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N A Woychik

Publications and source records attributed to N A Woychik.

12 recordsLinked to original sources
PubMed1992

Genes encoding transcription factor IIIA and the RNA polymerase common subunit RPB6 are divergently transcribed in Saccharomyces cerevisiae.

The gene encoding Saccharomyces cerevisiae transcription factor TFIIIA has been found adjacent to RPB6, a gene that specifies a subunit shared by nuclear RNA polymerases. Analysis of DNA upstream of the RPB6 gene revealed an open reading frame that predicts a protein, designated PZF1, with nine C2H2 zinc fingers. The presence of nine C2H2 zinc fingers in PZF1 protein, a hallmark of amphibian TFIIIA proteins, suggested that PZF1 might be a TFIIIA homologue. We found that purified recombinant PZF1 specifically binds the internal control region (ICR) of the 5S rRNA gene in S. cerevisiae. The presence of nine C2H2 zinc fingers, the specific binding to ICR DNA, and the similarity of the predicted molecular mass of PZF1 with that determined for purified yeast TFIIIA, together indicate that PZF1 is TFIIIA. The yeast and amphibian TFIIIA proteins share only a limited number of residues outside of those normally conserved in C2H2 zinc fingers; these conserved residues may provide clues to the sequence specificity of these proteins. The PZF1 gene was found to be single copy, transcribed into a 1.5-kilobase mRNA, and essential for yeast cell viability. Interestingly, the yeast RPB6 and TFIIIA coding sequences are divergently transcribed and are separated by only 233 base pairs, providing the potential for coregulated expression of components of RNA polymerases and the 5S rRNA component of ribosomes.

Amino Acid Sequence↗
PubMed1991

Yeast RNA polymerase II subunit RPB9 is essential for growth at temperature extremes.

The Saccharomyces cerevisiae RNA polymerase II subunit gene RPB9 was isolated and sequenced. RPB9 is a single copy gene on chromosome VII. The RPB9 sequence predicts a protein of 122 amino acids with a molecular mass of 14,200 Da. The yeast RPB9 subunit is similar in size and sequence to a protein encoded by DNA adjacent to the suppressor of the Hairy Wing gene in Drosophila melanogaster. Deletion of the RPB9 gene produced cells that were heat- and cold-sensitive. The RPB9 subunit, like the previously described RNA polymerase II subunit RPB4, is not essential for synthesis of mRNA, but is required for normal cell growth over a wide temperature range.

Amino Acid Sequence↗
PubMed1990

RNA polymerase II subunit RPB10 is essential for yeast cell viability.

The Saccharomyces cerevisiae gene encoding the smallest RNA polymerase II subunit, RPB10, was isolated and sequenced. The gene for this subunit is present in single copy and maps to chromosome XV, where two other yeast RNA polymerase II subunits, RPB2 and RPB8, reside. The RPB10 sequence predicts a protein only 46 amino acids in length with a molecular mass of 5400 daltons. Sporulation and tetrad analysis of diploid cells containing one copy of the RPB10 gene and one copy of HIS3 in place of the RPB10 gene revealed that the RPB10 subunit is essential for viability.

Amino Acid Sequence↗
PubMed1990

RNA polymerase II: subunit structure and function.

RNA polymerase II is the core of the complex apparatus that is responsible for the regulated synthesis of mRNA. A comprehensive knowledge of RNA polymerase II is essential to our understanding of the molecular mechanisms through which a variety of transcription factors regulate eukaryotic gene expression. The recent cloning of genes for all ten subunits of yeast RNA polymerase II has revealed intriguing similarities and differences between the eukaryotic RNA polymerase and its simpler prokaryotic counterpart. Epitope tagging and other experiments made possible by the cloning of these genes have provided a clearer picture of RNA polymerase II subunit composition, stoichiometry and function, and set the stage for further investigating the dialogue between RNA polymerase II and transcription factors.

Amino Acid Sequence↗
PubMed1990

Subunits shared by eukaryotic nuclear RNA polymerases.

RNA polymerases I, II, and III share three subunits that are immunologically and biochemically indistinguishable. The Saccharomyces cerevisiae genes that encode these subunits (RPB5, RPB6, and RPB8) were isolated and sequenced, and their transcriptional start sites were deduced. RPB5 encodes a 25-kD protein, RPB6, an 18-kD protein, and RPB8, a 16-kD protein. These genes are single copy, reside on different chromosomes, and are essential for viability. The fact that the genes are single copy, corroborates previous evidence suggesting that each of the common subunits is identical in RNA polymerases I, II, and III. Furthermore, immunoprecipitation of RPB6 coprecipitates proteins whose sizes are consistent with RNA polymerase I, II, and III subunits. Sequence similarity between the yeast RPB5 protein and a previously characterized human RNA polymerase subunit demonstrates that the common subunits of the nuclear RNA polymerases are well conserved among eukaryotes. The presence of these conserved and essential subunits in all three nuclear RNA polymerases and the absence of recognizable sequence motifs for DNA and nucleoside triphosphate-binding indicate that the common subunits do not have a catalytic role but are important for a function shared by the RNA polymerases such as transcriptional efficiency, nuclear localization, enzyme stability, or coordinate regulation of rRNA, mRNA, and tRNA synthesis.

Amino Acid Sequence↗
PubMed1989

RNA polymerase II subunit RPB4 is essential for high- and low-temperature yeast cell growth.

RPB4 encodes the fourth-largest RNA polymerase II subunit in Saccharomyces cerevisiae. The RPB4 gene was cloned and sequenced, and its identity was confirmed by amino acid sequence analysis of tryptic peptides from the purified subunit. The RPB4 DNA sequence predicted a protein of 221 amino acids with a molecular mass of 25,414 daltons. The central 100 amino acids of the RPB4 protein were found to be similar to a segment of the major sigma subunit in Escherichia coli RNA polymerase. Deletion of RPB4 produced cells that were heat and cold sensitive but could grow, albeit slowly, at intermediate temperatures. RNA polymerase II lacking the RPB4 subunit exhibited markedly reduced activity in crude extracts in vitro. The RPB4 subunit, although not essential for mRNA synthesis or enzyme assembly, was essential for normal levels of RNA polymerase II activity and indispensable for cell viability over a wide temperature range.

Amino Acid Sequence↗
PubMed1988

Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum.

Lysosomal enzymes are initially synthesized as precursor polypeptides which are proteolytically cleaved to generate mature forms of the enzymatically active protein. The identification of the proteinases involved in this process and their intracellular location will be important initial steps in determining the role of proteolysis in the function and targeting of lysosomal enzymes. Toward this end, axenically growing Dictyostelium discoideum cells were pulse radiolabeled with [35S]methionine and chased in fresh growth medium containing inhibitors of aspartic, metallo, serine, or cysteine proteinases. Cells exposed to the serine/cysteine proteinase inhibitors leupeptin and antipain and the cysteine proteinase inhibitor benzyloxycarbonyl-L-phenylalanyl-L-alanine-diazomethyl ketone (Z-Phe-AlaCHN2) were unable to complete proteolytic processing of the newly synthesized lysosomal enzymes, alpha-mannosidase and beta-glucosidase. Antipain and leupeptin treatment resulted in both a dramatic decrease in the efficiency of proteolytic processing, as well as a sevenfold increase in the secretion of alpha-mannosidase and beta-glucosidase precursors. However, leupeptin and antipain did not stimulate secretion of lysosomally localized mature forms of the enzymes suggesting that these inhibitors prevent the normal sorting of lysosomal enzyme precursors to lysosomes. In contrast to the results observed for cells treated with leupeptin or antipain, Z-Phe-AlaCHN2 did not prevent the cleavage of precursor polypeptides to intermediate forms of the enzymes, but greatly inhibited the production of the mature enzymes. The accumulated intermediate forms of the enzymes, however, were localized to lysosomes. Finally, fractionation of cell extracts on Percoll gradients indicated that the processing of radiolabeled precursor forms of alpha-mannosidase and beta-glucosidase to intermediate products began in cellular compartments intermediate in density between the Golgi complex and mature lysosomes. The generation of the mature forms, in contrast, was completed immediately upon or soon after arrival in lysosomes. Together these results suggest that different proteinases residing in separate intracellular compartments may be involved in generating intermediate and mature forms of lysosomal enzymes in Dictyostelium discoideum, and that the initial cleavage of the precursors may be critical for the proper localization of lysosomal enzymes.

Antipain↗
PubMed1987

A single mutation prevents the normal intracellular transport of multiple lysosomal proteins from the rough endoplasmic reticulum.

Dictyostelium discoideum strain HMW-426 has been previously shown to be defective in the proteolytic processing of the lysosomal enzyme precursor to alpha-mannosidase. We have now shown that the mutant is defective in the proteolytic processing of a second lysosomal enzyme, beta-glucosidase. Digestion of the HMW-426 alpha-mannosidase and beta-glucosidase precursors with endoglycosidase H revealed that the majority of oligosaccharide side chains on both precursors were sensitive to cleavage by this enzyme, indicating that both precursors fail to reach the Golgi apparatus. Subcellular fractionation experiments demonstrated that these two mutant precursors accumulated inside the lumen of the rough endoplasmic reticulum. The alpha-mannosidase precursor is conformationally altered, as evidenced by its abnormal protease susceptibility, suggesting that altered conformation is responsible for a generalized defect in transport of lysosomal protein precursors from the rough endoplasmic reticulum in the mutant.

