Cytosolic proteins of 21-23 kDa are methylated by kidney cortex membrane-associated C-terminal carboxyl methyltransferases.
We have studied the effect of a soluble fraction from kidney cortex on the C-terminal carboxyl methylation of 21-23 kDa proteins catalyzed by membrane-associated methyltransferases. Addition of soluble proteins to isolated luminal, antiluminal and intracellular membranes resulted in a large increase in the methylation of the membrane-associated 21-23 kDa substrates. Fractionation of the soluble extract from the cortex by Q-Sepharose anion exchange chromatography showed the presence of two distinct peaks of proteins presenting stimulating activities, eluting at 0.15 M (peak I) and 0.4 M (peak II) NaCl, respectively. Both peaks eluted as proteins of apparent molecular sizes of 40 kDa upon Superose 6 gel-filtration chromatography. No methylation activity towards N-acetyl-S-trans,trans-farnesylcysteine (AFC), a good substrate for C-terminal carboxyl methyltransferases, was associated with either peaks. In contrast, the increase in methylation induced by these proteins was strongly inhibited by AFC, suggesting that the methylation induced by these factors occurred on C-terminal isoprenylated cysteine residues. Both partially purified proteins competitively inhibited the methylation of AFC by the membrane-associated enzymes, suggesting that they may represent substrates for the methyltransferases. Immunoblotting of these partially purified soluble substrates with a rabbit polyclonal antibody directed against the small G-protein CDC42 showed the presence of this protein in peak I but not in peak II. Taken together, these results suggest the presence in a soluble fraction from the kidney of distinct methyl-accepting proteins, one of these being tentatively identified as the small G-protein CDC42.
Gingras
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Laboratoire de Membranologie, Université du Québec à Montréal, Canada.
Béliveau
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D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology*; D003600:Cytosol / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000032:analysis; D007672:Kidney Cortex / Q000201:enzymology*; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry*; D051381:Rats
D011506:Proteins; D008780:Methyltransferases; D019204:GTP-Binding Proteins