The TIS21 immediate-early gene and leukemia-associated BTG1 gene encode proteins with similar sequences. Two-hybrid analysis identified a protein that interacts with TIS21 and BTG1. Sequence motifs associated with S-adenosyl-L-methionine binding suggested this protein might have methyltransferase activity. A glutathione Stransferase (GST) fusion of the putative methyltransferase modifies arginine residues, in appropriate protein substrates, to form N G -monomethyl and N G ,N Gdimethylarginine (asymmetric). We term the proteinarginine N-methyltransferase (EC 2.1.1.23) gene "PRMT1," for protein-arginine methyltransferase 1. GST-TIS21 and GST-BTG1 fusion proteins qualitatively and quantitatively modulate endogenous PRMT1 activity, using control and hypomethylated RAT1 cell extracts as methyl-accepting substrates. PRMT1 message appears ubiquitous, and is constitutive in mitogen-stimulated cells. Modulation of PRMT1 activity by transiently expressed regulatory subunits may be an additional mode of signal transduction following ligand stimulation.The protein products of the immediate-early/primary response genes are thought to act as "third messengers," mediating phenotypic alterations in cells in response to ligands such as growth factors, hormones, neurotransmitters, cytokines, and neurotrophins. Many immediate-early genes encode transcription factors (e.g. Fos, Jun, Egr-1) that initiate transcriptional cascades required for proliferation or differentiation . Other ligand-induced immediate-early genes encode paracrine mediators of cellular communication whose products (e.g. prostaglandin synthase-2, inducible nitricoxide synthase, and cytokines such as MCP-1) modulate the behavior of neighboring cells (Smith and Herschman, 1995).Because immediate-early/primary response genes have been cloned on the basis of their induction characteristics, rather than the functions of their protein products, a number of these genes encode proteins whose biological roles have not yet been determined. One such immediate-early gene is TIS21. The TIS21 cDNA was cloned by differential screening, both from a cDNA library prepared from mitogen-treated, quiescent murine Swiss 3T3 cells (Fletcher et al., 1991) and from a cDNA library prepared from nerve growth factor-treated rat PC12 pheochromocytoma cells (Bradbury et al., 1991). The predicted rat and mouse TIS21 proteins differ at only four out of 158 amino acid residues. We demonstrated, by metabolic labeling followed by immunoprecipitation, that maximal TIS21 protein synthesis occurs within the first hour after exposure to ligand, both in mitogen-stimulated Swiss 3T3 cells and in nerve growth factor-stimulated PC12 cells (Varnum et al., 1994). Moreover, the half-life of both mitogen-and nerve growth factor-induced TIS21 protein is less than 15 min (Varnum et al., 1994). Despite substantial investigation into both the structure of the TIS21 gene and the induced expression of the TIS21 message and protein, no function has been identified for this protein.The human BTG1 gene was cloned and ...
We have identified the major enzymatic activity responsible for the S-adenosyl-L-methionine-dependent methylation of arginine residues (EC 2.1. Evidence for the posttranslational methylation of arginine residues in proteins was first provided by the presence of radioactive species chromatographing at positions near that of arginine in acid hydrolysates of isolated calf thymus nuclei incubated with S-adenosyl-L- [methyl-14
GTPases are widespread in directing cytoskeletal rearrangements and affecting cellular organization. How they do so is not well understood. Yeast cells divide by budding, which occurs in two spatially programmed patterns, axial or bipolar [1-3]. Cytoskeletal polarization to form a bud is governed by the Ras-like GTPase, Bud1/Rsr1, in response to cortical landmarks. Bud1 is uniformly distributed on the plasma membrane, so presumably its regulators, Bud5 GTPase exchange factor and Bud2 GTPase activating protein, impart spatial specificity to Bud1 action [4]. We examined the localizations of Bud5 and Bud2. Both Bud1 regulators associate with cortical landmarks designating former division sites. In haploids, Bud5 forms double rings that encircle the mother-bud neck and split upon cytokinesis so that each progeny cell inherits Bud5 at the axial division remnant. Recruitment of Bud5 into these structures depends on known axial landmark components. In cells undergoing bipolar budding, Bud5 associates with multiple sites, in response to the bipolar landmarks. Like Bud5, Bud2 associates with the axial division remnant, but rather than being inherited, Bud2 transiently associates with the remnant in late G1, before condensing into a patch at the incipient bud site. The relative timing of Bud5 and Bud2 localizations suggests that both regulators contribute to the spatially specific control of Bud1 GTPase.
