Enrichment of G-protein palmitoyltransferase activity in low density membranes: in vitro reconstitution of Galphai to these domains requires palmitoyltransferase activity.

Julianne T Dunphy,Wendy K Greentree,Maurine E Linder

doi:10.1074/jbc.m104275200

Julianne T Dunphy, Wendy K Greentree + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m104275200

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Abstract

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.

Highlights

Many signaling proteins are targeted to low density, sphingomyelin- and cholesterol-enriched membranes, called lipid rafts
We have previously shown that protein acyltransferase activity (PAT) activity for myristoylated G␣ subunits is highly enriched in the plasma membrane (PM) [25] where it facilitates the stable association of G␣ with the PM [26]
Fraction 4 represents the accumulation of low density membranes as indicated by the presence of caveolin (Fig. 1A) and the absence of the transferrin receptor (Fig. 1B), a marker for plasma membrane outside lipid rafts [15]

Summary

Introduction

Many signaling proteins are targeted to low density, sphingomyelin- and cholesterol-enriched membranes, called lipid rafts. 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. Prior to assaying PAT activity, the total cell lysate and fractions 9 and 10 were neutralized with 1 N HCl. G-protein Reconstitution into Low Density PM Domains—Cells were washed and scraped into high pH buffer as described above.

Results

Conclusion