y. However, trafficking of the membrane-bound vacuolar alkaline phosphatase remains normal. The endocytosis of uracil permease, used as an endocytic marker, is normal in vps55Delta cells, but its degradation is delayed and this marker transiently accumulates in late endosomal compartments. We also found that Vps55p is mainly localized in the late endosomes. Collectively, these results indicate that Vps55p is involved in late endosome to vacuole trafficking. Finally, we show that human OB-RGRP displays the same distribution as Vps55p and corrects the phenotypic defects of the vps55Delta strain. Therefore, the function of Vps55p has been conserved throughout evolution. This study highlights the importance of the multispanning Vps55p and OB-RGRP in membrane trafficking to the vacuole/lysosome of eukaryotic cells.