Protein methylation reactions can play important roles in cell physiology. After labeling intact Saccharomyces cerevisiae cells with S-adenosyl-L-[methyl-3 H]methionine, we identified a major methylated 49-kDa polypeptide containing [ 3 H]methyl groups in two distinct types of linkages. Peptide sequence analysis of the purified methylated protein revealed that it is eukaryotic elongation factor 1A (eEF1A, formerly EF-1␣), the protein that forms a complex with GTP and aminoacyltRNAs for binding to the ribosomal A site during protein translation. Previous studies have shown that eEF1A is methylated on several internal lysine residues to give mono-, di-, and tri-N-⑀-methyl-lysine derivatives. We confirm this finding but also detect methylation that is released as volatile methyl groups after base hydrolysis, characteristic of ester linkages. In cycloheximidetreated cells, methyl esterified eEF1A was detected largely in the ribosome and polysome fractions; little or no methylated protein was found in the soluble fraction. Because the base-labile, volatile [methyl-3 H]radioactivity of eEF1A could be released by trypsin treatment but not by carboxypeptidase Y or chymotrypsin treatment, we suggest that the methyl ester is present on the ␣-carboxyl group of its C-terminal lysine residue. From the results of pulse-chase experiments using radiolabeled intact yeast cells, we find that the N-methylated lysine residues of eEF1A are stable over 4 h, whereas the eEF1A carboxyl methyl ester has a half-life of less than 10 min. The rapid turnover of the methyl ester suggests that the methylation/demethylation of eEF1A at the Cterminal carboxyl group may represent a novel mode of regulation of the activity of this protein in yeast.Reversible covalent modification of proteins is a common mode of regulation in cell metabolism (1-3). A large number of protein phosphorylation and dephosphorylation reactions are involved in a variety of cell signaling and metabolic control reactions where specific kinases phosphorylate proteins using the ␥-phosphate group of ATP and dephosphorylation of these proteins occurs through the action of phosphatases (4). In a much smaller number of cases, methylation and demethylation reactions are also involved in cell signaling and, potentially, metabolic regulation (5). Methyltransferases use the methyl donor group on S-adenosylmethionine (AdoMet) 1 to methylate various substrates; methylesterases act to demethylate them.The yeast Saccharomyces cerevisiae contains the STE14 isoprenylcysteine methyltransferase that has been shown to methylate a wide range of proteins within the cell (6, 7). Its known substrates include the small G-proteins RAS1 and RAS2 and the secreted peptide a-factor (8 -10). The specific role of this methyltransferase is unknown, but it has been suggested that methylation of its products increases their hydrophobicity to help direct them to the membrane, decreases their rate of proteolytic degradation, and may modulate their protein-protein interactions involved in cell signaling (6).I...
Genomic studies in yeast have revealed that one eighth of genes are cell cycle regulated in their expression. Almost without exception, the significance of cell cycle periodic gene expression has not been tested. Given that many such genes are critical to cellular morphogenesis, we wanted to examine the importance of periodic gene expression to this process. The expression profiles of two genes required for the axial pattern of cell division, BUD3 and BUD10/AXL2/SRO4, are strongly cell cycle regulated. BUD3 is expressed close to the onset of mitosis. BUD10 is expressed in late G1. Through promotor-swap experiments, the expression profile of each gene was altered and the consequences examined. We found that an S/G2 pulse of BUD3 expression controls the timing of Bud3p localization, but that this timing is not critical to Bud3p function. In contrast, a G1 pulse of BUD10 expression plays a direct role in Bud10p localization and function. Bud10p, a membrane protein, relies on the polarized secretory machinery specific to G1 to be delivered to its proper location. Such a secretion-based targeting mechanism for membrane proteins provides cells with flexibility in remodeling their architecture or evolving new forms.
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