Enrichment of G-protein palmitoyltransferase activity in low density membranes: in vitro reconstitution of Galphai to these domains requires palmitoyltransferase activity.
Many signaling proteins are targeted to low density, sphingomyelin- and cholesterol-enriched membranes, also called lipid rafts. These domains organize receptor-mediated signaling events at the plasma membrane. Fatty acylation is one mechanism for targeting proteins to rafts. It was therefore of interest to determine if protein palmitoyltransferase activity is also present in these domains. In this study, protein palmitoyltransferase activity, assayed using G-protein alpha subunits as a substrate, was found to be highly enriched in low density membranes derived from cells that express caveolin as well as those that do not. Depletion of cellular cholesterol with the drug methyl-beta-cyclodextrin resulted in inhibition of palmitoyltransferase activity and a redistribution of the remaining activity to membranes of higher density. This effect was reversed by adding cholesterol to cyclodextrin-treated cells. When reconstituted into cell membranes, the population of purified recombinant G(alphai) that was palmitoylated was highly enriched in the low density membrane fractions, whereas the bulk unmodified G(alphai)-protein was largely excluded. This effect required palmitoyltransferase activity and was abolished if the palmitoylated cysteine was mutated. Thus, palmitoyltransferase facilitates the enrichment of fatty acylated signaling molecules in plasma membrane subdomains.
Dunphy
J T
JT
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Greentree
W K
WK
Linder
M E
ME
D000217:Acyltransferases / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D051242:Caveolin 1; D022461:Caveolins / Q000096:biosynthesis; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003505:Cyclodextrins / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics; D003902:Detergents / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D021962:Membrane Microdomains / Q000378:metabolism; D009154:Mutation; D019308:Palmitic Acid / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D003505:Cyclodextrins; D003902:Detergents; D011994:Recombinant Proteins; D019308:Palmitic Acid; D000217:Acyltransferases; C099553:G-protein palmitoyltransferase; D003545:Cysteine