Abstract
RAS2 protein of Saccharomyces cerevisiae undergoes post-translational modifications involving methyl esterification and palmitic acid addition, resulting in their association with the plasma membrane. In this paper, we provide evidence that two kinds of proteolytic events accompany the biosynthesis. This is shown by separating and characterizing three intracellular forms of RAS2 protein: precursor, intermediate, and mature (fatty acid-acylated) forms. N-Terminal sequencing has revealed that all three forms start with proline, which is the second amino acid expected from the RAS2 gene sequence. Thus, the first methionine is removed very early during the biosynthesis. Isolation and sequencing of C-terminal peptides indicate that three C-terminal amino acids present in the precursor form are removed in the intermediate and in the fatty acid acylated forms. C-Terminal proteolysis appears to accompany methyl esterification, since the methylation occurs with the intermediate and the fatty acid-acylated forms, but not with the precursor. Palmitic acid is identified as the major fatty acid attached to the fatty acid-acylated form.
Highlights
RASZ Protein of Succharomyces cereuisiae Undergoes Removal of Methionine at N Terminus and Removal of Three Amino Acids at C Terminus*
Using yeast RASl and RAS2 proteins as well as H-ras expressed in yeast, we have previously shown that the modification occurs in two distinct steps: 1) conversion of a precursor form to an intermediate form and 2) palmitic acid addition to the intermediate form producing a fatty acid-acylated form (Fujiyama and Tamanoi, 1986; Tamanoi et al, 1988)
Three Distinct Species of RAS2 Protein Can Be Identified during Its Biosynthesis-Based on pulse-chase with [35S]methionine and subcellular fractionation, we have previously proposed that RAS2 protein is synthesized first as a precursor which is converted to an intermediate form that migrates slightly faster than the precursor form on a SDS-polyacrylamide gel
Summary
Preparation of Labeled RAS2 Protein-To label RAS2 protein, yeast cells carrying plasmid YEp51-RAS2 which overproduce RASP protein in the presence of galactose (Fujiyama and Tamanoi, 1986). The 259-Sepharose fraction was mixed with trifluoroacetic acid After adding methanol and trifluoroacetic acid to 25% and l%, respectively, peptides were separated on a Cl8 or C4 HPLC column. Fatty acidacylated RAS2 protein isolated as above on a HPLC column was lyophilized, suspended in 8 M urea, and adjusted to 2 M urea in. Treatment with KOH was carried out by adding KOH to a final concentration of 0.1 M and incubating at 23 “C for 30 to 60 min. The extracted sample was dried in uacuo, dissolved in methanol containing authentic fatty acid markers, and injected into a C4 HPLC column. The fatty acids were eluted from the column with increasing concentrations of acetonitrile. RAS2 protein labeled with [ methyl-3H]methionine was purified by 259-Sepharose followed by HPLC as described above. Radioactive methanol from the methyl group of carboxyl methyl ester was assayed using liquid scintillation counter
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