Identification of Human VPS37C, a Component of Endosomal Sorting Complex Required for Transport-I Important for Viral Budding

Abstract

Endosomal sorting complex required for transport-I (ESCRT-I) is one of three defined protein complexes in the class E vacuolar protein sorting (VPS) pathway required for the sorting of ubiquitinated transmembrane proteins into internal vesicles of multivesicular bodies. In yeast, ESCRT-I is composed of three proteins, VSP23, VPS28, and VPS37, whereas in mammals only Tsg101(VPS23) and VPS28 were originally identified as ESCRT-I components. Using yeast two-hybrid screens, we identified one of a family of human proteins (VPS37C) as a Tsg101-binding protein. VPS37C can form a ternary complex with Tsg101 and VPS28 by binding to a domain situated toward the carboxyl terminus of Tsg101 and binds to another class E VPS factor, namely Hrs. In addition, VPS37C is recruited to aberrant endosomes induced by overexpression of Tsg101, Hrs, or dominant negative form of the class E VPS ATPase, VPS4. Enveloped viruses that encode PTAP motifs to facilitate budding exploit ESCRT-I as an interface with the class E VPS pathway, and accordingly, VPS37C is recruited to the plasma membrane along with Tsg101 by human immunodeficiency virus, type 1 (HIV-1) Gag. Moreover, direct fusion of VPS37C to HIV-1 Gag obviates the requirement for a PTAP motif to induce virion release. Depletion of VPS37C from cells does not inhibit murine leukemia virus budding, which is not mediated by ESCRT-I, however, if murine leukemia virus budding is engineered to be ESCRT-I-dependent, then it is inhibited by VPS37C depletion, and this inhibition is accentuated if VPS37B is simultaneously depleted. Thus, this study identifies VPS37C as a functional component of mammalian ESCRT-I. The multivesicular body (MVB)1 serves as an important organelle for the sorting of endocytosed and newly synthesized transmembrane proteins (1-3). A major decision governing the fate of such proteins is made when they either remain on the limiting membrane of the MVB or become internalized within the lumen of the MVB. Specifically, proteins that are sorted into luminal vesicles of the MVB are ultimately targeted to the lumen of the lysosome/vacuole for degradation (1-3). A number of protein factors that comprise the "class E" vacuolar protein sorting (VPS) machinery function at the MVB limiting membrane to select membrane proteins for luminal targeting and to induce the formation and budding of vesicles into the MVB lumen. Most of these class E VPS factors are components of protein complexes, and at least three "endosomal sorting complexes required for transport" (ESCRT-I, -II, or -III) (4 -6) are linked by an elaborate series of protein-protein interactions between their components and/or bridging factors (7-11). Cargo sorting and vesicle formation likely involves sequential recruitment of ESCRT-I, -II, and -III, and subsequent vesicle fission requires dissociation of ESCRT complexes from the MVB membrane, induced by an AAA-ATPase, VPS4 (12, 13). The series of recruitment events can be initiated, at least in part, by ubiquitinated cytoplasmic domains of transmembrane proteins, as we...

The Arabidopsis elch mutant reveals functions of an ESCRT component in cytokinesis

“…The third band, of approximately 24 kDa, contained the Arabidopsis VPS28-1 homolog. These data are consistent with studies in mammals where VPS37 is known to interact with TSG101 (Bache et al, 2004;Eastman et al, 2005;Stuchell et al, 2004). The interaction between VPS37-1 4685 RESEARCH ARTICLE Role of VPS23 in plant cytokinesis Pull-down assays were performed using ubiquitin coupled to agarose and the ELC-HA protein was detected on the western blot using an anti-HA antibody.…”

Exploring the ESCRTing machinery in eukaryotes

“…This shared protein motif and the low level of sequence identity were not significant enough to identify the VPS37 homologs in human, Drosophila, C. elegans and plants. However, recent experimental interaction studies have identified the four human orthologs of VPS37 (see Supplementary material Table S2 and Figure S2C) [40,43]. Using the human VPS37 sequences, we identified putative homologs in Drosophila and C. elegans and two putative Arabidopsis and rice homologs (see Supplementary material Table S2 and Figure S2C).…”

“…Therefore, VPS23 does not function as an ubiquitin conjugating enzyme and yet it is still able to bind ubiquitinated proteins. Via its C-terminal coiled coil structure, the human VPS23 homolog TSG101 interacts with VPS27, VPS28 [40] and VPS37 [41]. In rice and Arabidopsis, one and two putative VPS23 homologs are present, respectively (see Supplementary material Table S2 and Figure S2A).…”

Recruitment of Tom1L1/Srcasm to Endosomes and the Midbody by Tsg101

“…HIV-1 Gag was previously reported to colocalize with Tsg101 on endosomes (Eastman et al, 2005) (also see Fig. 4, A-A").…”