The protein synthesis elongation factor EF-la of Mucor racemosus hyphae contained eight or nine methylated amino acids per molecule, whereas the factor from sporangiospores was nonmethylated. During the course of spore germination, the specific activity of the factor in crude extracts increased sixfold. This increase in activity was accompanied by a constant level of EF-let-specific mRNA and a constant level of EF-loa protein.Methylation of the protein, however, accelerated during the germination process, in parallel with the increase in specific activity of the factor. We propose that the activity of EF-loa is regulated during germination through methylation of the protein and does not involve transcriptional regulation.Mucor racemosus is a dimorphic fungus that grows as a budding yeast in CO2 and as a mycelium in air. These alternative forms of growth, as well as the process of spore germination, are accompanied by differences in the translational system. Among these are differences in the rate of peptide bond formation (18,19), the extent of phosphorylation of ribosomal protein S-6 (11, 12), and the methylation of the elongation factor EF-la (9). In the latter case, we have found that of the 44 lysine residues, 8 or 9 are methylated when the organism grows in the mycelial form (9). The extent of methylation is less in the yeast form, and spores contain an EF-la that is essentially nonmethylated.In an effort to understand the regulation of EF-la synthesis and activity and the role EF-la may play in changes in the rate of peptide bond formation, we studied the acquisition of EF-la activity during spore germination. These studies were aided by obtaining a cDNA clone which made it possible to measure mRNA levels. We show here that EF-la protein and mRNA are made at constant levels during a period that EF-la activity is rising dramatically. Our conclusion is that activity of EF-la in M. racemosus is regulated through the methylation of the protein after synthesis.
MATERIALS AND METHODSOrganisms and culture conditions. M. racemosus (M. lusitanicus) ATCC 1216B was used in all experiments. Sporangiospores were prepared as described previously (20). Spores (2 x 105/ml) were germinated in YPG medium (2% glucose, 1% Bacto-Peptone, 0.3% yeast extract [pH 5.5] idodiphosphate was used in place of GTP, the reaction was stopped with 50 ,ul of 4% glutaraldehyde to stabilize the EF-1 complex bound to the ribosome. Polyuridylate-directed phenylalanine synthesis was conducted as described by Merrick (16), except that the buffer contained 50 mnM KCl and 0.8 mM spermidine.[3H]Phe-tRNA was prepared from yeast tRNA Phe (Boehringer Mannheim Biochemicals) and L-[2,6-3H]Phe (Amersham Corp.) (50 Ci/mmol) with Escherichia coli crude aminoacyl-tRNA synthetases (16). High-salt washed ribosomes were prepared as described by Larsen and Sypherd (11), except that NH4Cl was substituted for KCI. Elongation factor EF-la was prepared as previously described (9), and EF-2 was prepared from wheat germ as described by Legocki (13). Cell extracts for ...