The crystal structure of the C-terminal domain of Vps28 reveals a conserved surface required for Vps20 recruitment.
The endosomal sorting complex I required for transport (ESCRT-I) is composed of the three subunits Vps23/Tsg101, Vps28 and Vps37. ESCRT-I is recruited to cellular membranes during multivesicular endosome biogenesis and by enveloped viruses such as HIV-1 to mediate budding from the cell. Here, we describe the crystal structure of a conserved C-terminal domain from Sacharomyces cerevisiae Vps28 (Vps28-CTD) at 3.05 A resolution which folds independently into a four-helical bundle structure. Co-expression experiments of Vps28-CTD, Vps23 and Vps37 suggest that Vps28-CTD does not directly participate in ESCRT-I assembly and may thus act as an adaptor module for downstream interaction partners. We show through mutagenesis studies that Vps28-CTD employs its strictly conserved surface in the interaction with the ESCRT-III factor Vps20. Furthermore, we present evidence that Vps28-CTD is sufficient to rescue an equine infectious anaemia virus (EIAV) Gag late domain deletion. Vps28-CTD mutations abolishing Vps20 interaction in vitro also prevent the rescue of the EIAV Gag late domain mutant consistent with a potential direct Vps28-ESCRT-III Vps20 recruitment. Therefore, the physiological relevant EIAV Gag-Alix interaction can be functionally replaced by a Gag-Vps28-CTD fusion. Because both Alix and Vps28-CTD can directly recruit ESCRT-III proteins, ESCRT-III assembly coupled to Vps4 action may therefore constitute the minimal budding machinery for EIAV release.
Pineda-Molina
Estela
E
European Molecular Biology Laboratory (EMBL), 6 rue Jules Horowitz, 38042 Grenoble, France.
Belrhali
Hassan
H
Piefer
Andrew J
AJ
Akula
Indira
I
Bates
Paul
P
Weissenhorn
Winfried
W
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D003461:Crystallography; D056827:Endosomal Sorting Complexes Required for Transport; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology
D056827:Endosomal Sorting Complexes Required for Transport; D029701:Saccharomyces cerevisiae Proteins; C102954:VPS28 protein, S cerevisiae; D033921:Vesicular Transport Proteins