Stuchell Melissa D, Garrus Jennifer E, Müller Barbara, Stray Kirsten M, Ghaffarian Sanaz, McKinnon Rena, Kräusslich Hans-Georg, Morham Scott G, Sundquist Wesley I
Department of Biochemistry, University of Utah, Salt Lake City, Utah 84132-3201, USA.
J Biol Chem. 2004 Aug 20;279(34):36059-71. doi: 10.1074/jbc.M405226200. Epub 2004 Jun 23.
Efficient human immunodeficiency virus type 1 (HIV-1) budding requires an interaction between the PTAP late domain in the viral p6(Gag) protein and the cellular protein TSG101. In yeast, Vps23p/TSG101 binds both Vps28p and Vps37p to form the soluble ESCRT-I complex, which functions in sorting ubiquitylated protein cargoes into multivesicular bodies. Human cells also contain ESCRT-I, but the VPS37 component(s) have not been identified. Bioinformatics and yeast two-hybrid screening methods were therefore used to identify four novel human proteins (VPS37A-D) that share weak but significant sequence similarity with yeast Vps37p and to demonstrate that VPS37A and VPS37B bind TSG101. Detailed studies produced four lines of evidence that human VPS37B is a Vps37p ortholog. 1) TSG101 bound to several different sites on VPS37B, including a putative coiled-coil region and a PTAP motif. 2) TSG101 and VPS28 co-immunoprecipitated with VPS37B-FLAG, and the three proteins comigrated together in soluble complexes of the correct size for human ESCRT-I ( approximately 350 kDa). 3) Like TGS101, VPS37B became trapped on aberrant endosomal compartments in the presence of VPS4A proteins lacking ATPase activity. 4) Finally, VPS37B could recruit TSG101/ESCRT-I activity and thereby rescue the budding of both mutant Gag particles and HIV-1 viruses lacking native late domains. Further studies of ESCRT-I revealed that TSG101 mutations that inhibited PTAP or VPS28 binding blocked HIV-1 budding. Taken together, these experiments define new components of the human ESCRT-I complex and characterize several TSG101 protein/protein interactions required for HIV-1 budding and infectivity.
1型人类免疫缺陷病毒（HIV-1）的有效出芽需要病毒p6（Gag）蛋白中的PTAP晚期结构域与细胞蛋白TSG101之间相互作用。在酵母中，Vps23p/TSG101与Vps28p和Vps37p结合形成可溶性内体分选转运复合体-I（ESCRT-I），其作用是将泛素化蛋白货物分选到多泡体中。人类细胞中也含有ESCRT-I，但尚未鉴定出VPS37组分。因此，利用生物信息学和酵母双杂交筛选方法鉴定出四种与酵母Vps37p具有弱但显著序列相似性的新型人类蛋白（VPS37A-D），并证明VPS37A和VPS37B与TSG101结合。详细研究产生了四条证据表明人类VPS37B是Vps37p的直系同源物。1）TSG101与VPS37B上的几个不同位点结合，包括一个假定的卷曲螺旋区域和一个PTAP基序。2）TSG101和VPS28与VPS37B-FLAG共同免疫沉淀，这三种蛋白在人类ESCRT-I正确大小（约350 kDa）的可溶性复合物中一起迁移。3）与TGS101一样，在缺乏ATP酶活性的VPS4A蛋白存在的情况下，VPS37B被困在异常的内体区室中。4）最后，VPS37B可以募集TSG101/ESCRT-I活性，从而挽救缺乏天然晚期结构域的突变Gag颗粒和HIV-1病毒的出芽。对ESCRT-I的进一步研究表明，抑制PTAP或VPS28结合的TSG101突变会阻断HIV-1出芽。综上所述，这些实验确定了人类ESCRT-I复合物的新组分，并表征了HIV-1出芽和感染性所需的几种TSG101蛋白/蛋白相互作用。
