Identification of a precursor in the biosynthesis of the p21 transforming protein of harvey murine sarcoma virus.
The p21 transforming protein coded for by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV) migrates as a doublet band between 21,000 and 23,000 daltons during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lower band of the doublet is designated p21, and the upper band is designated pp21 since it comigrates with the phosphorylated form of p21. By pulse-labeling with [35S] methionine, we detected a p21 precursor, pro-p21, which migrated as if it was approximately 1,000 daltons larger than p21. The precursor-product relationship was established by pulse-chase experiments with [25S] methionine in the presence of 100 micrograms of cycloheximide per ml, which inhibited all de novo protein biosynthesis. Within 4 h, pro-p21 was completely chased into p21, and during the next 24 h pp21 accumulated. Thus, formation of pp21 from p21 did not require de novo protein synthesis. By subcellular fractionation into cytosol amd membrane fractions, we found that pro-p21 was synthesized in a non-membrane-bound state and that shortly after its complete synthesis, the p21 product was associated with the membrane fraction. By selective cleavage of p21 at a unique aspartic acid-proline residue with 70% formic acid or with Staphylococcus aureus V8 protease, we found that the intramolecular site of pro-p21 processing was located in the C-terminal portion of the pro-p21 molecule. The possibilities that the precursor was involved in the assembly of p21 into the plasma membrane and, alternatively, that the processing was a step in the activation of p21 biochemical activities are discussed.
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002472:Cell Transformation, Viral; D003600:Cytosol / Q000032:analysis; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000032:analysis; D051379:Mice; D015689:Oncogene Protein p21(ras); D011498:Protein Precursors / Q000302:isolation & purification* / Q000378:metabolism; D009053:Sarcoma Viruses, Murine / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis*
D011498:Protein Precursors; D014764:Viral Proteins; D015689:Oncogene Protein p21(ras)