Characterization of multiple multivesicular body sorting determinants within Sna3: a role for the ubiquitin ligase Rsp5.
A subset of proteins that transit the endosomal system are directed into the intralumenal vesicles of multivesicular bodies (MVBs). MVB formation is critical for a variety of cellular functions including receptor down-regulation, viral budding, antigen presentation, and the generation of lysosome-related organelles. Entry of transmembrane proteins into the intralumenal vesicles of a MVB is a highly regulated process that is positively modulated by covalent modification of cargoes with ubiquitin. To identify additional MVB sorting signals, we examined the previously described ubiquitination-independent MVB cargo Sna3. Although Sna3 ubiquitination is not essential, Sna3 MVB sorting is positively modulated by its ubiquitination. Examination of MVB sorting determinants within a form of Sna3 lacking all lysine residues identified two critical regions: an amino-terminal tyrosine-containing region and a carboxyl-terminal PPAY motif. This PPAY motif interacts with the WW domains of the ubiquitin ligase Rsp5, and mutations in either the WW or, surprisingly, the HECT domains of Rsp5 negatively impacted MVB targeting of lysine-minus Sna3. These data indicate that Rsp5 function is required for MVB targeting of Sna3 in a capacity beyond cargo ubiquitination. These results uncover a series of determinants impacting Sna3 MVB sorting, including unexpected roles for Rsp5.
Oestreich
Andrea J
AJ
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Aboian
Mariam
M
Lee
Jacqueline
J
Azmi
Ishara
I
Payne
Johanna
J
Issaka
Rachel
R
Davies
Brian A
BA
Katzmann
David J
DJ
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D025941:Protein Interaction Mapping; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D022161:Transport Vesicles / Q000737:chemistry / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins; C437323:Sna3 protein, S cerevisiae; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae