Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo.
BACKGROUND
Available in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation (for example, autoradiography with 3H-labeled anchor precursors) are time consuming (weeks/months), laborious and suffer from low sensitivity.


RESULTS
We describe a new technique for detecting prenyl anchors in N-terminally glutathione S-transferase (GST)-labeled constructs of target proteins expressed in vitro in rabbit reticulocyte lysate and incubated with 3H-labeled anchor precursors. Alternatively, hemagglutinin (HA)-labeled constructs expressed in vivo (in cell culture) can be used. For registration of the radioactive marker, we propose to use a thin layer chromatography (TLC) analyzer. As a control, the protein yield is tested by Western blotting with anti-GST- (or anti-HA-) antibodies on the same membrane that has been previously used for TLC-scanning. These protocols have been tested with Rap2A, v-Ki-Ras2 and RhoA (variant RhoA63L) including the necessary controls. We show directly that RasD2 is a farnesylation target.


CONCLUSIONS
Savings in time for experimentation and the higher sensitivity for detecting 3H-labeled lipid anchors recommend the TLC-scanning method with purified GST- (or HA-) tagged target proteins as the method of choice for analyzing their prenylation capabilities in vitro and in vivo and, possibly, also for studying the myristoyl and palmitoyl posttranslational modifications.
Benetka
Wolfgang
W
Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, A-1030 Vienna, Austria. benetka@imp.univie.ac.at
Koranda
Manfred
M
Maurer-Stroh
Sebastian
S
Pittner
Fritz
F
Eisenhaber
Frank
F
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051379:Mice; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011817:Rabbits; D012680:Sensitivity and Specificity
D011506:Proteins; D019204:GTP-Binding Proteins; C488189:Rasd2 protein, mouse; C442475:Rasd2 protein, rat