Alix (ALG-2-interacting protein X), a protein involved in apoptosis, binds to endophilins and induces cytoplasmic vacuolization.
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.
Chatellard-Causse
Christine
C
Laboratoire Neurodégénérescence et Plasticité, INSERM-UJF (EMI 0108), Hopital A. Michallon, CHU, BP 217, 38043 Grenoble Cedex 9, France.
Blot
Beatrice
B
Cristina
Nadine
N
Torch
Sakina
S
Missotten
Marc
M
Sadoul
Remy
R
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D016923:Cell Death; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D056827:Endosomal Sorting Complexes Required for Transport; D017353:Gene Deletion; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D000596:Amino Acids; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D056827:Endosomal Sorting Complexes Required for Transport; C117020:PDCD6IP protein, human; C492144:Pdcd6ip protein, mouse; D011994:Recombinant Proteins; C107640:SH3GL2 protein, human; C498434:Sh3gl2 protein, mouse