Direct Binding to Rsp5 Mediates Ubiquitin-independent Sorting of Sna3 via the Multivesicular Body Pathway

Abstract

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. INTRODUCTIONMultivesicular bodies (MVBs) are late endosomes containing lumenal vesicles that are formed by invagination of the endosomal membrane. The lumenal MVB vesicles are delivered into the hydrolytic interior of the lysosome upon fusion of the limiting MVB membrane with the lysosomal membrane (van Deurs et al., 1995;Futter et al., 1996;Mullock et al., 1998). Many cell-surface receptors that undergo down-regulation from the plasma membrane are sorted into MVB vesicles en route to being degraded in the lysosome, including members of the receptor tyrosine kinase family in metazoans and G protein-coupled receptors in the budding yeast Saccharomyces cerevisiae. In yeast, many biosynthetic proteins are also sorted into MVB vesicles during their transport from the Golgi to the vacuole, the functional equivalent of the lysosome (reviewed in Hicke and Dunn, 2003).Most integral membrane proteins require the attachment of a single ubiquitin (Ub) to their cytosolic domains in order to be sorted into the MVB pathway (Katzmann et al., 2001;Reggiori and Pelham, 2001;Urbanowski and Piper, 2001). Sorting of monoubiquitinated MVB cargoes is mediated by a highly conserved machinery comprised of class E Vps proteins, many of which assemble into distinct endosomal sorting complexes required for transport (ESCRTs) that contain Ub-binding domains (reviewed in Hurley and Emr, 2006). In addition to Ub-mediated cargo recognition, class E Vps proteins are generally required for MVB vesicle budding because cells in which class E Vps proteins are disrupted contain malformed late endosomes that lack lumenal vesicles (Rieder et al., 1996;Odorizzi et al., 1998;Doyotte et al., 2005).The class E Vps/ESCRT machinery is also required for the budding of many nonlytic enveloped viruses from infected host cells, which is topologically equival...

The Transmembrane Domain of Acid Trehalase Mediates Ubiquitin-independent Multivesicular Body Pathway Sorting

“…Along these lines, recent studies show that Sna3 can utilize both ubiquitin-dependent and -independent mechanisms for its MVB targeting. Sna3 ubiquitination is dependent on Rsp5, but the function of Rsp5 is also critical for the MVB sorting of Sna3 independent of cargo ubiquitination (McNatt et al, 2007;Oestreich et al, 2007). Here we found that Ath1 has certain properties that are similar to Sna3.…”

“…Recent studies showed that Sna3 is also ubiquitinated; however, without ubiquitination it still can utilize an alternative mechanism, which involves the function of Rsp5, to be sorted into the MVB pathway (McNatt et al, 2007;Oestreich et al, 2007). Here we showed that the acid trehalase, Ath1, had the same transport behavior as Sna3.…”

Decoding ubiquitin sorting signals for clathrin-dependent endocytosis by CLASPs

“…Ste6p, the a-factor transporter, is also multiply ubiquitylated and five acceptor lysine residues are known (Kelm et al, 2004). It has also been demonstrated very convincingly that Rsp5p, the E3 ligase that governs ubiquitylation of numerous transmembrane proteins at the yeast plasma membrane (Dunn and Hicke, 2001a;Dupre et al, 2004), is able to generate (Lys63-linked) polyubiquitin chains in a robust fashion (Kee et al, 2006;McNatt et al, 2007;Oestreich et al, 2007). This clearly raises the possibility that oligoubiquitin may be the predominant endocytic sorting signal in yeast.…”

Doa4 function in ILV budding is restricted through its interaction with the Vps20 subunit of ESCRT-III

“…Liquid cultures (100 ml) of BL21(DE3) transformed with Vps2, Vps20-HA, His 6 -Vps24, and Snf7 expression plasmids were grown and lysed as described previously (McNatt et al, 2007). Lysates were clarified of cell debris by centrifugation at 100,000 g for 30 min at 4˚C.…”