G-protein Palmitoyltransferase Activity Is Enriched in Plasma Membranes

doi:10.1074/jbc.271.12.7154

G-protein Palmitoyltransferase Activity Is Enriched in Plasma Membranes (*)

From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

** To whom correspondence should be addressed:
Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Ave., Box 8228, St. Louis, MO 63110
. Tel.: 314-362-6040; Fax: 314-362-7463.

Abstract

Heterotrimeric G proteins are covalently modified by lipids. Myristoylation of G-protein α subunits and prenylation of subunits are stable modifications. In contrast, palmitoylation of α subunits is dynamic and thus has the potential for regulating protein function. Indeed, receptor activation of G increases palmitate turnover on the α subunit, presumably by stimulating deacylation. The enzymes that catalyze reversible palmitoylation of G-protein α subunits have not been characterized. Here we report the identification of a palmitoyl-CoA:protein S-palmitoyltransferase activity that acylates G-protein α subunits in vitro. Palmitoyltransferase activity is membrane-associated and requires detergent for solubilization. The preferred G-protein substrate for the enzyme activity is the α subunit in the context of the heterotrimer. Both myristoylated and nonmyristoylated G-protein α subunits are recognized as substrates. The palmitoyltransferase activity demonstrates a modest preference for palmitoyl-CoA over other fatty acyl-CoA substrates. Palmitoyltransferase activity is high in plasma membrane and present at low or undetectable levels in Golgi, endoplasmic reticulum, and mitochondria of rat liver. The subcellular localization of this enzyme activity is consistent with a role for regulated cycles of acylation and deacylation accompanying activation of G-protein signal transduction pathways.

Footnotes

↵^§ Recipient of a Lucille P. Markey Pathway predoctoral fellowship.
↵^¶ Supported by Training Grant T32GM07067 to Washington University.
↵* This work was supported in part by United States Public Health Service Grant GM50556, the Diabetes Research and Training Center at Washington University, and the Monsanto-Searle/Washington University Biomedical Research Program. 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.
↵¹ The abbreviations used are:

T

transducin α subunit

T

transducin β subunits

rG

recombinant G

N-myristoyltransferase

myristoyl-CoA:protein N-myristoyltransferase

PAT

palmitoyl-CoA:protein S-palmitoyltransferase

protease inhibitors

0.1 mM phenylmethylsulfonyl fluoride, 21 μg/ml TPCK (N-tosyl-L-phenylalanine chloromethyl ketone), 21 μg/ml TLCK (N-p-tosyl-L-lysine chloromethyl ketone), 1.8 μg/ml aprotinin, 3.2 μg/ml leupeptin, 3.2 μg/ml lima bean trypsin inhibitor

BCA

bicinchoninic acid

Mes

2-(4-morpholino)ethanesulfonic acid

DTT

dithiothreitol

PAGE

polyacrylamide gel electrophoresis

INT

2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride

GTPS

guanosine 5′-(-thio)triphosphate

HPLC

high pressure liquid chromatography

ER

endoplasmic reticulum.
↵² To confirm that the radioactivity incorporated into myristoylated rGin vitro was palmitate, the protein was excised from the gel and treated with base to cleave thioester bonds. Hydrolysates were resolved by reversed phase HPLC, and fatty acids were identified by co-elution with fatty acid standards. The radioactivity released from myristoylated rG was identified as palmitic acid (data not shown). These data also demonstrate that in vitro palmitoylation of the protein is alkaline-sensitive, consistent with a thioester linkage.
- Received December 7, 1995.
- Revision received January 16, 1996.