Isoprenylated proteins in cultured cells: Subcellular distribution and changes related to altered morphology and growth arrest induced by mevalonate deprivation

Abstract

In the presence of lovastatin (mevinolin), an inhibitor of endogenous mevalonate synthesis, C1300 murine neuroblastoma cells incorporated (2‐¹⁴C)mevalonate into several discrete polypeptides that were separable by SDS‐PAGE. The electrophoretic pattern of the labeled proteins did not vary substantially when cells were homogenized with Ca⁺⁺, Mg⁺⁺, high concentrations of NaCl or phosphatase inhibitor, or when cells were lysed immediately in trichloroacetic acid. When cells that had been prelabeled with (¹⁴C)mevalonate were incubated with lovastatin and simultaneously deprived of exogenous mevalonate, there was a 50–60% decline in the concentration of protein‐bound isoprenoid label within 17 h. In contrast, there was little change in the radioactivity in the sterol, dolichol, or ubiquinone fractions. The time course of the decline in mevalonate‐derived label in cellular polypeptides paralleled the onset of neurite outgrowth and preceded the decline of DNA synthesis, suggesting that a decreased intracellular concentration of protein‐bound isoprenoid groups may contribute to the well‐documented effects of mevalonate deprivation on cell morphology and cell cycling. Fractionation of neuroblastoma cells by differential centrifugation and sucrose density‐gradient centrifugation revealed that mevalonate‐labeled proteins of 53 kDA, 22–26 kDa, and 17 kDa were concentrated in the cytosol. Proteins migrating at 45 kDa were found in both the soluble and particulate fractions, including those enriched in mitochondria and plasma membrane. The isoprenylated proteins migrating at approximately 66 kDa were localized exclusively in the nuclear fraction. When chromatin was removed from the nuclei by extraction with 2 M NaCl, the 66 kDa isoprenylated proteins remained associated with the residual components of the nuclear matrix and lamina. Isoprenylated proteins with electrophoretic mobilities similar to those observed in neuroblastoma cells were detected in a variety of established cell lines. However, there was considerable variation among cell lines in the overall efficiency of protein labeling with (¹⁴C)mevalonate and in the prominence and mobilities of specific labeled proteins in the 45–70 kDa range. Comparisons of paired transformed vs. nontransformed fibroblast cell lines suggested that the profile of mevalonate‐labeled proteins in a given cell line is not altered by malignant transformation. The finding that electrophoretically distinct isoprenylated proteins are localized in discrete subcellular compartments indicates that they do not represent dissociated subunits of a single multimeric protein complex, and provides a possible explanation for the diverse effects of mevalonate deprivation. The presence of these proteins in a wide variety of mammalian cells suggests that they play a fundamental role in cell structure and/or regulation.