Higher-Order Oligomerization Targets Plasma Membrane Proteins and HIV Gag to Exosomes

Fang, Yi; Wu, Ning; Gan, Xin; Yan, Wei; Morrell, James C.; Gould, Stephen J.

doi:10.1371/journal.pbio.0050158

Cited by 350 publications

(357 citation statements)

References 64 publications

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“…The WD40 domain controls this behavior, likely by mediating the formation of higher-order oligomers. It has been reported that high-order oligomerization and membrane-binding motifs artificially target cytosolic proteins to membrane patches, which accumulate at the lagging edge in polarized cells (48)(49)(50)(51)(52). Oligomerizing membrane microdomain components also enrich at the rear of polarized neutrophils or T cells (53)(54)(55)(56)(57).…”

Section: Discussionmentioning

confidence: 99%

Novel protein Callipygian defines the back of migrating cells

Swaney

Borleis

Iglesias

et al. 2015

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

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Asymmetric protein localization is essential for cell polarity and migration. We report a novel protein, Callipygian (CynA), which localizes to the lagging edge before other proteins and becomes more tightly restricted as cells polarize; additionally, it accumulates in the cleavage furrow during cytokinesis. CynA protein that is tightly localized, or "clustered," to the cell rear is immobile, but when polarity is disrupted, it disperses throughout the membrane and responds to uniform chemoattractant stimulation by transiently localizing to the cytosol. These behaviors require a pleckstrin homology-domain membrane tether and a WD40 clustering domain, which can also direct other membrane proteins to the back. Fragments of CynA lacking the pleckstrin homology domain, which are normally found in the cytosol, localize to the lagging edge membrane when coexpressed with full-length protein, showing that CynA clustering is mediated by oligomerization. Cells lacking CynA have aberrant lateral protrusions, altered leading-edge morphology, and decreased directional persistence, whereas those overexpressing the protein display exaggerated features of polarity. Consistently, actin polymerization is inhibited at sites of CynA accumulation, thereby restricting protrusions to the opposite edge. We suggest that the mutual antagonism between CynA and regions of responsiveness creates a positive feedback loop that restricts CynA to the rear and contributes to the establishment of the cell axis.chemotaxis | polarity | asymmetry | Dictyostelium | protein localization

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Section: Discussionmentioning

confidence: 99%

Novel protein Callipygian defines the back of migrating cells

Swaney

Borleis

Iglesias

et al. 2015

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

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“…This final step is thought to be mediated by ESCRT-III, possibly in conjuction with the ATPase VPS4 (33,49). In the conception of these experiments, we had anticipated that inefficient release of Gag puncta into the GUV interior might be seen even in the absence of ESCRTs, because high levels of Gag overexpression have been shown to drive budding (50). If ESCRT-III assembly were sufficient for scission, as seen for the yeast proteins, we would have expected to see a large increase in VLP release upon completion of ESCRTassembly.…”

Section: Discussionmentioning

confidence: 99%

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.

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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...

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“…Additionally, plasma membrane anchors induce vesicle budding directly from the plasma membrane of highly oligomeric proteins, such as the HIV Gag protein and the yeast cytoplasmic protein Tya [123,124]. An evaluation of different membrane anchors for Tya demonstrated that myristoylation was the most effective enhancer of the formation of shedding vesicles, whereas other types such as, phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate-binding domains, prenylation/palmitoylation tag or the type-1 plasma membrane protein CD43 did not cause this effect [124].…”

Section: Other Ptms In Evsmentioning

confidence: 99%