The calcium-binding protein ALG-2 promotes endoplasmic reticulum exit site localization and polymerization of Trk-fused gene (TFG) protein.
Apoptosis-linked gene 2 (ALG-2), which is a gene product of PDCD6, is a 22-kDa Ca2+-binding protein. Accumulating evidence points to a role for ALG-2 as a Ca2+-responsive adaptor protein. On binding to Ca2+, ALG-2 undergoes a conformational change that facilitates its interaction with various proteins. It also forms a homodimer and heterodimer with peflin, a paralog of ALG-2. However, the differences in cellular roles for the ALG-2 homodimer and ALG-2/peflin heterodimer are unclear. In the present study, we found that Trk-fused gene (TFG) protein interacted with the ALG-2 homodimer. Immunostaining analysis revealed that TFG and ALG-2 partially overlapped at endoplasmic reticulum exit sites (ERES), a platform for COPII-mediated protein transport from the endoplasmic reticulum. Time-lapse live-cell imaging demonstrated that both green fluorescent protein-fused TFG and mCherry-fused ALG-2 are recruited to ERES after thapsigargin treatment, which raises intracellular Ca2+levels. Furthermore, overexpression of ALG-2 induced the accumulation of TFG at ERES. TFG has an ALG-2-binding motif and deletion of the motif decreased TFG binding to ALG-2 and shortened its half-life at ERES, suggesting a critical role for ALG-2 in retaining TFG at ERES. We also demonstrated, by in vitro cross-linking assays, that ALG-2 promoted the polymerization of TFG in a Ca2+-dependent manner. Collectively, the results suggest that ALG-2 acts as a Ca2+-sensitive adaptor to concentrate and polymerize TFG at ERES, supporting a potential role for ALG-2 in COPII-dependent trafficking from the endoplasmic reticulum.
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan.
Takashi
T
Kanadome
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan.
Hideki
H
Shibata
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Japan.
Keiko
K
Kuwata
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan.
Terunao
T
Takahara
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan.
Masatoshi
M
Maki
D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D022181:COP-Coated Vesicles / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D019284:Thapsigargin / Q000494:pharmacology; D059008:Time-Lapse Imaging; D000097573:Red Fluorescent Protein; D033921:Vesicular Transport Proteins
D051017:Apoptosis Regulatory Proteins; D002118:Calcium; D002135:Calcium-Binding Proteins; D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D019284:Thapsigargin; D000097573:Red Fluorescent Protein; C097753:PDCD6 protein, human; C121810:PEF1 protein, human; C096163:TFG protein, human; D033921:Vesicular Transport Proteins
ALG-2; Ca2+-binding protein; Trk-fused gene (TFG) protein; endoplasmic reticulum exit sites; protein-protein interaction