Changes in Protein Isoprenylation during the Growth of Suspension-Cultured Tobacco Cells.

Isoprenylation facilitates the association of proteins with intracellular membranes and/or other proteins. In mammalian and yeast cells, isoprenylated proteins are involved in signal transduction, cell division, organization of the cytoskeleton, and vesicular transport. Recently, protein isoprenylation has been demonstrated in higher plants, but little is currently known about the functions of isoprenylated plant proteins. We report that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (lovastatin) or prenyl:protein transferases (perilly alcohol) severely impair the growth of cultured tobacco (Nicotiana tabacum) cells but only when added within the first 2 d following transfer to fresh medium, before any increase in culture volume is detectable. This "window" of sensitivity to inhibitors of protein isoprenylation correlates temporally with an increase in [14C]mevalonate incorporation into tobacco cell proteins in vitro. We have also observed a marked increase in farnesyl:protein transferase activity at this early time in the growth of tobacco cultures. In contrast, type I geranylgeranyl:protein transferase activity does not change significantly during culture growth. Although these events coincide with the replication of DNA, I [mu]M lovastatin-treated cells are capable of DNA synthesis, suggesting that lovastatin-induced cell growth arrest is not due to inhibition of DNA replication. Together, these data support the hypothesis that protein isoprenylation is necessary for the early stages of growth of tobacco cultures.

Novel isoprenylated proteins identified by an expression library screen.

Isoprenylated proteins are involved in eukaryotic cell growth and signal transduction. The protein determinant for prenylation is a short carboxyl-terminal motif containing a cysteine, to which the isoprenoid is covalently attached via thioether linkage. To date, isoprenylated proteins have almost all been identified by demonstrating the attachment of an isoprenoid to previously known proteins. Thus, many isoprenylated proteins probably remain undiscovered. To identify novel isoprenylated proteins for subsequent biochemical study, colony blots of a Glycine max cDNA expression library were [3H]farnesyl-labeled in vitro. Proteins identified by this screen contained several different carboxyl termini that conform to consensus farnesylation motifs. These proteins included known farnesylated proteins (DnaJ homologs) and several novel proteins, two of which contained six or more tandem repeats of a hexapeptide having the consensus sequence (E/G)(G/P)EK(P/K)K. Thus, plants contain a diverse array of genes encoding farnesylated proteins, and our results indicate that fundamental differences in the identities of farnesylated proteins may exist between plants and other eukaryotes. Expression library screening by direct labeling can be adapted to identify isoprenylated proteins from other organisms, as well as proteins with other post-translational modifications.