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

J Biol Chem. 2008 Apr 11;283(15):9623-32. doi: 10.1074/jbc.M800717200. Epub 2008 Feb 6.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing / physiology*
  • Amino Acid Substitution*
  • Annexin A7 / genetics
  • Annexin A7 / metabolism
  • Annexins / genetics
  • Annexins / metabolism
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism*
  • Binding Sites / genetics
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism*
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cell Line
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Endosomal Sorting Complexes Required for Transport
  • Phospholipid Transfer Proteins / genetics
  • Phospholipid Transfer Proteins / metabolism*
  • Protein Binding / genetics
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Vesicular Transport Proteins / genetics
  • Vesicular Transport Proteins / metabolism

Substances

  • Annexin A7
  • Annexins
  • Apoptosis Regulatory Proteins
  • Calcium-Binding Proteins
  • Carrier Proteins
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Endosomal Sorting Complexes Required for Transport
  • PDCD6 protein, human
  • PDCD6IP protein, human
  • PLSCR3 protein, human
  • PLSCR4 protein, human
  • Phospholipid Transfer Proteins
  • Protein Isoforms
  • Recombinant Fusion Proteins
  • SEC31A protein, human
  • Transcription Factors
  • Tsg101 protein
  • Vesicular Transport Proteins