Protein methylation in cerebellar synaptosomes.
Synaptosomes from five regions of adult rat brain were isolated, analyzed for methyl acceptor proteins, and probed for methyltransferases by photoaffinity labeling. Methylated proteins of 17 and 35 kDa were observed in all regions, but cerebellar synaptosomes were enriched in a 21-26-kDa family of methyl acceptor proteins and contained a unique major methylated protein of 52 kDa and a protein of 50 kDa, which was methylated only in the presence of EGTA. When cerebellar and liver subcellular fractions were compared, the cytosolic fractions of each tissue contained methylated proteins of 17 and 35 kDa; liver membrane fractions contained few methylated proteins, whereas cerebellar microsomes had robust methylation of the 21-26-kDa group. Differential centrifugation of lysed cerebellar synaptosomes localized the 17- and 35-kDa methyl acceptor proteins to the synaptoplasm, the 21-26-kDa family to the synaptic membranes, and the 52-kDa to synaptic vesicles. The 21-26-kDa family was identified as GTP-binding proteins by [alpha-32P]GTP overlay assay; these proteins contained a putative methylated carboxyl cysteine, based on the presence of volatile methyl esters and the inhibition of methylation by acetylfarnesylcysteine. The 52-kDa methylated protein also contained volatile methyl esters, but did not bind [alpha-32P]GTP. When synaptosomes were screened for putative methyltransferases by S-adenosyl-L-[methyl-3H]methionine photoaffinity labeling, a protein of 24 kDa was detected only in cerebellum, and this labeled protein was localized to synaptic membranes.
Wright
L S
LS
Department of Pediatrics, University of Wisconsin Medical School, Madison 53705.
Siegel
F L
FL
D000345:Affinity Labels; D000818:Animals; D002531:Cerebellum / Q000378:metabolism* / Q000648:ultrastructure; D004533:Egtazic Acid / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D008745:Methylation; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010777:Photochemistry; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D013570:Synaptic Membranes / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism*
D000345:Affinity Labels; D009419:Nerve Tissue Proteins; D004533:Egtazic Acid; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins