Identification of isoprenyl modified proteins metabolically labeled with [3H]farnesyl- and [3H]geranylgeranyl-pyrophosphate.
Here we describe a direct approach for two-dimensional (2-D) gel mapping of proteins that are modified by post-translational isoprenylation in mammalian cells. Briefly, transformed human amnion cells (AMA) and transfected COS-1 cells were metabolically labeled with either [3H]farnesyl-pyrophosphate or [3H]geranylgeranyl-pyrophosphate following treatment with lovastatin, which blocks the synthesis of mevalonic acid. The proteins were then separated by 2-D gel electrophoresis and electrotransferred to nitrocellulose filters. The membranes were immersed in dimethyl ether, containing 10% of 2,5-diphenyloxazole prior to fluorography. Over 40 [3H]farnesyl-labeled proteins and over 25 [3H]geranylgeranylated proteins were identified on the 2-D autoradiograms. Several [3H]farnesyl-labeled proteins exhibited the same coordinates (M(r) and pI) as their [3H]geranylgeranylated counterparts, raising the possibility that they may be substrates for both farnesyl and geranylgeranyl transferase(s). The approach offers high resolution of both farnesylated and geranylgeranylated proteins and it may serve as a powerful tool for the identification of hitherto unknown prenylated proteins as well as for the determination of prenylated protein levels, type of isoprenoid modification, and possible changes in protein prenyltransferase activity.
Gromov
P S
PS
Department of Medical Biochemistry, Aarhus University, Denmark. gromov@biobase.dk
Madsen
P
P
Celis
J E
JE
D000650:Amnion / Q000166:cytology; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D004122:Dimethylallyltranstransferase / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D008148:Lovastatin / Q000494:pharmacology; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D012717:Sesquiterpenes; D014316:Tritium
D004791:Enzyme Inhibitors; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D014316:Tritium; C004808:farnesyl pyrophosphate; D008148:Lovastatin; D004122:Dimethylallyltranstransferase; C002963:geranylgeranyl pyrophosphate