Cells incorporate
isoprenoid products derived from
mevalonate (MVA) into several unique
proteins. The aim of this study was to delineate the effects of blocking MVA synthesis on the covalent isoprenylation of these
proteins in murine
erythroleukemia cells. Inhibition of
protein synthesis with
cycloheximide prevented the incorporation of [3H]MVA into
proteins, suggesting that isoprenylation normally occurs immediately after synthesis of the
polypeptides. However, incubation of cells with
lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl
coenzyme A (
HMG-CoA) reductase, for as little as 1 h prior to addition of
cycloheximide rendered the isoprenylation step insensitive to
cycloheximide.
Lovastatin had no apparent effect on the stability of the isoprenylated
proteins, but the development of
cycloheximide insensitivity during the
lovastatin preincubation was dependent on synthesis of new
protein during that period. Addition of 50-200 microM MVA to the culture medium eliminated the effects of preincubation with
lovastatin. Preincubation of cells with
25-hydroxycholesterol, which suppresses the synthesis and enhances the degradation of
HMG-CoA reductase but is not a competitive
enzyme inhibitor, did not induce
cycloheximide-insensitivity of the isoprenylation reaction. The results suggest that blocking MVA synthesis with
lovastatin causes a rapid depletion of
isoprenoid groups available for
protein modification. Consequently, there is an accumulation of non-isoprenylated substrate
proteins. Shifts in the ratio of modified vs. unmodified
proteins in response to MVA availability may have implications for the changes in cell morphology, cell proliferation and
HMG-CoA reductase gene expression that occur when cells are subjected to MVA deprivation.