Direct binding to Rsp5 mediates ubiquitin-independent sorting of Sna3 via the multivesicular body pathway.
The sorting of most integral membrane proteins into the lumenal vesicles of multivesicular bodies (MVBs) is dependent on the attachment of ubiquitin (Ub) to their cytosolic domains. However, Ub is not required for sorting of Sna3, an MVB vesicle cargo protein in yeast. We show that Sna3 circumvents Ub-mediated recognition by interacting directly with Rsp5, an E3 Ub ligase that catalyzes monoubiquitination of MVB vesicle cargoes. The PPAY motif in the C-terminal cytosolic domain of Sna3 binds the WW domains in Rsp5, and Sna3 is polyubiquitinated as a consequence of this association. However, Ub does not appear to be required for transport of Sna3 via the MVB pathway because its sorting occurs under conditions in which its ubiquitination is impaired. Consistent with Ub-independent function of the MVB pathway, we show by electron microscopy that the formation of MVB vesicles does not require Rsp5 E3 ligase activity. However, cells expressing a catalytically disabled form of Rsp5 have a greater frequency of smaller MVB vesicles compared with the relatively broad distribution of vesicles seen in MVBs of wild-type cells, suggesting that the formation of MVB vesicles is influenced by Rsp5-mediated ubiquitination.
McNatt
Matthew W
MW
Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309-0347, USA.
McKittrick
Ian
I
West
Matthew
M
Odorizzi
Greg
G
D020816:Amino Acid Motifs; D002384:Catalysis; D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D025821:Polyubiquitin / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D022161:Transport Vesicles / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D043743:Ubiquitin-Protein Ligase Complexes / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; C437323:Sna3 protein, S cerevisiae; D025821:Polyubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae