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. 1990 Jan 19;247(4940):320-2.
doi: 10.1126/science.2296721.

Identification of geranylgeranyl-modified proteins in HeLa cells

C C Farnsworth  1 , M H Gelb, J A Glomset
Affiliations

Identification of geranylgeranyl-modified proteins in HeLa cells

C C Farnsworth et al. Science. .

Abstract

Previous studies have shown that animal cells contain isoprenoid-modified proteins and that one of these proteins, lamin B, contains a thioether-linked farnesyl group that is attached to cysteine. In the present study, a novel isoprenoid-modification was identified by labeling HeLa cells with [3H]mevalonic acid and analyzing proteolytic digests of the total cell protein. Radioactive fragments were purified from these digests and treated with Raney nickel. The released, labeled material was analyzed by gas-liquid chromatography (GC) and mass spectrometry (MS). This approach revealed that an all-trans geranylgeranyl group was a major isoprenoid modification.

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Figures

Fig. 1
Fig. 1
Size-exclusion chromatography of proteolytic hydrolysates of HeLa cell total proteins on Sephadex LH-20. Cells were labeled for 36 hours with [5-3H]mevalonic acid in the presence of 30 µM mevinolin, harvested, washed with phosphate-buffered saline, and extracted with lipid solvents. Cell pellets were then successively digested with proteases, and labeled digestion products were concentrated and purified by step elution from DEAE Sephacel. The eluted material was then passed through Sephadex LH-20 in 20% formic acid in ethanol at a flow rate of 0.25 nl/mmin, and 1.0-ml fractions were collected (4). Peak A, which corresponded to 1000-dalton material, contained 74% of the recovered label. Peak B, which corresponded to 500-dalton material, contained 22% of the recovered label. Recovery of the total applied label was 78%. Comparable chromatograms were obtained in six separate experiments.
Fig. 2
Fig. 2
Gas chromatographic (GC) analyses of labeled, Raney nickel-released material from proteolytic fragments of HeLa cell proteins. Tritium-labeled material corresponding to peak A (Fig. 1) was treated with Raney nickel and extracted with pentane as described in Table 1. (A) Pentane-soluble label was then concentrated, and one-half of it was analyzed. (B) The remainder was hydrogenated over platinum and analyzed. (C) A mixture of all eight possible cis-trans isomers of 2,6,10, 14-tetramethyl-2,6,10,14-hexadecatetraene, which yielded eight distinct peaks when analyzed by capillary GC, was cochromatographed with the all-trans isomer (major peak). (D) Authentic phytane was also analyzed. Analyses were performed on a 15-m DB-5 megabore column with a temperature program that started at 80°C for 2 min, then increased 2°C per minute up to 220°C. Peak mass was detected by flame ionization, and radio-activity was detected with a Flow-One Beta Detector (Radiomatic Instrument Co., Tampa, Florida) (4). In this system the all-trans and nearest cis-containing tetraene isomers were separated sufficiently (0.4 min) to ensure unambiguous identification of the protein-derived material as the all-trans tetraene. The all-trans tetraene was synthesized from all-trans geranylgeraniol by conversion to the bromide followed by treatment with superhydride (9). The isomeric mixture of tetraenes was synthesized by a Wittig reaction between commercially available famesyl acetone (mixture of all possible cis-trans isomers) and the ylide derived from (ethyl)triphenylphosphonium bromide (4). The structures of all of the synthetic compounds were verified by high-resolution proton nuclear magnetic resonance.
Fig. 3
Fig. 3
Electron ionization spectra of Raney nickel—released material from proteolytic fragments of HeLa cell proteins. Samples were prepared from 3 liters of cultured cells as described in Figs. 1 and 2, then analyzed by GC-MS on a 30-m DB-5 column, before or after hydrogenation (3). (A) Nonhydrogenated, peak A–derived material corresponding to the major peak of radioactivity in Fig. 2A. (B) Hydrogenated, peak A–derived material corresponding to the peak of radioactivity in Fig. 2B. (C) All-trans 2,6,10,14-tetramethyl-2,6,10,14-hexadecatriene. (D) Phytane. All spectra have been background corrected, and the spectra in (A) and (C) have been enhanced. Minor differences between spectra of protein-derived samples and standards are likely due to the much smaller amounts of naturally derived materials analyzed.
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