Studies on the cellular location, physical properties and endogenously attached lipids of acylated proteins in human squamous-carcinoma cell lines

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Abstract
The location of cell proteins with covalently attached lipid was examined in two human squamous-carcinoma cell lines. Cells were labelled with either palmitic acid or myristic acid and disrupted by sonication, followed by differential centrifugation of the cell lysates. SDS/polyacrylamide-gel electrophoresis of the resulting cell fractions indicated that most of the palmitate-labelled proteins were found in cell membranes, whereas most of the myristate-labelled proteins were found in the cytosol, although some were located in cell membranes. Experiments with lipid-labelled proteins extracted with the phase-separable detergent Triton X-114 showed that palmitate-labelled proteins behaved as hydrophobic proteins, partitioning into the lower phase of the detergent, whereas most of the myristate-labelled proteins remained in the upper phase. Although one of these cell lines expressed large amounts of epidermal-growth-factor receptor, this could not be labelled by either myristic acid or palmitic acid, whereas transferrin receptor was labelled by palmitic acid. The lipids normally attached to cell proteins in these two human squamous-carcinoma cell lines were characterized by labelling the cells with [3H]acetate. The labelled cell proteins were exhaustively extracted with organic solvents, and subjected to sequential alkaline and acid hydrolyses to release the attached lipids, which were then analysed by h.p.l.c. Most of the lipid released by the alkaline treatment chromatographed as palmitic acid or stearic acid, whereas the subsequent acid treatment released myristic acid as well as some palmitic acid and stearic acid. No other fatty acids apart from these were found attached to cell proteins.