Direct Binding to Rsp5p Regulates Ubiquitination-independent Vacuolar Transport of Sna3p

Abstract

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.

Dual Sorting of the Saccharomyces cerevisiae Vacuolar Protein Sna4p

“…No ubiquitin signals are seen when using Sna4p K128R or Sna4p lacking the PY motif, indicating that Sna4p is polyubiquitylated on its target K128 residue by ubiquitin ligase Rsp5p binding to the PY motif of Sna4p. A similar control mechanism of ubiquitylation is observed for Sna3p, since it carries a PY motif that mediates its interaction with Rsp5p, resulting in its Rsp5p-dependent polyubiquitylation (20,25,31,37). Both Sna3p and Sna4p possess a PY motif in their cytosolic region, suggesting that they may have similar functions in the MVB pathway.…”

“…2A), contains a PPxY sequence in its C-terminal region that fits the PY motif recognized by WW domain-containing proteins, such as the E3 ubiquitin ligase Rsp5p/Npi1p (14,37). We introduced mutations in the PPPY motif of Sna4p or removed the PPPY sequence and examined the localization of these Sna4p variants fused to GFP expressed in WT cells.…”

“…We found that Sna4p physically interacts with Rsp5p and that its PY motif is important in this process. Most direct substrates of Rsp5p have a PY motif (12), and a number of these have been shown to physically interact with Rsp5p via their PY motif, e.g., Rod1p and Rog3p (1), Bsd2p (15), and Sna3p (20,25,31,37). The interaction between Sna4p and Rsp5p is completely inhibited when the PY motif is deleted from Sna4p, suggesting that Sna4p binds Rsp5p directly.…”

“…Sna3p is ubiquitylated by Rsp5p and modified by K63-linked ubiquitin chains. Sna3p-Rsp5p interaction was found to be dependent on the Sna3p PPxY motif and on the WW2/WW3 domains of Rsp5p (20,25,31,37).…”

Human Immunodeficiency Virus Type 1 Nef Protein Targets CD4 to the Multivesicular Body Pathway

“…5 and 6). Thus, downregulated CD4 might be another example of a minority of cargoes such as the delta opioid receptor (36) and the interleukin-2 receptor ␤ chain (79) in mammals and Sna3p (58,60,63,78) and Ath1p (38) in yeast, which are sorted to the MVB pathway in an ESCRT-dependent but apparently ubiquitination-independent manner. Does CD4 lysine ubiquitination then have any physiological role?…”