Acetylglucosaminidase↗
PubMed1987

A late developmental change in lysosomal enzyme sulfation specific to newly synthesized proteins in Dictyostelium discoideum.

During development in Dictyostelium discoideum, several lysosomal glycosidases undergo changes in post-translational modification that are thought to involve differences in the extent of sulfation or phosphorylation, and appear to be required for the maintenance of cellular enzyme activity late in development. We have used monoclonal antibodies specific to the lysosomal enzyme alpha-mannosidase-1 to study the major late (12 hr) developmental change in the modification system. Pulse-chase experiments performed both early and late in development reveal that the substrate for the late form of modification is restricted to newly synthesized alpha-mannosidase-1 precursor protein. We have identified one modification difference between the two developmentally distinct isozymes of alpha-mannosidase-1: 35SO4 pulse-chase data show that the newly synthesized "late" enzyme precursor is significantly undersulfated in comparison with the enzyme synthesized early in development. This apparent lack of sulfation is associated with the lack of acquisition of endoglycosidase H resistance. By contrast, an aggregation-deficient mutant, which is defective with regard to the accumulation of alpha-mannosidase-1 activity late in development, synthesizes the "early" sulfated form of the enzyme throughout development. We conclude that the late developmental change in post-translational modification specifically involves one of the biochemical steps in which the N-linked oligosaccharide side chains of the newly synthesized alpha-mannosidase-1 precursor are modified by sulfation.

Acetylglucosaminidase↗
PubMed1986

A conformationally altered precursor to the lysosomal enzyme alpha-mannosidase accumulates in the endoplasmic reticulum in a mutant strain of Dictyostelium discoideum.

The mutant strain of Dictyostelium discoideum, HMW-437, contains a mutation in the structural gene coding for the lysosomal enzyme alpha-mannosidase. Unlike the wild type strain, Ax3, this strain fails to proteolytically process or secrete the 140,000-dalton alpha-mannosidase precursor. The level of sulfate incorporation into the mutant precursor was significantly lower when compared to the wild type precursor. In addition, the mutant precursor was entirely sensitive to endoglycosidase H. Subcellular fractionation of HMW-437 membranes indicated that the majority of the alpha-mannosidase precursor sedimented in a region of the gradient corresponding to the rough endoplasmic reticulum. This accumulation within the rough endoplasmic reticulum did not appear to result from gross conformational changes which lead to aggregation. Trypsin digestion of radioactively labeled Ax3 and HMW-437 precursors demonstrated that there were differences in susceptibility to protease cleavage between the wild type and mutant alpha-mannosidase precursor molecules, suggesting that a minor conformational change could contribute to the accumulation of the mutant precursor inside the endoplasmic reticulum.

Acetylglucosaminidase↗
PubMed1985

Lysosomal enzyme inactivation associated with defects in post-translational modification during development in Dictyostelium discoideum.

The developmental accumulation of lysosomal alpha-mannosidase-1 activity in Dictyostelium discoideum is controlled at the level of de novo enzyme precursor biosynthesis. Aggregation-deficient mutants are defective with regard to the accumulation of alpha-mannosidase-1 activity beyond 8-16 h of development. We used enzyme-specific monoclonal antibodies to show that the activity defect in aggregation-deficient strains is not due to a lack of alpha-mannosidase-1-precursor synthesis or processing, or to preferential degradation of the mature enzyme protein. Instead, the defect is a result of enzyme inactivation: cells of aggregation-deficient strains contain significant amounts of inactive alpha-mannosidase-1 protein late in development. The alpha-mannosidase-1 inactivation phenotype is associated with a more general defect in lysosomal enzyme modification. A change in the post-translational modification system occurs during normal slime-mold development, as shown by differences in enzyme isoelectric point, antigenicity, and thermolability. We found that this change in modification does not occur in mutant strains blocked early in development. We propose a model in which pleiotropic mutations in early aggregation-essential genes can indirectly affect the accumulation of alpha-mannosidase-1 activity by preventing the expression of a developmentally controlled change in the post-translational modification system, a change which is required for the stability of several lysosomal enzymes late in development.

Enzyme Activation↗