Negative membrane curvature catalyzes nucleation of endosomal sorting complex required for transport (ESCRT)-III assembly

The endosomal sorting complexes required for transport (ESCRT) machinery functions in HIV-1 budding, cytokinesis, multivesicular body biogenesis, and other pathways, in the course of which it interacts with concave membrane necks and bud rims. To test the role of membrane shape in regulating ESCRT assembly, we nanofabricated templates for invaginated supported lipid bilayers. The assembly of the core ESCRT-III subunit CHMP4B/Snf7 is preferentially nucleated in the resulting 100-nm-deep membrane concavities. ESCRT-II and CHMP6 accelerate CHMP4B assembly by increasing the concentration of nucleation seeds. Superresolution imaging was used to visualize CHMP4B/Snf7 concentration in a negatively curved annulus at the rim of the invagination. Although Snf7 assemblies nucleate slowly on flat membranes, outward growth onto the flat membrane is efficiently nucleated at invaginations. The nucleation behavior provides a biophysical explanation for the timing of ESCRT-III recruitment and membrane scission in HIV-1 budding.The endosomal sorting complexes required for transport (ESCRTs) are an ancient and conserved system for membrane scission (1, 2). ESCRT membrane remodeling activities are important in the budding of HIV-1 and other viruses from host cell membranes (3); cytokinesis (4); lysosomal transport (5); and more recently discovered functions that include membrane repair, exosome biogenesis, and nuclear envelope reformation (6). The ESCRTs are unique in that they promote membrane budding and sever membrane necks by working from the inner face of the bud (1, 2).The ESCRTs consist of the upstream complexes ESCRT-I, ESCRT-II, and ALIX, which recognize cargo, the ESCRT-III complex responsible for membrane scission, and the AAA+ ATPase VPS4, which releases and recycles ESCRT-III (1, 2). In this study, we focus on the human ESCRT-III subunit CHMP4B, which is considered a core component of the membrane scission machinery and is essential for HIV-1 budding (7). Its yeast counterpart is Snf7. CHMP4 can be recruited and activated through two different pathways in human cells. The first proceeds through ESCRT-I, ESCRT-II, and CHMP6, and the second through ALIX (3). The ESCRT-II– and CHMP6-dependent pathway functions downstream of ESCRT-I, which is in turn the essential link between HIV-1 Gag and the ESCRTs (3). Although there is uncertainty over whether ESCRT-II itself is essential in HIV-1 budding, the most recent virological data suggest that ESCRT-II is important for the efficient release of HIV-1 (8). Moreover, ESCRT-II and CHMP6 were required to bridge Gag and ESCRT-I to the rest of ESCRT-III in a reconstituted system (9).Most concepts of ESCRT recruitment to HIV-1 budding sites have focused on protein–protein interactions between the PTAP and YPXL late domain motifs of the Gag p6 domain and ESCRT-I and ALIX, respectively (3). ESCRT-I is recruited to HIV-1 budding sites simultaneously with Gag (10). However, ESCRT-III is recruited after a time lag and only to Gag that has already assembled on the plasma membrane (10, 11). In principle, either the oligomerization of Gag or its membrane association might trigger ESCRT-III recruitment to Gag-ESCRT-I assemblies. In one recent report, ESCRT-III assembly was visualized by superresolution light microscopy within the center of the Gag shell (12). This observation led to a model for virus scaffolding of ESCRT-III assembly, which downplayed the direct role of membrane shape. Another group, also using superresolution imaging, noted a displacement of the ESCRT-III localization closer to the plasma membrane than the mean position of Gag (13), consistent with ESCRT-III localization predominantly to the bud neck (3). The latter model implies that ESCRT-III could be a coincidence detector, responsive both to the presence of upstream interacting proteins and to membrane shape. Whereas an abundant literature describes the role of viral late domains and other protein interactions in ESCRT recruitment, almost no data are available on the role of membrane curvature in initiating ESCRT-III assembly.In this study, we set out to characterize the recruitment and assembly of purified ESCRT complexes on membranes of a defined geometry approximating that of an early stage HIV-1 budding site. At early stages, budding profiles with broad necks have been visualized in thin-section EM and in cryo-EM tomograms (14–17). During the process of their formation, HIV-1 budding intermediates are 50–100 nm deep and slightly over 100 nm wide. The well-developed methods for studying protein interactions with positively curvature membranes (18) cannot be applied to this type of geometry. Here, we used a focused ion beam to fabricate a 100-nm-deep invaginated template for negative curvature, which approximates the shape of a nascent HIV-1 bud. When coated with a supported lipid bilayer, we term this structure an invaginated supported lipid bilayer (invSLB). We went on to measure CHMP4B/Snf7 assembly in real time, which allowed us to dissect in vitro, and in real time, the role of membrane shape in the nucleation and growth of ESCRT polymers.     

