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. 1987 Nov;6(11):3353–3357. doi: 10.1002/j.1460-2075.1987.tb02656.x

Dynamic fatty acylation of p21N-ras.

A I Magee 1, L Gutierrez 1, I A McKay 1, C J Marshall 1, A Hall 1
PMCID: PMC553790  PMID: 3322807

Abstract

To study the acylation of p21N-ras with palmitic acid we have used cells which express the human N-ras gene to high levels under control of the steroid-inducible MMTV--LTR promoter. Addition of [3H]palmitate to these cells resulted in detectable incorporation of label into p21N-ras within 5 min, which continued linearly for 30-60 min. Inhibition of protein synthesis for up to 24 h before addition of [3H]palmitate had no effect on acylation of p21N-ras, suggesting that this can occur as a late post-translational event. Acylated p21N-ras with a high SDS--PAGE mobility is found only in the membrane fraction, whereas approximately 50% of the [35S]methionine-labelled p21N-ras is cytoplasmic and has a lower mobility. Conversion of the acylated high mobility form to a deacylated form of slightly lower mobility can be achieved with neutral hydroxylamine, which is known to cleave thioesters. This treatment also results in partial removal of p21N-ras from the membranes. A remarkably high rate of turnover of the palmitate moiety can be demonstrated by pulse--chase studies (t1/2 approximately 20 min in serum-containing medium) which cannot be attributed to protein degradation. The data suggest an active acylation--deacylation cycle for p21N-ras, which may be involved in its proposed function as a signal transducing protein.

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