Abstract
ALG-2-interacting protein X (Alix), also known as AIP1, is a cytoplasmic protein ubiquitously expressed and concentrated in phagosomes and exosomes. Alix may regulate apoptosis since it binds apoptosis-linked gene 2 (ALG-2), a Ca2+-binding protein necessary for cell death, and also overexpression of its C-terminal half (Alix-CT) blocks death induced by several stimuli. This part of Alix contains a long proline-rich domain containing several potential SH3-binding sites. Using Alix as bait in a yeast two-hybrid system to screen a mouse brain library, we have found that SH3p4, SH3p8, and SH3p13, collectively known as endophilins, bind to Alix. Co-immunoprecipitations and overlay experiments allowed us to demonstrate that endophilins bind to Alix-CT through an SH3/proline-rich domain interaction. We have narrowed the region of Alix interacting with endophilins down to 14 amino acids containing a PXRPPPP consensus sequence, also present in synaptojanin and germinal center kinase-like kinase, allowing their interaction to endophilins. We further show that overexpression of Alix-CT, which blocks cell death, leads to cytoplasmic vacuolization into tubulo-vesicular structures delineated by Alix-CT. This vacuolization phenomenon is greatly enhanced upon co-expression with endophilins and may be part of the protecting mechanism afforded by Alix-CT.
Highlights
apoptosis-linked gene 2 (ALG-2)-interacting protein X (Alix), known as AIP1, is a cytoplasmic protein ubiquitously expressed and concentrated in phagosomes and exosomes
A cDNA encoding full-length ALG-2-interacting protein X (Alix) was fused to the DNA binding domain of the GAL4 transcription factor and used to screen a mouse brain cDNA library fused to the GAL4 activation domain (the same library as that used to search for ALG-2-interacting proteins in Missotten et al [1])
By using Alix as bait in a yeast two-hybrid screen, we found that it binds to Src homology domain 3 (SH3) domain-containing proteins, a result that was predictable in view of the multiple potential SH3 binding motives of the long Alix proline-rich domain (PRD)
Summary
ALG-2-interacting protein X; ALG-2, apoptosis-linked gene 2; AIP1, ALG-2-interacting protein 1; SH3, Src homology domain 3; PRD, proline-rich domain; SETA, SH3 domain expressed in tumorigenic astrocytes; GST, glutathione S-transferase; ER, endoplasmic reticulum; HEK, human embryonic kidney cells; aa, amino acid(s); TBS, Tris-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Alix possesses no obvious enzymatic signature, but its last 150 amino acids are rich in proline (32%), tyrosine, and glutamine residues and contain several Src homology domain 3 (SH3) binding motives (PXXP) and two WW binding domains (PPXY). This 150-amino acid-long proline-rich domain (PRD) interacts with the second SH3 domain of SETA (SH3 domain expressed in tumorigenic astrocytes), described as Ruk [7,8,9]. When the same Alix-CT is co-expressed with endophilins, the vacuoles/tubules are drastically enlarged and can even form very large spherical vacuoles with a diameter of up to several microns
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