Isopentenoid synthesis in embryonic Drosophila cells: prenylated protein profile and prenyl group usage.
It has been established that vertebrates and yeasts modified a unique subset of polypeptides with farnesyl and geranylgeranyl residues. This observation has been extended to Drosophila Kc cells. [3H]Mevalonate was incorporated into 54 Kc cell peptides (18-92 kDa). As reported for mammalian cells, most of the labeled peptides had molecular weights between 21 and 27 kDa. C18 radio-HPLC tryptic digest profiles for delipidized, [3H]mevalonate-labeled (a) insect (Drosophila and Spodoptera frugiperda) and mammalian (Chinese hamster ovary met 18-2b) cells, (b) Kc cell nuclear lamin, and (c) a 23.5-kDa purified Kc cell GTP-binding protein were compared and analyzed. [35S]Cysteine-labeled Kc cells yielded a tryptic digest radio-HPLC profile which was congruent with that for [3H]mevalonate-labeled cells. A significant fraction (30-33%) of the doubly labeled tryptic peptides were eluted with greater than or equal to 93% acetonitrile. Kc cell nuclear lamin tryptic digests yielded a single 3H-labeled product which migrated as S-farnesylcysteine. The Kc cell 23.5-kDa GTP-binding protein's 3H-labeled oligopeptide(s)/amino acid(s) was geranylgeranylated and its tryptic digest profile was representative of prenylated proteins whose oligopeptides eluted with greater than or equal to 93% acetonitrile. Moreover, the 3H-labeled oligopeptide/amino acid profiles plus prenyl group patterns for [3H]mevalonate-labeled Kc and mammalian cell total extracts were similar. Collectively, these observations supported a prenylated protein spectrum and prenyl group usage as highly conserved eukaryotic cellular characteristics.
Havel
C M
CM
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Fisher
P
P
Watson
J A
JA
D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D004224:Diterpenes / Q000378:metabolism*; D004330:Drosophila / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D006868:Hydrolysis; D008798:Mevalonic Acid / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*
D004224:Diterpenes; D011506:Proteins; C065977:geranylgeranylcysteine; C057785:S-farnesylcysteine; D003545:Cysteine; D008798:Mevalonic Acid