Alix regulates cortical actin and the spatial distribution of endosomes
Department of Biochemistry, the Norwegian Radium Hospital, Montebello, Oslo.Journal of Cell Science (Impact Factor: 5.43). 07/2005; 118(Pt 12):2625-35. DOI: 10.1242/jcs.02382
Alix/AIP1 is a proline-rich protein that has been implicated in apoptosis, endocytic membrane trafficking and viral budding. To further elucidate the functions of Alix, we used RNA interference to specifically suppress its expression. Depletion of Alix caused a striking redistribution of early endosomes from a peripheral to a perinuclear location. The redistribution of endosomes did not affect transferrin recycling or degradation of endocytosed epidermal growth factor receptors, although the uptake of transferrin was mildly reduced when Alix was downregulated. Quantitative immunoelectron microscopy showed that multivesicular endosomes of Alix-depleted cells contained normal amounts of CD63, whereas their levels of lysobisphosphatidic acid were reduced. Alix depletion also caused an accumulation of unusual actin structures that contained clathrin and cortactin, a protein that couples membrane dynamics to the cortical actin cytoskeleton. Our results suggest that Alix functions in the actin-dependent intracellular positioning of endosomes, but that it is not essential for endocytic recycling or for trafficking of membrane proteins between early and late endosomes in non-polarised cells.
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- "Meanwhile, another Bro1 protein, HD-PTP, plays a part in CHMP4 recruitment during EGFR sorting (Ali et al., 2013). The selective role that HD-PTP appears to have over Alix in ubiquitylated MVB cargo sorting (Bowers et al., 2006; Cabezas et al., 2005; Doyotte et al., 2008) is underlined by its selective binding to the ESCRT-0 subunit signal transducing adaptor molecule (STAM), and to UBAP1 (Ali et al., 2013; Stefani et al., 2011). A further ESCRT component with selective function is IST1, an ESCRT-III subunit that is essential for cytokinesis but not for EGFR sorting or virus budding (Agromayor et al., 2009; Bajorek et al., 2009). "
ABSTRACT: ESCRT-I is essential for the multivesicular body (MVB) sorting of ubiquitinated cargo such as epidermal growth factor receptor, as well as for divergent cellular functions such as cell division and retroviral budding. ESCRT-I has four subunits; TSG101, VPS28, VPS37 and MVB12. There are several members of VPS37 and MVB12 families in mammalian cells, and their differential incorporation into ESCRT-I could provide function-specific variants of the complex. However, it remains unclear whether these different forms of VPS37 and MVB12 combine randomly or generate selective pairings within ESCRT-I, and what the mechanistic basis for such pairing would be. Here we show that the incorporation into ESCRT-I of two MVB12 members, UBAP1 and MVB12A, is highly selective with respect to their VPS37 partners. We map the selective assembly of UBAP1/VPS37A to the core ESCRT-I binding domain of VPS37A. In contrast, selective integration of UBAP1 requires both the minimal ESCRT-I binding region and a neighbouring predicted helix. The biochemical specificity in ESCRT-I assembly is matched by functional specialisation, since siRNA-mediated depletion of UBAP1, but not MVB12A or MVB12B, disrupts ubiquitin-dependent sorting at the MVB.Journal of Cell Science 11/2013; 127(3). DOI:10.1242/jcs.140673 · 5.43 Impact Factor
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- "The plasmid pCMV-FLAG-ALIX was kindly provided by Jörg Votteler (University of Erlangen, Germany). The small interfering RNAs (siRNA) used to down regulate Tsg101 has been previously described [6,38] as well as the Alix specific siRNAs [39,40]. As non-target siRNA the “AllStars Neg. "
ABSTRACT: Late assembly (L)-domains are protein interaction motifs, whose dysfunction causes characteristic budding defects in enveloped viruses. Three different amino acid motifs, namely PT/SAP, PPXY and YPXnL have been shown to play a major role in the release of exogenous retroviruses. Although the L-domains of exogenous retroviruses have been studied comprehensively, little is known about these motifs in endogenous human retroviruses. Using a molecular clone of the human endogenous retrovirus K113 that had been engineered to reverse the presumed non-synonymous postinsertional mutations in the major genes, we identified three functional L-domains of the virus, all located in the Gag p15 protein. A consensus PTAP tetrapeptide serves as the core of a main L-domain for the virus and its inactivation reduces virus release in HEK 293T cells by over 80%. Electron microscopy of cells expressing the PTAP mutant revealed predominantly late budding structures and budding chains at the plasma membrane. The fact that this motif determines subcellular colocalization with Tsg101, an ESCRT-I complex protein known to bind to the core tetrapeptide, supports its role as an L-domain. Moreover, two YPXnL motifs providing additional L-domain function were identified in the p15 protein. One is adjacent to the PTAP sequence and the other is in the p15 N-terminus. Mutations in either motif diminishes virus release and induces an L-domain phenotype while inactivation of all three L-domains results in a complete loss of particle release in HEK 293T cells. The flexibility of the virus in the use of L-domains for gaining access to the ESCRT machinery is demonstrated by overexpression of Tsg101 which rescues the release of the YPXnL mutants. Similarly, overexpression of Alix not only enhances release of the PTAP mutant by a factor of four but also the release of a triple mutant, indicating that additional cryptic YPXnL domains with a low affinity for Alix may be present. No L-domain activity is provided by the proline-rich peptides at the Gag C-terminus. Our data demonstrate that HERV-K(HML-2) release is predominantly mediated through a consensus PTAP motif and two auxiliary YPXnL motifs in the p15 protein of the Gag precursor.Retrovirology 11/2013; 10(1):140. DOI:10.1186/1742-4690-10-140 · 4.19 Impact Factor
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- "Similarly, ALIX, which interacts with the ESCRT-I subunit TSG101 (Strack et al., 2003; von Schwedler et al., 2003) and the ESCRT-III subunit CHMP4 (Fisher et al., 2007; Usami et al., 2007), is also required during cytokinesis and virus budding at the plasma membrane. Unlike ESCRTs, however, ALIX does not play a role in ubiquitin-dependent lysosome targeting of the EGF receptor (Cabezas et al., 2005; Doyotte et al., 2008; Luyet et al., 2008; Schmidt et al., 2004), but perhaps in the ubiquitin-independent, ESCRT-III-dependent sorting of the GPCR PAR1 (Dores et al., 2012). We found that ALIX interacts with the unconventional phospholipid LBPA (Matsuo et al., 2004), which is detected only in late, but not early, endosomes, and is abundant in ILVs (Kobayashi et al., 1999). "
ABSTRACT: ALIX plays a role in nucleocapsid release during viral infection, as does lysobisphosphatidic acid (LBPA). However, the mechanism remains unclear. Here we report that LBPA is recognized within an exposed site in ALIX Bro1 domain predicted by MODA, an algorithm for discovering membrane-docking areas in proteins. LBPA interactions revealed a strict requirement for a structural calcium tightly bound near the lipid interaction site. Unlike other calcium- and phospholipid-binding proteins, the all-helical triangle-shaped fold of the Bro1 domain confers selectivity for LBPA via a pair of hydrophobic residues in a flexible loop, which undergoes a conformational change upon membrane association. Both LBPA and calcium binding are necessary for endosome association and virus infection, as are ALIX ESCRT binding and dimerization capacity. We conclude that LBPA recruits ALIX onto late endosomes via the calcium-bound Bro1 domain, triggering a conformational change in ALIX to mediate the delivery of viral nucleocapsids to the cytosol during infection.Developmental Cell 05/2013; 25(4). DOI:10.1016/j.devcel.2013.04.003 · 9.71 Impact Factor
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