Regulation of ALIX during Exocytic Vesicle Release and Assembly of ESCRT Proteins on the Plasma Membrane

Abstract

Endosomal sorting complexes required for transport (ESCRTs) are protein complexes that facilitate the release of budding vesicles into extracellular environment. ESCRTs are essential in most cellular processes requiring fission of budding membrane, including multi-vesicular body formation, envelope virus release, and exosome release. During HIV-1 budding, HIV-1 Gag assembles on the plasma membrane and drives the outward deformation of the membrane toward forming vesicles. HIV-1 virus like particles (VLPs) can be generated by expressing Gag alone in the cytosol, these VLPs are consistent of ∼2000 copies of Gag and form vesicles with an average diameter of 120 nanometers and their assembly on the plasma membrane take ∼20-30 minutes. HIV-1 utilizes ESCRTs extensively for the release of progeny virions into extracellular space and VLPs serve as a good model system for studying ESCRT recruitment on the plasma membrane. Gag has two late domain motifs which interact with TSG101 and ALIX, two of the early ESCRT components. Given the stoichiometry of Gag and its steady accumulation in the forming VLPs, it has been expected that ALIX would be recruited into the forming VLPs along with Gag during the assembly. Using live imaging to observe real time assembly of VLPs in the presence of fluorescently tagged ALIX proteins, we have observed that ALIX is recruited transiently at the end of Gag assembly and is mostly recycled after VLPs release. This observation implies high spatial and temporal regulation for recruitment of ALIX during budding. We have further dissected the essential interactions that govern ALIX recruitment, showed the interaction of ALIX Bro1 domain with CHMP4 is critical for recruitment of ALIX and proposed the emerging regulatory mechanism essential for recruitment of ALIX to the plasma membrane during vesicle budding.