Isoprenoid modification of G25K (Gp), a low molecular mass GTP-binding protein distinct from p21ras.
Cultured murine erythroleukemia (MEL) cells synthesize a number of low molecular mass GTP-binding proteins that undergo post-translational modification by isoprenoids. We used two-dimensional electrophoresis and immunoblotting to show that a 23-24-kDa protein labeled by the isoprenoid precursor [3H]mevalonate was specifically recognized by an antibody to G25K (Gp), a low molecular mass GTP-binding protein originally purified from placental, platelet, and brain membranes. Several isoelectric variants of G25K were detected in MEL cells, and all were radiolabeled with [3H]mevalonte. The G25K-immunoreactive protein did not cross-react with pan-ras antibody. Although mature p21ras is known to be localized in the cell membrane, most of the isoprenylated G25K was found in the 100,000 x g supernatant fraction when cells were lysed in buffer without detergent. Blocking isoprenoid synthesis by incubation of MEL cells with lovastatin resulted in a decrease in the concentration of G25K in the particulate fraction and a corresponding increase in immunodetectable protein in the soluble fraction. Lovastatin treatment also produced shifts in the electrophoretic mobilities of the G25K isoforms on two-dimensional gels. These observations are consistent with the idea that isoprenylation plays a permissive role in the association of G25K with the cell membrane or other organelles. However, the high proportion of soluble isoprenylated G25K in MEL cells under normal culture conditions suggests that the role of the isoprenoid modification may be more complex than simply serving as a structural anchor for stable insertion of proteins into the lipid bilayer.
Maltese
W A
WA
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
Sheridan
K M
KM
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute; D007942:Leukemia, Experimental; D008148:Lovastatin / Q000494:pharmacology; D008798:Mevalonic Acid / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D016283:Proto-Oncogene Proteins p21(ras) / Q000302:isolation & purification
D008148:Lovastatin; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D008798:Mevalonic Acid