title: Identification of Alix-type and Non-Alix-type ALG-2-binding sites in human phospholipid scramblase 3: differential binding to an alternatively spliced isoform and amino acid-substituted mutants.
abstract: ALG-2, a prototypic member of the penta-EF-hand protein family, interacts with Alix at its C-terminal Pro-rich region containing four tandem PXY repeats. Human phospholipid scramblase 3 (PLSCR3) has a similar sequence (ABS-1) in its N-terminal region. In the present study, we found that ALG-2 interacts with PLSCR3 expressed in HEK293 cells in a Ca(2+)-dependent manner by co-immunoprecipitation, pulldown with glutathione S-transferase (GST) fused ALG-2 and an overlay assay using biotin-labeled ALG-2. The GST fusion protein of an alternatively spliced isoform of ALG-2, GST-ALG-2(DeltaGF122), pulled down green fluorescent protein (GFP)-fused PLSCR3 but not GFP Alix. Deletion of a region containing ABS-1 was not sufficient to abrogate the binding. A second ALG-2-binding site (ABS-2) was essential for interaction with ALG-2(DeltaGF122). Real-time interaction analyses with a surface plasmon resonance biosensor using synthetic oligopeptides and recombinant proteins corroborated direct Ca(2+)-dependent binding of ABS-1 to ALG-2 and that of ABS-2 to ALG-2 as well as to ALG-2(DeltaGF122). The sequence of ABS-2 contains multiple prolines and two phenylalanines, among which Phe(49) was found to be critical, because its substitution with Ala or Tyr caused a loss of binding ability by pulldown assays using oligopeptide-immobilized beads. ALG-2-interacting proteins were classified into two groups based on binding ability to ALG-2(DeltaGF122): (i) isoform-non-interactive (ABS-1) types, including Alix, annexin A7, annexin A11, and TSG101 and (ii) isoform-interactive (ABS-2) types including PLSCR3, PLSCR4 and Sec31A. GST-pulldown assays using single amino acid-substituted ALG-2 mutants revealed differences in binding specificities between the two groups, suggesting structural flexibility in ALG-2-ligand complex formation.
authors.affiliation: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Shibata
authors.forename: Hironori
authors.initials: H
authors.lastname: Suzuki
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kakiuchi
authors.forename: Tatsutoshi
authors.initials: T
authors.lastname: Inuzuka
authors.forename: Haruna
authors.initials: H
authors.lastname: Yoshida
authors.forename: Takako
authors.initials: T
authors.lastname: Mizuno
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Maki
mesh_terms: D017398:Alternative Splicing / Q000502:physiology*; D019943:Amino Acid Substitution; D017310:Annexin A7 / Q000235:genetics / Q000378:metabolism; D017302:Annexins / Q000235:genetics / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D049294:Phospholipid Transfer Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism; D000099177:Tsg101 Protein
chemical_list: D017310:Annexin A7; D017302:Annexins; D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D049294:Phospholipid Transfer Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D033921:Vesicular Transport Proteins; D000099177:Tsg101 Protein; C097753:PDCD6 protein, human; C117020:PDCD6IP protein, human; C493424:PLSCR3 protein, human; C493425:PLSCR4 protein, human; C507455:SEC31A protein, human