Distinct domains within Vps35p mediate the retrieval of two different cargo proteins from the yeast prevacuolar/endosomal compartment.
Resident membrane proteins of the trans-Golgi network (TGN) of Saccharomyces cerevisiae are selectively retrieved from a prevacuolar/late endosomal compartment. Proper cycling of the carboxypeptidase Y receptor Vps10p between the TGN and prevacuolar compartment depends on Vps35p, a hydrophilic peripheral membrane protein. In this study we use a temperature-sensitive vps35 allele to show that loss of Vps35p function rapidly leads to mislocalization of A-ALP, a model TGN membrane protein, to the vacuole. Vps35p is required for the prevacuolar compartment-to-TGN transport of both A-ALP and Vps10p. This was demonstrated by phenotypic analysis of vps35 mutant strains expressing A-ALP mutants lacking either the retrieval or static retention signals and by an assay for prevacuolar compartment-to-TGN transport. A novel vps35 allele was identified that was defective for retrieval of A-ALP but functional for retrieval of Vps10p. Moreover, several other vps35 alleles were identified with the opposite characteristics: they were defective for Vps10p retrieval but near normal for A-ALP localization. These data suggest a model in which distinct structural features within Vps35p are required for associating with the cytosolic domains of each cargo protein during the retrieval process.
Nothwehr
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Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211, USA. nothwehr@biosci.mpb.missouri.edu
Bruinsma
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Strawn
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D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D003001:Cloning, Molecular; D011992:Endosomes / Q000502:physiology*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014617:Vacuoles / Q000502:physiology*; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C076106:VPS35 protein, S cerevisiae; D033921:Vesicular Transport Proteins