Open Access Highly Accessed Methodology article

Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo

Wolfgang Benetka1, Manfred Koranda1, Sebastian Maurer-Stroh12, Fritz Pittner3 and Frank Eisenhaber1*

Author affiliations

1 Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, A-1030 Vienna, Austria

2 VIB – SWITCH lab, Pleinlaan 2, B-1050 Brussels, Belgium

3 University Vienna, Department of Biochemistry, Dr.-Bohr-Gasse 9, A-1030 Vienna, Austria

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Citation and License

BMC Biochemistry 2006, 7:6  doi:10.1186/1471-2091-7-6

Published: 28 February 2006

Abstract

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.

Conclusion

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.