Search PubMed⌕ Search

Biomedical subjects

N C Martin

Publications and source records attributed to N C Martin.

At least 37 records · Page 2Linked to original sources
PubMed1997

Mitochondrial RNase P: the RNA family grows.

Recent results of biochemical approaches and genome sequencing approaches has extended the members of the family of mitochondrial RNase P RNA genes. So far all of them are AU rich, and most of their secondary structures are easier to predict than was the structure of the first mitochondrial RNase P RNA from S. cerevisiae. The recently sequenced protozoan R. americana mitochondrial gene displays many of the evolutionarily conserved primary sequence and secondary structure attributes of the well characterized bacterial RNase P RNAs. Continued addition of RNAs to this data base should allow increasingly informative alignments and an understanding of what structural elements are dispensable in the smallest mitochondrial RNAs. The only protein subunits identified to date are Rpm2p from S.cerevisiae and the homologous protein from S. douglasii.

Animals↗
PubMed1996

Mechanisms leading to and the consequences of altering the normal distribution of ATP(CTP):tRNA nucleotidyltransferase in yeast.

CCA1 codes for mitochondrial, cytosolic, and nuclear ATP(CTP):tRNA nucleotidyltransferase. Studies reported here examine the mechanisms leading to and the consequences of altering the distribution of this important tRNA processing enzyme. We show that the majority of Cca1p-I, translated from the first in-frame ATG, is in mitochondria but surprisingly, there is a small contribution to nuclear and cytosolic tRNA processing by this form as well. The majority of Cca1p-II and Cca1p-III, translated from ATG2 and ATG3, respectively, is in the cytosol but both are also located in the nucleus for processing precursors. Altering the cytosolic/nuclear distribution of Cca1p by fusing the SV40 nuclear localization signal to the 5' end of CCA1 causes a growth defect and results in the accumulation of end-shortened tRNAs in the cytosol. These results suggest an important role for Cca1p in the cytosol of eukaryotes, presumably in the repair of 3' CCA termini. These experiments also demonstrate that individual tRNAs are affected differently by reduced cytosolic nucleotidyltransferase and that cells resuming exponential growth are more severely affected than those continuing exponential growth.

Base Sequence↗
PubMed1996

Yeast mitochondrial RNase P RNA synthesis is altered in an RNase P protein subunit mutant: insights into the biogenesis of a mitochondrial RNA-processing enzyme.

Rpm2p is a protein subunit of Saccharomyces cerevisiae yeast mitochondrial RNase P, an enzyme which removes 5' leader sequences from mitochondrial tRNA precursors. Precursor tRNAs accumulate in strains carrying a disrupted allele of RPM2. The resulting defect in mitochondrial protein synthesis causes petite mutants to form. We report here that alteration in the biogenesis of Rpm1r, the RNase P RNA subunit, is another consequence of disrupting RPM2. High-molecular-weight transcripts accumulate, and no mature Rpm1r is produced. Transcript mapping reveals that the smallest RNA accumulated is extended on both the 5' and 3' ends relative to mature Rpm1r. This intermediate and other longer transcripts which accumulate are also found as low-abundance RNAs in wild-type cells, allowing identification of processing events necessary for conversion of the primary transcript to final products. Our data demonstrate directly that Rpm1r is transcribed with its substrates, tRNA met f and tRNAPro, from a promoter located upstream of the tRNA met f gene and suggest that a portion also originates from a second promoter, located between the tRNA met f gene and RPM1. We tested the possibility that precursors accumulate because the RNase P deficiency prevents the removal of the downstream tRNAPro. Large RPM1 transcripts still accumulate in strains missing this tRNA. Thus, an inability to process cotranscribed tRNAs does not explain the precursor accumulation phenotype. Furthermore, strains with mutant RPM1 genes also accumulate precursor Rpm1r, suggesting that mutations in either gene can lead to similar biogenesis defects. Several models to explain precursor accumulation are presented.

Alleles↗
PubMed1995

Interaction of cysteine proteinases with recombinant kininogen domain 2, expressed in Escherichia coli.

The calpain-binding domain 2 of the kininogens, the major plasma inhibitors of cysteine proteinases, was expressed in Escherichia coli. Expression of soluble protein was optimal at 15 degrees C and was augmented by growing the bacteria in sorbitol and betaine. The recombinant domain showed high affinity (Ki 0.3-1 nM) for cathepsin L and papain, and a somewhat lower affinity (Ki approximately 15 nM) for calpain. The binding to cathepsin H was substantially weaker, and no inhibition of actinidin and cathepsin B was detected. The affinity for cathepsin L was comparable to that reported for the domain isolated from plasma L-kininogen, whereas the affinities for papain and calpain were about tenfold lower. The latter difference may be due to the recombinant domain being nonglycosylated.

Animals↗
PubMed1995

Location of N2,N2-dimethylguanosine-specific tRNA methyltransferase.

