The Penta-EF-Hand Protein ALG-2 Interacts with a Region Containing PxY Repeats in Alix/AIP1, Which Is Required for the Subcellular Punctate Distribution of the Amino-Terminal Truncation Form of Alix/AIP1

• Published in: Journal of biochemistry (Tokyo) Vol. 135; no. 1; pp. 117 - 128
• Main Authors: Shibata, Hideki, Yamada, Keiko, Mizuno, Takako, Yorikawa, Chiharu, Takahashi, Hiroshi, Satoh, Hirokazu, Kitaura, Yasuyuki, Maki, Masatoshi
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.01.2004
• Subjects: ALG-2; Alix/AIP1; Amino Acid Sequence; Apoptosis Regulatory Proteins; calcium-binding protein; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Cycle Proteins; Cell Line; EF Hand Motifs; EF-hand; Endosomal Sorting Complexes Required for Transport; GFP; green fluorescent protein; HEK; HeLa Cells; hemagglutinin; human embryonic kidney; Humans; mAb; Molecular Sequence Data; monoclonal antibody; pAb; PBS; PEF; penta-EF-hand; phosphate buffered saline; Point Mutation; polyclonal antibody; polyproline II; polyvinylidene difluoride; PPII; Protein Binding; Protein Structure, Tertiary - genetics; Proteins - genetics; Proteins - metabolism; protein–protein interaction; PVDF; Repetitive Sequences, Amino Acid; SH3; src homology 3; Subcellular Fractions - metabolism; TBS; Tris-buffered saline; Two-Hybrid System Techniques
• ISSN: 0021-924X
• DOI: 10.1093/jb/mvh014

ALG-2 is a Ca2+-binding protein that belongs to the penta-EF-hand protein family and associates with several proteins, including annexin VII, annexin XI, and Alix/AIP1, in a Ca2+-dependent manner. The yeast two-hybrid system and a biotin-tagged ALG-2 overlay assay were carried out to characterize the interaction between ALG-2 and Alix. The region corresponding to amino acid residues 794 to 827 in the carboxy-terminal proline-rich region of Alix was sufficient to confer the ability to interact directly with ALG-2. This region includes four-tandem PxY repeats. Alanine substitutions indicated that seven proline residues in this region, four in the PxY repeats, and four tyrosine residues in the PxY repeats are crucial for the binding affinity with ALG-2. Endogenous ALG-2 was co-immunoprecipitated in the presence of Ca2+ with FLAG-tagged Alix or FLAG-tagged AlixΔEBS, a deletion mutant lacking the endophilin binding consensus sequence, but not with FLAG-tagged AlixΔABS, another mutant lacking the region comprising amino acids 798–841, from the lysates of HEK293 cells transfected with each FLAG-tagged protein expression construct. FLAG-tagged ALG-2 overexpressed in HEK293 cells was also co-immunoprecipitated with Alix in a Ca2+-dependent fashion, whereas FLAG-tagged ALG-2E47A/E114A, a Ca2+-binding deficient mutant of ALG-2, was not detected in the immunoprecipitates of Alix even in the presence of Ca2+. Fluorescent microscopic analyses using the carboxy-terminal half of Alix fused with green fluorescent protein (GFP-AlixCT) revealed that endogenous ALG-2 in HeLa cells exhibits a dot-like pattern overlapping with exogenously expressed GFP-AlixCT, and the distribution of GFP-AlixCTΔABS is observed diffusely in the cytoplasm. These results indicate the requirement of ABS in Alix for the efficient accumulation of AlixCT and raise the possibility that ALG-2 participates in membrane trafficking through a Ca2+-dependent interaction with Alix.