Mutation of the Highly Conserved Ser-40 of the HIV-1 p6 Gag Protein to Phe Causes the Formation of a Hydrophobic Patch,Enhances Membrane Association,and Polyubiquitination of Gag

The HIV-1 p6 Gag protein contains two late assembly (l-) domains that recruit proteins of the endosomal sorting complex required for transport (ESCRT) pathway to mediate membrane fission between the nascent virion and the cell membrane. It was recently demonstrated that mutation of the highly conserved Ser-40 to Phe (S40F) disturbs CA-SP1 processing, virus morphogenesis, and infectivity. It also causes the formation of filopodia-like structures, while virus release remains unaffected. Here, we show that the mutation S40F, but not the conservative mutation to Asp (S40D) or Asn (S40N), augments membrane association, K48-linked polyubiquitination, entry into the 26S proteasome, and, consequently, enhances MHC-I antigen presentation of Gag derived epitopes. Nuclear magnetic resonance (NMR) structure analyses revealed that the newly introduced Phe-40, together with Tyr-36, causes the formation of a hydrophobic patch at the C-terminal α-helix of p6, providing a molecular rationale for the enhanced membrane association of Gag observed in vitro and in HIV-1 expressing cells. The extended exposure of the S40F mutant to unidentified membrane-resident ubiquitin E3-ligases might trigger the polyubiquitination of Gag. The cumulative data support a previous model of a so far undefined property of p6, which, in addition to MA, acts as membrane targeting domain of Gag.     

Ubiquitination is associated with lysosomal degradation of cell surface-resident ATP-binding cassette transporter A1 (ABCA1) through the endosomal sorting complex required for transport (ESCRT) pathway

ATP-binding cassette transporter A1 (ABCA1) plays an essential role in the biogenesis of high-density lipoprotein in liver and in the prevention of foam cell formation in macrophages by mediating the efflux of cellular cholesterol and phospholipids to apolipoprotein A-I (apoA-I). Our current study investigated the mechanism of degradation of cell surface-resident ABCA1, focusing on ubiquitination. A coimmunoprecipitation study indicated the presence of ubiquitinated ABCA1 in the plasma membrane of the human hepatoma cell line, HuH-7, of cells from mouse liver, and of macrophages differentiated from the human acute monocytic leukemia cell line, THP-1 (THP-1 macrophages). In HuH-7 cells, degradation of cell surface-resident ABCA1 was inhibited by the overexpression of a dominant-negative form of ubiquitin. Moreover, the disruption of the endosomal sorting complex required for transport (ESCRT) pathway, a dominant mechanism for ubiquitination-mediated lysosomal degradation, by the knockdown of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), significantly delayed the degradation of cell surface-resident ABCA1. This was accompanied by an increase in ABCA1 expression as well as in apoA-I-mediated [3H]-cholesterol efflux function. The effect of HRS knockdown was also observed after calpain inhibitor treatment, which is reported to retard ABCA1 degradation. The induction of ABCA1 by HRS knockdown was confirmed in THP-1 macrophages. CONCLUSION: Together with the fact that lysosomal inhibitor treatments increased ABCA1 expression in HuH-7 and THP-1 macrophages, these results suggest that ubiquitination mediates the lysosomal degradation of cell surface-resident ABCA1 through the ESCRT pathway, and thereby controls the expression and cholesterol efflux function of ABCA1. This mechanism seems to mediate ABCA1 degradation independently of the calpain-involving pathway. The modulation of ABCA1 ubiquitination could thus be a potential new therapeutic target for antiatherogenic drugs.     

