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Journal of Cell Science 114, 4105-4115 (2001)
© 2001 The Company of Biologists Limited

RESEARCH ARTICLE

Direct targeting of cis-Golgi matrix proteins to the Golgi apparatus

Shin-ichiro Yoshimura1,2, Nobuhiro Nakamura2,*, Francis A. Barr3, Yoshio Misumi4, Yukio Ikehara4, Hiroshi Ohno2, Masao Sakaguchi1 and Katsuyoshi Mihara1

1 Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan
2 Cancer Research Institute, Kanazawa University, Kanazawa 920-0934, Japan
3 Department of Cell Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18a, Martinsried, D-82152, Germany
4 Department of Biochemistry, Fukuoka University School of Medicine, Fukuoka 814-0180, Japan

*Author for correspondence (e-mail: osaru3{at}kenroku.kanazawa-u.ac.jp)

Accepted August 4, 2001

The targeting route of newly synthesized GM130 and GRASP65 to the Golgi apparatus was investigated by three different approaches. First, localization of pulse labeled GM130 and GRASP65 in normal rat kidney (NRK) cells was traced by subcellular fractionation followed by immunoprecipitation. Immediately after the pulse labeling, GM130 and GRASP65 were found in the Golgi but not in the endoplasmic reticulum (ER) membrane fractions, whereas a control Golgi membrane protein was still found in the ER membrane fractions. Second, epitope tagged GM130 and GRASP65 were expressed in NRK cells by plasmid microinjection into the nuclei and their localization was analyzed by immunofluorescence. When ER to Golgi transport was inhibited by prior microinjection of a GTP-restricted mutant of Sar1 protein into the cytosol, the expressed GM130 and GRASP65 showed clear Golgi localization. Last, binding of GM130 and GRASP65 to the membranes was analyzed in vitro. In vitro synthesized GM130 and GRASP65 specifically bound to purified Golgi membranes but not to microsomal membranes. The bound GM130 and GRASP65 were found to form a complex with pre-existing counterparts on the Golgi membrane. These results strongly suggested that GM130 and GRASP65 are directly targeted to the Golgi membrane without initial assembly on the ER and subsequent vesicular transport to the Golgi apparatus.

Key words: Golgi, Membrane binding, Vesicular transport, Subcellular fractionation, Microinjection, Mutant Sar1p


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© The Company of Biologists Ltd 2001