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Ubiquitin modification of endosomal membrane proteins is a signal for active inclusion into the Multivesicular Body (MVB) pathway, resulting in lysosomal degradation. However, the endosome represents a dynamic site of protein sorting with a majority of proteins destined for recycling, rather than MVB targeting. Substrate recognition by ubiquitin ligases is therefore highly regulated. We have investigated substrate recognition by the Nedd4 ortholog Rsp5 as a model for understanding ligasesubstrate interactions. Rsp5 interacts directly with its substrate Cps1 via a novel interaction mode. Perturbation of this mode of interaction revealed a compensatory role for the Rsp5 adaptor Bsd2. These results highlight the ability of Rsp5 to interact with substrates via multiple modalities, suggesting additional mechanisms of regulating this interaction and relevant outcomes.The post-translational addition of ubiquitin to protein substrates is a regulatory modification of remarkable scope in eukaryotic biology. Cellular processes as diverse as protein degradation, protein trafficking, DNA repair, and nuclear signaling are regulated by ubiquitination, and as a consequence, numerous pathologies and developmental defects have been linked to defects in the ubiquitin system (reviewed in Refs. 1-3). A cascade of reactions culminates in the formation of an isopeptide bond between the C-terminal glycine of ubiquitin and an acceptor amine within the substrate. Ubiquitin modification is tightly regulated, with the third enzyme in the ubiquitination cascade, the ubiquitin ligase, responsible for substrate selection (reviewed in Refs. 4, 5).Ubiquitin modification of endosomal transmembrane proteins has previously been demonstrated to play a major role in targeting proteins into multivesicular bodies (MVBs) 3 en route to lysosomal degradation (6 -9). Entry into intralumenal vesicles during MVB sorting is tightly regulated, and Carboxypeptidase S (Cps1) has served as a model MVB cargo in analyses demonstrating the role of ubiquitin modification as a positive cis-acting MVB sorting determinant (6 -9). Considerable evidence supports a model wherein the HECT ubiquitin ligase Rsp5 plays the major role in targeting a number of MVB cargoes, including Cps1, into this pathway in Saccharomyces cerevisiae (9 -20). Rsp5 is the yeast ortholog of Nedd4 family ligases, all of which contain WW protein interaction domains involved in substrate recognition (21). These WW domains participate in substrate recognition either directly through "PY" motifs within the substrates (9, 18 -20, 22-26) or indirectly via adaptors that contain PY motifs (27-29). Bsd2 is one such cofactor that has been implicated in Cps1 ubiquitination and subsequent MVB targeting (17, 30). However, we have previously observed a direct interaction between Rsp5 and Cps1 in vitro (9), suggesting that the interactions leading to Cps1 ubiquitination may be more complicated. Cps1 contains MVB targeting information within the amino acid sequence "PVEKAPR" (6), which does not possess a PY mot...

Heterologous expression of HIV-1 Gag in a variety of host cells results in its packaging into virus-like particles (VLPs) that are subsequently released into the extracellular milieu. This phenomenon represents a useful tool for probing cellular factors required for viral budding and has contributed to the discovery of roles for ubiquitin ligases and the endosomal sorting complexes required for transport (ESCRTs) in viral budding. These factors are highly conserved throughout eukaryotes and have been studied extensively in the yeast Saccharomyces cerevisiae, a model eukaryote previously utilized as a host for the production of VLPs. We used heterologous expression of HIV Gag in yeast spheroplasts to examine the role of ESCRTs and associated factors (Rsp5, a HECT ubiquitin ligase of the Nedd4 family; Bro1, a homolog of Alix; and Vps4, the AAA-ATPase required for ESCRT function in all contexts/organisms investigated) in the generation of VLPs. Our data reveal: 1) characterized Gag-ESCRT interaction motifs (late domains) are not required for VLP budding, 2) loss of function alleles of the essential HECT ubiquitin ligase Rsp5 do not display defects in VLP formation, and 3) ESCRT function is not required for VLP formation from spheroplasts. These results suggest that the egress of HIV Gag from yeast cells is distinct from the most commonly described mode of exit from mammalian cells, instead mimicking ESCRT-independent VLP formation observed in a subset of mammalian cells. As such, budding of Gag from yeast cells appears to represent ESCRT-independent budding relevant to viral replication in at least some situations. Thus the myriad of genetic and biochemical tools available in the yeast system may be of utility in the study of this aspect of viral budding.

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