Targeting of Sna3p to the endosomal pathway depends on its interaction with Rsp5p and multivesicular body sorting on its ubiquitylation.
Rsp5p is an ubiquitin (Ub)-protein ligase of the Nedd4 family that carries WW domains involved in interaction with PPXY-containing proteins. It plays a key role at several stages of intracellular trafficking, such as Ub-mediated internalization of endocytic cargoes and Ub-mediated sorting of membrane proteins to internal vesicles of multivesicular bodies (MVBs), a process that is crucial for their subsequent targeting to the vacuolar lumen. Sna3p is a membrane protein previously described as an Ub-independent MVB cargo, but proteomic studies have since shown it to be an ubiquitylated protein. Sna3p carries a PPXY motif. We observed that this motif mediates its interaction with Rsp5p WW domains. Mutation of either the Sna3p PPXY motif or the Rsp5p WW3 domain or reduction in the amounts of Rsp5 results in the mistargeting of Sna3p to multiple mobile vesicles and prevents its sorting to the endosomal pathway. This sorting defect appears to occur prior to the defect displayed in rsp5 mutants by other MVB cargoes, which are correctly sorted to the endosomal pathway but missorted to the vacuolar membrane instead of the vacuolar lumen. Sna3p is polyubiquitylated on one target lysine, and a mutant Sna3p lacking its target lysine displays defective MVB sorting. Sna3p undergoes Rsp5-dependent polyubiquitylation, with K63-linked Ub chains.
Stawiecka-Mirota
Marta
M
Institut Jacques Monod, CNRS, Universités Paris VI et VII, Département de Biologie Cellulaire, 2 Place Jussieu 75005 Paris, France.
Pokrzywa
Wojciech
W
Morvan
Joelle
J
Zoladek
Teresa
T
Haguenauer-Tsapis
Rosine
R
Urban-Grimal
Danièle
D
Morsomme
Pierre
P
D001665:Binding Sites; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism*; D047468:Immunoprecipitation; D008239:Lysine / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics / Q000378:metabolism*
D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; C437323:Sna3 protein, S cerevisiae; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae; D008239:Lysine