scholarly journals Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast.

1994 ◽  
Vol 125 (2) ◽  
pp. 283-298 ◽  
Author(s):  
B Singer-Krüger ◽  
H Stenmark ◽  
A Düsterhöft ◽  
P Philippsen ◽  
J S Yoo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Lucifer Yellow ◽  
Protein Sorting ◽  
Personal Communication ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Endocytic Pathway ◽  
Phenotypic Characterization ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Protein

The small GTPase rab5 has been shown to represent a key regulator in the endocytic pathway of mammalian cells. Using a PCR approach to identify rab5 homologs in Saccharomyces cerevisiae, two genes encoding proteins with 54 and 52% identity to rab5, YPT51 and YPT53 have been identified. Sequencing of the yeast chromosome XI has revealed a third rab5-like gene, YPT52, whose protein product exhibits a similar identity to rab5 and the other two YPT gene products. In addition to the high degree of identity/homology shared between rab5 and Ypt51p, Ypt52p, and Ypt53p, evidence for functional homology between the mammalian and yeast proteins is provided by phenotypic characterization of single, double, and triple deletion mutants. Endocytic delivery to the vacuole of two markers, lucifer yellow CH (LY) and alpha-factor, was inhibited in delta ypt51 mutants and aggravated in the double ypt51ypt52 and triple ypt51ypt52ypt53 mutants, suggesting a requirement for these small GTPases in endocytic membrane traffic. In addition to these defects, the here described ypt mutants displayed a number of other phenotypes reminiscent of some vacuolar protein sorting (vps) mutants, including a differential delay in growth and vacuolar protein maturation, partial missorting of a soluble vacuolar hydrolase, and alterations in vacuole acidification and morphology. In fact, vps21 represents a mutant allele of YPT51 (Emr, S., personal communication). Altogether, these data suggest that Ypt51p, Ypt52p, and Ypt53p are required for transport in the endocytic pathway and for correct sorting of vacuolar hydrolases suggesting a possible intersection of the endocytic with the vacuolar sorting pathway.

scholarly journals Involvement of Specific COPI Subunits in Protein Sorting from the Late Endosome to the Vacuole in Yeast

2006 ◽  
Vol 27 (2) ◽  
pp. 526-540 ◽  
Author(s):  
Galina Gabriely ◽  
Rachel Kama ◽  
Jeffrey E. Gerst
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Protein Sorting ◽  
Multivesicular Body ◽  
Carboxypeptidase Y ◽  
Direct Role ◽  
Vacuolar Protein Sorting ◽  
Early Secretory Pathway ◽  
Physical Interactions ◽  
Vacuolar Protein

ABSTRACT Although COPI function on the early secretory pathway in eukaryotes is well established, earlier studies also proposed a nonconventional role for this coat complex in endocytosis in mammalian cells. Here we present results that suggest an involvement for specific COPI subunits in the late steps of endosomal protein sorting in Saccharomyces cerevisiae. First, we found that carboxypeptidase Y (CPY) was partially missorted to the cell surface in certain mutants of the COPIB subcomplex (COPIb; Sec27, Sec28, and possibly Sec33), which indicates an impairment in endosomal transport. Second, integral membrane proteins destined for the vacuolar lumen (i.e., carboxypeptidase S [CPS1]; Fur4, Ste2, and Ste3) accumulated at an aberrant late endosomal compartment in these mutants. The observed phenotypes for COPIb mutants resemble those of class E vacuolar protein sorting (vps) mutants that are impaired in multivesicular body (MVB) protein sorting and biogenesis. Third, we observed physical interactions and colocalization between COPIb subunits and an MVB-associated protein, Vps27. Together, our findings suggest that certain COPI subunits could have a direct role in vacuolar protein sorting to the MVB compartment.

scholarly journals An evolutionary switch in the specificity of an endosomal CORVET tether underlies formation of regulated secretory vesicles in the ciliate Tetrahymena thermophila

2017 ◽  
Author(s):  
Daniela Sparvoli ◽  
Elisabeth Richardson ◽  
Hiroko Osakada ◽  
Xun Lan ◽  
Masaaki Iwamoto ◽  
...  
Keyword(s):  
Tetrahymena Thermophila ◽  
Secretory Granules ◽  
Protein Sorting ◽  
Endocytic Pathway ◽  
Forward Genetics ◽  
Vacuolar Protein Sorting ◽  
Protein Receptor ◽  
Early Endosomes ◽  
Class C ◽  
Vacuolar Protein

SummaryIn the endocytic pathway of animals, two related complexes, called CORVET (Class C Core Vacuole/Endosome Transport) and HOPS (Homotypic fusion and protein sorting), act as both tethers and fusion factors for early and late endosomes, respectively. Mutations in CORVET or HOPS lead to trafficking defects and contribute to human disease including immune dysfunction. HOPS and CORVET are conserved throughout eukaryotes but remarkably, in the ciliate Tetrahymena thermophila, the HOPS-specific subunits are absent while CORVET-specific subunits have proliferated. VPS8 (Vacuolar Protein Sorting), a CORVET subunit, expanded to 6 paralogs in Tetrahymena. This expansion correlated with loss of HOPS within a ciliate subgroup including the Oligohymenophorea, which contains Tetrahymena. As uncovered via forward genetics, a single VPS8 paralog in Tetrahymena (VPS8A) is required to synthesize prominent secretory granules called mucocysts. More specifically, ∆vps8a cells fail to deliver a subset of cargo proteins to developing mucocysts, instead accumulating that cargo in vesicles also bearing the mucocyst sorting receptor, Sor4p. Surprisingly, although this transport step relies on CORVET, it does not appear to involve early endosomes. Instead, Vps8a associates with the late endosomal/lysosomal marker Rab7, indicating target specificity switching occurred in CORVET subunits during the evolution of ciliates. Mucocysts belong to a markedly diverse and understudied class of protist secretory organelles called extrusomes. Our results underscore that biogenesis of mucocysts depends on endolysosomal trafficking, revealing parallels with invasive organelles in apicomplexan parasites and suggesting that a wide array of secretory adaptations in protists, like in animals, depend on mechanisms related to lysosome biogenesis.AbbreviationsLRO(Lysosome-related organelle)HOPS(homotypic fusion and protein sorting complex)CORVET(Class C core Vacuole/Endosome Transport)VPS(vacuolar protein sorting)GRL(granule lattice)GRT(granule tip)Igr(Induced upon granule regeneration)SNARE(Soluble NSF attachment protein receptor)LECA(last eukaryotic common ancestor)

scholarly journals A Novel RING Finger Protein Complex Essential for a Late Step in Protein Transport to the Yeast Vacuole

1997 ◽  
Vol 8 (11) ◽  
pp. 2307-2327 ◽  
Author(s):  
Stephanie E. Rieder ◽  
Scott D. Emr
Keyword(s):  
Protein Complex ◽  
Protein Transport ◽  
Protein Sorting ◽  
Ring Finger ◽  
Endocytic Pathway ◽  
Vacuolar Protein Sorting ◽  
Oligomeric Protein ◽  
Late Step ◽  
Class C ◽  
Vacuolar Protein

Protein transport to the lysosome-like vacuole in yeast is mediated by multiple pathways, including the biosynthetic routes for vacuolar hydrolases, the endocytic pathway, and autophagy. Among the more than 40 genes required for vacuolar protein sorting (VPS) inSaccharomyces cerevisiae, mutations in the four class CVPS genes result in the most severe vacuolar protein sorting and morphology defects. Herein, we provide complementary genetic and biochemical evidence that the class C VPSgene products (Vps18p, Vps11p, Vps16p, and Vps33p) physically and functionally interact to mediate a late step in protein transport to the vacuole. Chemical cross-linking experiments demonstrated that Vps11p and Vps18p, which both contain RING finger zinc-binding domains, are components of a hetero-oligomeric protein complex that includes Vps16p and the Sec1p homologue Vps33p. The class C Vps protein complex colocalized with vacuolar membranes and a distinct dense membrane fraction. Analysis of cells harboring a temperature-conditionalvps18 allele (vps18tsf) indicated that Vps18p function is required for the biosynthetic, endocytic, and autophagic protein transport pathways to the vacuole. In addition,vps18tsfcells accumulated multivesicular bodies, autophagosomes, and other membrane compartments that appear to represent blocked transport intermediates. Overproduction of either Vps16p or the vacuolar syntaxin homologue Vam3p suppressed defects associated with vps18tsfmutant cells, indicating that the class C Vps proteins and Vam3p may functionally interact. Thus we propose that the class C Vps proteins are components of a hetero-oligomeric protein complex that mediates the delivery of multiple transport intermediates to the vacuole.