In vitro reconstitution of the photosystem I light-harvesting complex LHCI-730: heterodimerization is required for antenna pigment organization

Here we describe the in vitro reconstitution of photosystem I light-harvesting complexes with pigments and proteins (Lhca1 and Lhca4) obtained by overexpression of tomato Lhca genes in Escherichia coli. Using Lhca1 and Lhca4 individually for reconstitution results in monomeric pigment-proteins, whereas a combination thereof yields a dimeric complex. Interactions of the apoproteins is highly specific, as reconstitution of either of the two constituent proteins in combination with a light-harvesting protein of photosystem II does not result in dimerization. The reconstituted Lhca1/4, but not complexes obtained with either Lhca1 or Lhca4 alone, closely resembles the native LHCI-730 dimer from tomato leaves with regard to spectroscopic properties, pigment composition, and stoichiometry. Monomeric complexes of Lhca1 or Lhca4 possess lower pigment/protein ratios, indicating that interactions of the two subunits not only facilitates pigment reorganization but also recruitment of additional pigments. In addition to higher averages of chlorophyll a/b ratios in monomeric complexes than in LHCI-730, comparative fluorescence and CD spectra demonstrate that heterodimerization involves preferential ligation of more chlorophyll b.     

Super-Resolution STED Microscopy-Based Mobility Studies of the Viral Env Protein at HIV-1 Assembly Sites of Fully Infected T-Cells

The ongoing threat of human immunodeficiency virus (HIV-1) requires continued, detailed investigations of its replication cycle, especially when combined with the most physiologically relevant, fully infectious model systems. Here, we demonstrate the application of the combination of stimulated emission depletion (STED) super-resolution microscopy with beam-scanning fluorescence correlation spectroscopy (sSTED-FCS) as a powerful tool for the interrogation of the molecular dynamics of HIV-1 virus assembly on the cell plasma membrane in the context of a fully infectious virus. In this process, HIV-1 envelope glycoprotein (Env) becomes incorporated into the assembling virus by interacting with the nascent Gag structural protein lattice. Molecular dynamics measurements at these distinct cell surface sites require a guiding strategy, for which we have used a two-colour implementation of sSTED-FCS to simultaneously target individual HIV-1 assembly sites via the aggregated Gag signal. We then compare the molecular mobility of Env proteins at the inside and outside of the virus assembly area. Env mobility was shown to be highly reduced at the assembly sites, highlighting the distinct trapping of Env as well as the usefulness of our methodological approach to study the molecular mobility of specifically targeted sites at the plasma membrane, even under high-biosafety conditions.     

The Ku-like protein from Saccharomyces cerevisiae is required in vitro for the assembly of a stable multiprotein complex at a eukaryotic origin of replication.

We have previously shown that three distinct DNA-binding activities, in crude form, are necessary for the ATP-dependent assembly of a specific and stable multiprotein complex at a yeast origin of replication. Here we show the purification of one of these DNA binding activities, referred to as origin binding factor 2 (OBF2). The purified protein is a heterodimer composed of two polypeptides with molecular mass values of 65 and 80 kDa as determined by SDS/PAGE. Purified OBF2 not only binds DNA but also supports the formation of a protein complex at essential sequences within the ARS121 origin of replication. Interestingly, OBF2 binds tightly and nonspecifically to both duplex DNA and single-stranded DNA. The interaction with duplex DNA occurs at the termini. N-terminal sequencing of the 65-kDa subunit has revealed that this polypeptide is identical to the previously identified HDF1 peptide, a yeast homolog of the small subunit of the mammalian Ku autoantigen. Although the potential involvement of Ku in DNA metabolic events has been proposed, this is the first requirement for a Ku-like protein in the assembly of a protein complex at essential sequences within a eukaryotic origin of replication.