Endosomal Sorting Complex Required for Transport (ESCRT) Complexes Induce Phase-separated Microdomains in Supported Lipid Bilayers

Bouřa, Evžen; Ivanov, Vassili; Carlson, Lars A.; Mizuuchi, Kiyoshi; Hurley, James H.

doi:10.1074/jbc.m112.378646

Cited by 64 publications

(60 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This would be consistent with a possible role for the acidic lipid-binding MABP domain of MVB12 (42). These results also parallel the 3-4 order of magnitude increase in affinity between ESCRTs and ubiquitin when the latter is tethered to a membrane (47). Indeed, the effective approximately 3 order of magnitude increase in affinity seen for the ESCRT-I:Gag interaction represents one of the most dramatic differences between the more realistic context of this study and the solution analysis of binary interactions between small fragments.…”

Section: Recruitment Ofsupporting

confidence: 76%

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

Carlson

Hurley

2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Most membrane-enveloped viruses depend on host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release. HIV-1 is the prototypic ESCRT-dependent virus. The direct interactions between HIV-1 and the early ESCRT factors TSG101 and ALIX have been mapped in detail. However, the full pathway of ESCRT recruitment to HIV-1 budding sites, which culminates with the assembly of the late-acting CHMP4, CHMP3, CHMP2, and CHMP1 subunits, is less completely understood. Here, we report the biochemical reconstitution of ESCRT recruitment to viral assembly sites, using purified proteins and giant unilamellar vesicles. The myristylated full-length Gag protein of HIV-1 was purified to monodispersity. Myr-Gag forms clusters on giant unilamellar vesicle membranes containing the plasma membrane lipid PIð4,5ÞP 2 . These Gag clusters package a fluorescent oligonucleotide, and recruit early ESCRT complexes ESCRT-I or ALIX with the appropriate dependence on the Gag PTAP and LYPðXÞ n L motifs. ALIX directly recruits the key ESCRT-III subunit CHMP4. ESCRT-I can only recruit CHMP4 when ESCRT-II and CHMP6 are present as intermediary factors. Downstream of CHMP4, CHMP3 and CHMP2 assemble synergistically, with the presence of both subunits required for efficient recruitment. The very late-acting factor CHMP1 is not recruited unless the pathway is completed through CHMP3 and CHMP2. These findings define the minimal sets of components needed to complete ESCRT assembly at HIV-1 budding sites, and provide a starting point for in vitro structural and biophysical dissection of the system. confocal microscopy | host-pathogen interaction | membrane traffic | virus budding M ost membrane-enveloped viruses utilize host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release (1-3). This dependence was first described for HIV-1, where mutation of an ESCRTbinding motif (late domain) in the viral protein Gag was shown to stall virus bud release at a late stage in virus assembly (4, 5). Late domains have subsequently been found in other retroviruses and numerous other virus families including, e.g., flavi-, filo-and rhabdoviruses (6). Indeed, the only well-characterized case of ESCRT-independent release of a membrane-enveloped virus is influenza virus (7). In normal cell physiology, ESCRTs function in cytokinesis, formation of intralumenal vesicles in multivesicular endosomes, and vesicle release from the plasma membrane (8-10). These seemingly disparate processes all involve membrane budding away from the cytoplasm, and ESCRTs are the only described protein machinery to perform vesicle formation with this topology, which is analogous to virus release.The initial events in HIV-1 ESCRT recruitment are well characterized. The structural protein of HIV-1, Gag, binds earlyacting ESCRT factors through two late-domain motifs in its C-terminal p6 domain: a PTAP sequence that interacts with the TSG101 subunit of ESCRT-I (11-16) and a LYPðXÞ n L motif that interacts with the ESCRT...

show abstract

Section: Recruitment Ofsupporting

confidence: 76%

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

Carlson

Hurley

2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Surprisingly, given the well described and important functions, mice lacking PI4K IIα kinase activity initially appear normal and only later manifest neurodegeneration [41]. It is well documented that proteins can regulate the physical properties and/or shape of the biological membranes [42][43][44][45]. However, the structural and functional analysis of PI4K IIα suggests that in fact the membrane regulates the activity of PI4K IIα enzyme [46][47][48].…”

Section: Type II Pi4ksmentioning

confidence: 94%

Phosphatidylinositol 4-kinases: Function, structure, and inhibition

Bouřa

Nencka

2015

Experimental Cell Research

Self Cite

130

107

View full text Add to dashboard Cite

“…Bilayers can segregate their lipids laterally to form cholesterol-rich microdomains called lipid rafts (112), and both MVB vesicles and HIV virions are cholesterol rich (16, 17, 113). Cholesterol is required for ESCRT-II self-assembly on supported lipid bilayers, and the resulting assemblies induce phase separation into liquid-ordered domains (114). This is potentially important because phase separation between liquid-ordered and -disordered regions can create line tension that promotes membrane scission (17).…”

Section: Escrt:membrane Interactionsmentioning

confidence: 99%

Membrane Fission Reactions of the Mammalian ESCRT Pathway

McCullough

Colf

Sundquist³

2013

Annu. Rev. Biochem.

220

288

View full text Add to dashboard Cite

The endosomal sorting complexes required for transport (ESCRT) pathway was initially defined in yeast genetic screens that identified the factors necessary to sort membrane proteins into intraluminal endosomal vesicles. Subsequent studies have revealed that the mammalian ESCRT pathway also functions in a series of other key cellular processes, including formation of extracellular microvesicles, enveloped virus budding, and the abscission stage of cytokinesis. The core ESCRT machinery comprises Bro1 family proteins and ESCRT-I, ESCRT-II, ESCRT-III, and VPS4. Site-specific adaptors recruit these soluble factors to assemble on different cellular membranes, where they carry out membrane fission reactions. ESCRT-III proteins form filaments that draw membranes together from the cytoplasmic face, and mechanistic models have been advanced to explain how ESCRT-III filaments and the VPS4 ATPase can work together to catalyze membrane fission.

show abstract

Endosomal Sorting Complex Required for Transport (ESCRT) Complexes Induce Phase-separated Microdomains in Supported Lipid Bilayers

Cited by 64 publications

References 47 publications

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

Phosphatidylinositol 4-kinases: Function, structure, and inhibition

Membrane Fission Reactions of the Mammalian ESCRT Pathway

Contact Info

Product

Resources

About