scholarly journals An Interorganellar Bidding War: Vps13 Localization by Competitive Organelle-Specific Adaptors

Contact ◽  
2018 ◽  
Vol 1 ◽  
pp. 251525641881462
Author(s):  
Samantha K. Dziurdzik ◽  
Björn D.M. Bean ◽  
Elizabeth Conibear
Keyword(s):  
Endoplasmic Reticulum ◽  
Recent Work ◽  
Protein Sorting ◽  
Repeat Region ◽  
Vacuolar Protein Sorting ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Prospore Membrane ◽  
Membrane Contact ◽  
Vacuolar Protein

Membrane contact sites are regulated through the controlled recruitment of constituent proteins. Yeast vacuolar protein sorting 13 (Vps13) dynamically localizes to membrane contact sites at endosomes, vacuoles, mitochondria, and the endoplasmic reticulum under different cellular conditions and is recruited to the prospore membrane during meiosis. Prior to our recent work, the mechanism for localization at contact sites was largely unknown. We identified Ypt35 as a novel Vps13 adaptor for endosomes and the nucleus-vacuole junction. Furthermore, we discovered a conserved recruitment motif in Ypt35 and found related motifs in the prospore membrane and mitochondrial adaptors, Spo71 and Mcp1, respectively. All three adaptors compete for binding to a six-repeat region of Vps13, suggesting adaptor competition regulates Vps13 localization. Here, we summarize and discuss the implications of our work, highlighting key outstanding questions.

MP005INCREASED LYSOSOMAL DEGRADATION OF AQP2 IN MPKCCDC14 CELLS WITH KNOCKDOWN OF VACUOLAR PROTEIN SORTING-ASSOCIATED PROTEIN 35

2016 ◽  
Vol 31 (suppl_1) ◽  
pp. i345-i345
Author(s):  
Hyo-Jung Choi ◽  
Mi Suk Lee ◽  
Dasom Kim ◽  
Eui-Jung Park ◽  
Yu-Jung Lee ◽  
...  
Keyword(s):  
Protein Sorting ◽  
Lysosomal Degradation ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Protein

scholarly journals The HOPS complex mediates autophagosome–lysosome fusion through interaction with syntaxin 17

2014 ◽  
Vol 25 (8) ◽  
pp. 1327-1337 ◽  
Author(s):  
Peidu Jiang ◽  
Taki Nishimura ◽  
Yuriko Sakamaki ◽  
Eisuke Itakura ◽  
Tomohisa Hatta ◽  
...  
Keyword(s):  
Protein Sorting ◽  
Endocytic Pathway ◽  
Mass Spectrometry Analysis ◽  
Autophagic Flux ◽  
Interacting Protein ◽  
Spectrometry Analysis ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Vacuolar Protein ◽  
Hops Complex

Membrane fusion is generally controlled by Rabs, soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs), and tethering complexes. Syntaxin 17 (STX17) was recently identified as the autophagosomal SNARE required for autophagosome–lysosome fusion in mammals and Drosophila. In this study, to better understand the mechanism of autophagosome–lysosome fusion, we searched for STX17-interacting proteins. Immunoprecipitation and mass spectrometry analysis identified vacuolar protein sorting 33A (VPS33A) and VPS16, which are components of the homotypic fusion and protein sorting (HOPS)–tethering complex. We further confirmed that all HOPS components were coprecipitated with STX17. Knockdown of VPS33A, VPS16, or VPS39 blocked autophagic flux and caused accumulation of STX17- and microtubule-associated protein light chain (LC3)–positive autophagosomes. The endocytic pathway was also affected by knockdown of VPS33A, as previously reported, but not by knockdown of STX17. By contrast, ultraviolet irradiation resistance–associated gene (UVRAG), a known HOPS-interacting protein, did not interact with the STX17–HOPS complex and may not be directly involved in autophagosome–lysosome fusion. Collectively these results suggest that, in addition to its well-established function in the endocytic pathway, HOPS promotes autophagosome–lysosome fusion through interaction with STX17.

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