Decoding the intrinsic mechanism that prohibits ALIX interaction with ESCRT and viral proteins.
The adaptor protein ALIX [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] links retroviruses to ESCRT (endosomal sorting complex required for transport) machinery during retroviral budding. This function of ALIX requires its interaction with the ESCRT-III component CHMP4 (charged multivesicular body protein 4) at the N-terminal Bro1 domain and retroviral Gag proteins at ... the middle V domain. Since cytoplasmic or recombinant ALIX is unable to interact with CHMP4 or retroviral Gag proteins under non-denaturing conditions, we constructed ALIX truncations and mutations to define the intrinsic mechanism through which ALIX interactions with these partner proteins are prohibited. Our results demonstrate that an intramolecular interaction between Patch 2 in the Bro1 domain and the TSG101 (tumour susceptibility gene 101 protein)-docking site in the proline-rich domain locks ALIX into a closed conformation that renders ALIX unable to interact with CHMP4 and retroviral Gag proteins. Relieving the intramolecular interaction of ALIX, by ectopically expressing a binding partner for one of the intramolecular interaction sites or by deleting one of these sites, promotes ALIX interaction with these partner proteins and facilitates ALIX association with the membrane. Ectopic expression of a GFP (green fluorescent protein)-ALIX mutant with a constitutively open conformation, but not the wild-type protein, increases EIAV (equine infectious anaemia virus) budding from HEK (human embryonic kidney)-293 cells. These findings predict that relieving the autoinhibitory intramolecular interaction of ALIX is a critical step for ALIX to participate in retroviral budding.
Mesh Terms:
Calcium-Binding Proteins, Cell Cycle Proteins, Cell Membrane, DNA-Binding Proteins, Endosomal Sorting Complexes Required for Transport, Gene Products, gag, HEK293 Cells, Humans, Hydrophobic and Hydrophilic Interactions, Infectious Anemia Virus, Equine, Mutant Proteins, Oncogene Protein pp60(v-src), Proline-Rich Protein Domains, Protein Interaction Domains and Motifs, Recombinant Fusion Proteins, Recombinant Proteins, Retroviridae Proteins, Transcription Factors, Vesicular Transport Proteins, Virus Release
Calcium-Binding Proteins, Cell Cycle Proteins, Cell Membrane, DNA-Binding Proteins, Endosomal Sorting Complexes Required for Transport, Gene Products, gag, HEK293 Cells, Humans, Hydrophobic and Hydrophilic Interactions, Infectious Anemia Virus, Equine, Mutant Proteins, Oncogene Protein pp60(v-src), Proline-Rich Protein Domains, Protein Interaction Domains and Motifs, Recombinant Fusion Proteins, Recombinant Proteins, Retroviridae Proteins, Transcription Factors, Vesicular Transport Proteins, Virus Release
Biochem. J.
Date: Dec. 15, 2010
PubMed ID: 20929444
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