Direct binding to Rsp5p regulates ubiquitination-independent vacuolar transport of Sna3p.
The sorting of integral membrane proteins such as carboxypeptidase S (Cps1p) into the luminal vesicles of multivesicular bodies (MVBs) in Saccharomyces cerevisiae requires ubiquitination of their cytosolic domains by the ubiquitin ligases Rsp5p and/or Tul1p. An exception is Sna3p, which does not require ubiquitination for entry into MVBs. The mechanism underlying this ubiquitination-independent MVB sorting pathway has not yet been characterized. Here, we show that Sna3p sorting into the MVB pathway depends on a direct interaction between a PPAY motif within its C-terminal cytosolic tail and the WW domains of Rsp5p. Disruption of this interaction inhibits vacuolar targeting of Sna3p and causes its accumulation in a compartment that overlaps only partially with MVBs. Surprisingly, Sna3p does require a functional ubiquitin-ligase HECT domain within Rsp5p; however, the dependence of Sna3p on HECT domain activity is distinct from that of Cps1p. Last, we show that Sna3p requires neither Tul1p nor the transmembrane adaptor protein Bsd2p for its MVB sorting. Our data demonstrate that Sna3p follows a novel ubiquitination-independent, but Rsp5p-mediated, sorting pathway to the vacuole.
Watson
Hadiya
H
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Bonifacino
Juan S
JS
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D001693:Biological Transport, Active; D002268:Carboxypeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D005800:Genes, Fungal; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D025801:Ubiquitin / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
C495386:BSD2 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C437323:Sna3 protein, S cerevisiae; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D002268:Carboxypeptidases; C019836:CPS1 protein, S cerevisiae; C469287:RSP5 protein, S cerevisiae