Production of N-lauroylated G protein alpha-subunit in Sf9 insect cells: the type of N-acyl group of Galpha influences G protein-mediated signal transduction.
The alpha-subunit of rod photoreceptor G protein transducin (G(t1)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 G(t1)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 G(t1)alpha isoform from the retinal rod cells. Here we found that G(t1)alpha/G(i1)alpha chimera (G(t/i)alpha) expressed in Sf9 cells is also heterogeneously modified by myristate (approximately 90%) and laurate (approximately 10%), raising the possibility that the N-acyl group of recombinant G(t/i)alpha may be manipulated by modifying culture media. In fact, addition of myristic acid to the medium decreased the relative content of lauroylated G(t/i)alpha to an undetectable level, whereas exogenously added lauric acid significantly increased the relative content of lauroylated G(t/i)alpha in a concentration-dependent manner. By culturing the G(t/i)alpha-virus infected Sf9 cells with fatty acids, we obtained four different preparations of N-acylated G(t/i)alpha, in which the relative abundance of lauroylated isoform was 0%, 20%, 33% and approximately 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 G(t/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 G(t1)alpha is composed of isoforms having functionally heterogeneous signaling properties.
Hashimoto
Yuichi
Y
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033.
Matsuda
Takahiko
T
Matsuura
Yoshiharu
Y
Haga
Tatsuya
T
Fukada
Yoshitaka
Y
D000215:Acylation; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002384:Catalysis; D002417:Cattle; D002460:Cell Line; D004789:Enzyme Activation; D005227:Fatty Acids / Q000032:analysis / Q000737:chemistry; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D018411:Spodoptera / Q000166:cytology* / Q000821:virology
D005227:Fatty Acids; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D011993:Recombinant Fusion Proteins