1. A group of 21 to 24-kDa proteins of pheochromocytoma (PC-12) cells was found in blot overlay assays to bind specifically [α-32P]GTP. Binding was inhibited by GTP analogues but not by ATP. Such small GTP-binding proteins were found in the cytosolic and in the particulate fraction of the cells, but they were unevenly distributed: about 75% of the small GTP-binding proteins were localized within the particulate fraction of the cells. Separation of these proteins by two-dimensional gel electrophoresis revealed the existence of seven distinct [α-32P]GTP-binding proteins. 2. Targeting of the small GTP-binding proteins to the particulate fraction of PC-12 cells requires modification by isoprenoids, since depleting the cells of the isoprenoid precursor mevalonic acid (MVA) by the use of lovastatin resulted in a 50% decrease in membrane-bound small GTP-binding proteins, with a proportionate increase in the cytosolic form. This blocking effect of lovastatin was reversed by exogenously added MVA. 3. In addition, metabolic labeling of PC-12 cells with [3H]MVA revealed incorporation of [3H]MVA metabolites into the cluster of 21 to 24-kDa proteins in a form typical of isoprenoids; the label was not removed from the proteins by hydroxylamine, and labeling was enhanced in cells incubated with lovastatin. The latter effect reflects a decrease in the isotopic dilution of the exogenously added [3H]MVA, as the addition of exogenous MVA reversed the effect of lovastatin on [3H]MVA-metabolite incorporation into the 21 to 24-kDa proteins. 4. Additional experiments demonstrated that isoprenylation is required not only for membrane association of small GTP-binding proteins, but also for their further modification by a methylation enzyme. This was evident in experiments in which the cells were metabolically labeled with [methyl-3H]methionine, a methylation precursor. The group of 21 to 24-kDa proteins was labeled with a methyl-3H group in a form typical of C-terminal-cysteinyl carboxylmethyl esters. Their methylation was blocked by the methylation inhibitors methylthioadenosine (MTA), 3-deazadenosine and homocysteine thiolactone as well as by lovastatin. MVA reversed the lovastatin block of methylation. 5. Two-dimensional gel analysis of the [3H]methylated proteins detected seven methylated small GTP-binding proteins that correspond to the isoprenylated proteins. Levels of the small GTP-binding proteins as well as isoprenylation and methylation were reduced by cycloheximide. 6. Distribution of the methylated proteins between particulate and cytosolic fractions was found to be similar to that of the small GTP-binding proteins (i.e., a 4:1 ratio). Membrane association of these proteins does not require methylation, as it was not blocked by MTA. Isoprenylated 21 to 24-kDa proteins were found to be equally distributed between particulate and cytosol, even though most of the methylated small GTP-binding proteins are membrane bound and their membrane binding depends on isoprenoid modification. 7. The data are consistent with a scheme of events in which small GTP-binding proteins of PC-12 cells are isoprenylated shortly after translation and this modification is a prerequisite for their membrane association and methylation. The excess of isoprenylated 21 to 24-kDa proteins in the cytosol may reflect proteins that were localized in the membranes and underwent demethylation and dissociation from the membranes. 8. Our results suggest that the known requirements of isoprenoids for normal cell cycling is related, inter alia, to a requirement for isoprenylation of small GTP-binding proteins.
- G proteins
- protein carboxylmethylation