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.
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.
Shibata
Hideki
H
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan. shibabou@agr.nagoya-u.ac.jp
Inuzuka
Tatsutoshi
T
Yoshida
Haruna
H
Sugiura
Hirofumi
H
Wada
Ikuo
I
Maki
Masatoshi
M
D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D001665:Binding Sites; D022181:COP-Coated Vesicles; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins; D059008:Time-Lapse Imaging / Q000379:methods*; D033921:Vesicular Transport Proteins / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; C097753:PDCD6 protein, human; D011993:Recombinant Fusion Proteins; C507455:SEC31A protein, human; D033921:Vesicular Transport Proteins