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J. Biochem, 2003, Vol. 134, No. 3 415-426
© 2003 Japanese Biochemical Society

CELL

Post-Translational Targeting of a Tail-Anchored Green Fluorescent Protein to the Endolpasmic Reticulum

Ryuichi Masaki*,1, Keiichi Kameyama2 and Akitsugu Yamamoto§,1

1 Department of Physiology, and Division of Cell Biology, Liver Research Center, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka 570-8506; and 2 Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193

Microsomal aldehyde dehydrogenase (msALDH) is a tail-anchored protein localized to the cytoplasmic face of the endoplasmic reticulum (ER). The carboxyl-terminal 35 amino acids of msALDH possess ER-targeting sequences in addition to a hydrophobic membrane-spanning domain. To study the mechanism for ER targeting of this protein in vivo, we took advantage of a green fluorescent protein-msALDH fusion protein containing the last 35 amino acids of msALDH [GFPALDH(35)]. When expressed from cDNA in COS-7 cells, the fusion protein was localized to the ER. We then prepared a recombinant fusion protein and injected it into the cytoplasm of COS-7 cells. The injected protein was correctly localized to the ER after a 30-min incubation at 37°C. However, a recombinant fusion protein that contained only the transmembrane domain of msALDH failed to be targeted to the ER. When the assay was carried out at 4°C, the recombinant GFPALDH(35) remained in the cytoplasm. Moreover, incubation of COS-7 cells under conditions of ATP depletion resulted in the cytoplasmic distribution of the injected protein. These results indicate that GFPALDH(35) is targeted to the ER post-translationally via an ATP-dependent pathway. This microinjection system worked effectively in different mammalian cell types, suggesting a common mechanism for ER targeting of the tail-anchored protein.

* To whom correspondence should be addressed. Tel: +81-6-6993-9422, Fax: +81-6-6993-5319, E-mail: masaki{at}takii.kmu.ac.jp

§ Present address: Department of Bio-Science, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829.


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