ABSTRACT

Monoclonal antibody, LAS-2, directed against the subunit of transducin (G), inhibited G-dependent, pertussis toxin-catalyzed ADP ribosylation of G and was specific for G. Immunoblotting studies on proteolytic fragments of G were consistent with an amino-terminal epitope. To define the antibody recognition site, recombinant G was synthesized in Escherichia coli cotransfected with or without yeast N-myristoyltransferase. Amino-terminal fatty acylation of G, verified by use of radiolabeled fatty acid, was required for immunoreactivity. LAS-2 did not react with a chimeric protein consisting of residues 1-9 of G and the remainder G, regardless of its myristoylation. Immunoreactivity was observed when amino acids 1-17 of G were present in a G chimera and the protein was amino-terminally myristoylated; there was no reactivity without myristoylation. It appears that the LAS-2 epitope requires both G-specific sequence in amino acids 10-17 and a fatty acyl group in proximity to these residues. These results are consistent with the hypothesis that the myristoyl group is essential for protein structure; conceivably it ``folds back'' on and stabilizes the amino-terminal structure of G, as opposed to protruding from an amino-terminal -helix and serving as an amino-terminal membrane anchor.

FOOTNOTES

*

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: NIH, Bldg. 10, Rm. 5N-307, 10 Center Dr., MSC 1434, Bethesda, MD 20892-1434. Tel.: 301-496-1254; Fax: 301-402-1610.

¶

Present address: VA Medical Center, 3710 SW U. S. Veterans Hospital Rd., Portland, OR 97035.

()

The abbreviations used are: G protein, heterotrimeric guanine nucleotide-binding protein; PAGE, polyacrylamide gel electrophoresis; TPCK, L-tosyl-amido-2-phenylethyl chloromethyl ketone; G, transducin; G, subunit of transducin; G, subunit of transducin; G, subunit of the abundant G protein obtained from bovine brain; G, subunit of the inhibitory G protein of the adenylyl cyclase system; G, subunit of the stimulatory G protein of the adenylyl cyclase system; PCR, polymerase chain reaction; rG, recombinant G protein.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				E. Kostenis, F.-Y. Zeng, and J. Wess Functional Characterization of a Series of Mutant G Protein alpha q Subunits Displaying Promiscuous Receptor Coupling Properties J. Biol. Chem., July 10, 1998; 273(28): 17886 - 17892. [Abstract] [Full Text] [PDF]

				J. M. Justice, J. J. Murtagh , Jr., J. Moss, and M. Vaughan Hydrophobicity and Subunit Interactions of Rod Outer Segment Proteins Investigated Using Triton X-114 Phase Partitioning J. Biol. Chem., July 28, 1995; 270(30): 17970 - 17976. [Abstract] [Full Text] [PDF]