Identification and characterization of a novel Golgi protein, GCP60, that interacts with the integral membrane protein giantin.
We demonstrated previously that the integral membrane protein giantin has the Golgi localization signal at the COOH-terminal cytoplasmic domain (Misumi, Y., Sohda, M., Tashiro, A., Sato, H., and Ikehara, Y. (2001) J. Biol. Chem. 276, 6867-6873). In the present study, using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein interacting with giantin. The 3.6-kilobase mRNA encoding a 528-amino acid protein of 60 kDa designated GCP60 was ubiquitously expressed and was especially abundant in the testis and ovary. Immunofluorescence and immunoelectron microscopy confirmed that GCP60 was co-localized with giantin in the Golgi complex. GCP60 was found to be a peripheral protein associated with the Golgi membrane, where a COOH-terminal domain of GCP60 interacts with the COOH-terminal cytoplasmic domain of giantin. Overexpression of the COOH-terminal domain of GCP60 caused disassembly of the Golgi structure and blocked protein transport from the endoplasmic reticulum to the Golgi. Taken together, these results suggest that GCP60 is involved in the maintenance of the Golgi structure by interacting with giantin, affecting protein transport between the endoplasmic reticulum and the Golgi.
Sohda
M
M
Department of Biochemistry, School of Medicine and Advanced Materials Institute, Fukuoka University, Jonan-ku, Fukuoka 814-0180, Japan.
Misumi
Y
Y
Yamamoto
A
A
Yano
A
A
Nakamura
N
N
Ikehara
Y
Y
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D016466:CHO Cells; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000737:chemistry* / Q000378:metabolism; D000077614:Golgi Matrix Proteins; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D014018:Tissue Distribution; D014162:Transfection; D020798:Two-Hybrid System Techniques
C449459:ACBD3 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018076:DNA, Complementary; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; C087574:macrogolgin