A Crescent-Shaped ALIX Dimer Targets ESCRT-III CHMP4 Filaments

Abstract

ALIX recruits ESCRT-III CHMP4 and is involved in membrane remodeling during endosomal receptor sorting, budding of some enveloped viruses and cytokinesis. We show that ALIX dimerizes via the middle domain (ALIX-V) in solution. Structural modeling based on small angle X-ray scattering (SAXS) data reveal an elongated crescent shaped conformation for dimeric ALIX lacking the proline rich domain (ALIXBRO1-V). Mutations at the dimerization interface prevent dimerization and induce an open elongated monomeric conformation of ALIX-V as determined by SAXS modeling. ALIX dimerizes in vivo and dimeric ALIX co-localizes with CHMP4B upon co-expression. We show further that ALIX dimerization affects HIV-1 budding. C-terminally truncated activated CHMP4B retaining the ALIX binding site forms linear, circular and helical filaments in vitro, which can be bridged by ALIX. Our data suggest that dimeric ALIX represents the active form that interacts with ESCRT-III CHMP4 polymers and functions as a scaffolding protein during membrane remodeling processes.

ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

“…To test whether At-ALIX also forms dimers, we performed yeast two-hybrid assays using full-length and truncated versions of At-ALIX comprising the Bro1 domain (amino acids 1 to 413) or the coiled coils plus the Pro-rich region (amino acids 405 to 846) ( Figure 3F). The C-terminal portion of At-ALIX (amino acids 405 to 846) interacted with all protein versions in at least one bait/prey orientation ( Figure 3G), pointing to a role for this domain in intramolecular interactions and/or dimerization of At-ALIX, as observed in mammals (Pires et al, 2009). To confirm At-ALIX dimerization in vivo, tandem affinity purification (TAP) assays were performed using soluble protein extracts from Arabidopsis lines expressing an N-terminal fusion of At-ALIX to the TAPa tag .…”

“…In mammals, ALIX dimerizes in vitro and in vivo via the V domain, the central region of ALIX composed of two coiled coils. For ALIX dimerization, the V domain must be open, which allows antiparallel interaction of the ALIX V domain arms (Pires et al, 2009) (Figure 3E). To test whether At-ALIX also forms dimers, we performed yeast two-hybrid assays using full-length and truncated versions of At-ALIX comprising the Bro1 domain (amino acids 1 to 413) or the coiled coils plus the Pro-rich region (amino acids 405 to 846) ( Figure 3F).…”

Identification and Structural Characterization of the ALIX-Binding Late Domains of Simian Immunodeficiency Virus SIV mac239 and SIV agmTan-1

“…Second, activated ALIX is dimeric (27), which should enhance binding avidity to oligomeric Gag assemblies. Third, ALIX can associate with ubiquitin, and ubiquitylation of Gag (or associated proteins) could therefore enhance ALIX recruitment (18).…”

Alix Protein Is Substrate of Ozz-E3 Ligase and Modulates Actin Remodeling in Skeletal Muscle

“…Relevant to this study are the findings that Alix also participates in cytoskeleton remodeling by binding with F-actin, ␣-actinin, cortactin, and focal adhesion kinase (14,21). These multiple and seemingly diverse interactions of Alix are made possible by the primary structure of the protein that includes at least three distinct protein-protein interaction domains as follows: the N-terminal Bro1 domain; a middle region called the V-domain and containing a coiled-coil motif; and a C-terminal prolinerich region (PRR), 5 which is a potential docking site for proteins containing Src homology 3 domains (22)(23)(24). Both the Bro1 and the PRR domains of Alix bind F-actin, whereas the N-terminal half of its V-domain interacts with ␣-actinin and cortactin.…”