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J. Biochem, 2004, Vol. 135, No. 3 319-329
© 2004 The Japanese Biochemical Society

BIOCHEMISTRY

Production of N-Lauroylated G Protein {alpha}-Subunit in Sf9 Insect Cells: The Type of N-Acyl Group of G{alpha} Influences G Protein–Mediated Signal Transduction

Yuichi Hashimoto1, Takahiko Matsuda*,1, Yoshiharu Matsuura2, Tatsuya Haga3 and Yoshitaka Fukada§,1

1 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033; 2 Research Center for Emerging Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871; and 3 Institute for Biomolecular Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588

The {alpha}-subunit of rod photoreceptor G protein transducin (Gt1{alpha}) is heterogeneously modified at the N-terminus by a mixture of acyl groups, laurate (C12:0), myristate (C14:0), and two unsaturated fatty acids (C14:1 and C14:2). Although the N-fatty acylation of Gt1{alpha} plays important roles in protein-protein and protein-membrane interactions in light signaling, the biological significance of the heterogeneous acylation remains unclear due to the difficulty in isolating each Gt1{alpha} isoform from the retinal rod cells. Here we found that Gt1{alpha}/Gi1{alpha} chimera (Gt/i{alpha}) expressed in Sf9 cells is also heterogeneously modified by myristate (~90%) and laurate (~10%), raising the possibility that the N-acyl group of recombinant Gt/i{alpha} may be manipulated by modifying culture media. In fact, addition of myristic acid to the medium decreased the relative content of lauroylated Gt/i{alpha} to an undetectable level, whereas exogenously added lauric acid significantly increased the relative content of lauroylated Gt/i{alpha} in a concentration-dependent manner. By culturing the Gt/i{alpha}-virus infected Sf9 cells with fatty acids, we obtained four different preparations of N-acylated Gt/i{alpha}, in which the relative abundance of lauroylated isoform was 0%, 20%, 33% and ~70%, respectively. Functional analysis of these proteins showed that an increase in the relative content of the lauroylated isoform remarkably slowed down the steady-state GTP hydrolysis rate of Gt/i{alpha}; the steady-state GTPase activity of the lauroylated isoform was estimated to be one order of magnitude lower than that of the myristoylated isoform. These results suggest that the retinal Gt1{alpha} is composed of isoforms having functionally heterogeneous signaling properties.

* Present address: Department of Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA, USA.

§ To whom correspondence should be addressed. Tel/Fax: +81-3-5802-8871, E-mail: sfukada{at}mail.ecc.u-tokyo.ac.jp


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[Abstract] [Full Text] [PDF]


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