Abstract
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.
Highlights
Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation
Sna3p physically interacts with Rsp5p via its PY motif Rsp5p is a modular protein with an N-terminal C2 domain that may interact with membranes, three tryptophan-rich WW domains involved in protein–protein interactions with proline-rich peptides and a C-terminal catalytic HECT domain (Figure 1A)
Sna3p ubiquitylation is required for its multivesicular bodies (MVBs) sorting We investigated the role of Sna3p ubiquitylation in its MVB sorting both by biochemical and by immunofluorescence approaches using Sna3p tagged with hemagglutinin protein (HA), which does not carry any Lys residue
Summary
Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation. Ubiquitin is required for sorting of membrane proteins to internal vesicles, which invaginate, budding into the lumen of late endosomes, resulting in the formation of multivesicular bodies (MVBs) [2]. These proteins are delivered to the vacuolar lumen after fusion of MVBs with the vacuole/ lysosome. Multivesicular body sorting involves a machinery conserved from yeast to humans, composed of several endosomal sorting complexes required for transport (ESCRTs), some of which contain a subunit carrying an Ub-binding domain [3] These complexes are thought to act sequentially in recognition of ubiquitylated cargoes. Two proteomic studies reported that Sna3p is ubiquitylated (target K125) [14,15]
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