The ALG-2 binding site in Sec31A influences the retention kinetics of Sec31A at the endoplasmic reticulum exit sites as revealed by live-cell time-lapse imaging

Biosci Biotechnol Biochem. 2010;74(9):1819-26. doi: 10.1271/bbb.100215. Epub 2010 Sep 7.


ALG-2, a member of the penta-EF-hand protein family, interacts Ca²+-dependently with a COPII component, Sec31A. In this study, we first established HeLa cells stably expressing green fluorescent protein-fused ALG-2 (GFP-ALG-2) and red fluorescent protein-fused Sec31A (Sec31A-RFP). After inducing Ca²+-mobilization, the cytoplasmic distribution of GFP-ALG-2 changed from a diffuse to a punctate pattern, which extensively overlapped with the Sec31A-RFP-positive structures, indicating that ALG-2 is recruited to the endoplasmic reticulum exit sites (ERES) in living cells. Next, overlay experiments with biotin-labeled ALG-2 were done to dissect the ALG-2 binding site (ABS). They revealed that a sequence comprising amino acid residues 839-851 in the Pro-rich region was necessary and sufficient for direct binding to ALG-2. Finally, fluorescence recovery after photobleaching analysis indicated that the ABS deletion reduced the high-affinity population of Sec31A to the ERES, suggesting that the ABS is one of the key determinants of the retention kinetics of Sec31A at ERES.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis Regulatory Proteins / metabolism*
  • Binding Sites
  • COP-Coated Vesicles
  • Calcium-Binding Proteins / metabolism*
  • Endoplasmic Reticulum / metabolism*
  • HeLa Cells
  • Humans
  • Kinetics
  • Luminescent Proteins
  • Protein Binding
  • Protein Transport
  • Recombinant Fusion Proteins
  • Time-Lapse Imaging / methods*
  • Vesicular Transport Proteins / metabolism*


  • Apoptosis Regulatory Proteins
  • Calcium-Binding Proteins
  • Luminescent Proteins
  • PDCD6 protein, human
  • Recombinant Fusion Proteins
  • SEC31A protein, human
  • Vesicular Transport Proteins