Most steps in the maturation of nuclear coded tRNAs occur in the nucleus in eukaryotic cells, but little is known as to the intranuclear location of this RNA maturation pathway. Indirect immunofluorescence experiments using antibody to N2,N2 dimethylguanosine-specific tRNA methyltransferase, a tRNA processing enzyme, and to Nup1p, a nuclear pore protein, show that both locate to the nuclear periphery in wild type cells. Staining of the nuclear membrane is more uniform with anti-Trm1p than the punctate staining observed with antibodies recognizing Nup1p. Biochemical fractionation experiments comparing fractionation of Trm1p with Nup1p, tRNA splicing ligase, and tRNA splicing endonuclease show that Trm1p behaves more like the known peripheral nuclear membrane proteins, Nup1p and tRNA splicing ligase, than like the integral membrane protein, tRNA splicing endonuclease. Cells overproducing Trm1p also concentrate it to the nuclear periphery. Thus, the site(s) of interaction of Trm1p are not easily saturable and are likely to be in excess to Trm1p. Trm1p is shared by mitochondria and the nucleus. Cells transformed with a gene coding Trm1p with a mutant nuclear targeting signal display cytoplasmic staining and an enzyme with increased solubility when compared to the solubility of wild type enzyme. Thus, mutations that prevent the enzyme from entering the nucleus result in an increase in its cytosolic but not mitochondrial concentration suggesting that the mitochondrial/nuclear distribution of Trm1p is not due solely to competition of mitochondrial and nuclear targeting information.

Amino Acid Sequence↗
PubMed1995

Mutations altering the mitochondrial-cytoplasmic distribution of Mod5p implicate the actin cytoskeleton and mRNA 3' ends and/or protein synthesis in mitochondrial delivery.

The Saccharomyces cerevisiae MOD5 gene encodes proteins that function in three subcellular locations: mitochondria, the cytoplasm, and nuclei (M. Boguta, L.A. Hunter, W.-C. Shen, E. C. Gillman, N. C. Martin, and A. K. Hopper, Mol. Cell. Biol. 14:2298-2306, 1994; E. C. Gillman, L. B. Slusher, N. C. Martin, and A. K. Hopper, Mol. Cell. Biol. 11:2382-2390, 1991). A mutant allele of MOD5 encoding a protein (Mod5p-I,KR6) located predominantly in mitochondria was constructed. Mutants defective in delivering Mod5p-I,KR6 to mitochondria were sought by selecting cells with increased cytosolic activity of this protein. Twenty-five mutants defining four complementation groups, mdp1, mdp2, mdp3, and mdp4, were found. They are unable to respire at 34 degrees C or to grow on glucose medium at 38 degrees C. Cell fractionation studies showed that mdp1, mdp2, and mdp3 mutants have an altered mitochondrial-cytoplasmic distribution of Mod5p. mdp2 can be suppressed by ACT1, the actin-encoding gene. The actin cytoskeleton organization is also aberrant in mdp2 cells. MDP2 is the same as VRP1 (S. F. H. Donnelly, M. J. Picklington, D. Pallotta, and E. Orr, Mol. Microbiol. 10:585-596, 1993). MDP3 is identical to PAN1, which encodes a protein that interacts with mRNA 3' ends and affects initiation of protein synthesis (A. B. Sachs and J. A. Deardoff, Cell 70:961-973, 1992). These results implicate the actin cytoskeleton and mRNA 3' ends and/or protein synthesis as being as important for protein distribution in S. cerevisiae as they are for distribution of cytosolic proteins in higher eukaryotes. This provides the potential to apply genetic and molecular approaches to study gene products and mechanisms involved in this type of protein distribution. The selection strategy also offers a new approach for identifying gene products involved in the distribution of proteins to their subscellular destinations.

Actins↗
PubMed1995

RPM2, independently of its mitochondrial RNase P function, suppresses an ISP42 mutant defective in mitochondrial import and is essential for normal growth.

RPM2 is identified here as a high-copy suppressor of isp42-3, a temperature-sensitive mutant allele of the mitochondrial protein import channel component, Isp42p. RPM2 already has an established role as a protein component of yeast mitochondrial RNase P, a ribonucleoprotein enzyme required for the 5' processing of mitochondrial precursor tRNAs. A relationship between mitochondrial tRNA processing and protein import is not readily apparent, and, indeed, the two functions can be separated. Truncation mutants lacking detectable RNase P activity still suppress the isp42-3 growth defect. Moreover, RPM2 is required for normal fermentative yeast growth, even though mitochondrial RNase P activity is not. The portion of RPM2 required for normal growth and suppression of isp42-3 is the same. We conclude that RPM2 is a multifunctional gene. We find Rpm2p to be a soluble protein of the mitochondrial matrix and discuss models to explain its suppression of isp42-3.

Amino Acid Sequence↗
PubMed1994

Interplay of heterogeneous transcriptional start sites and translational selection of AUGs dictate the production of mitochondrial and cytosolic/nuclear tRNA nucleotidyltransferase from the same gene in yeast.

ATP (CTP):tRNA nucleotidyltransferase catalyzes the addition of the CCA end to tRNAs. In yeast, nucleotidyltransferase is encoded by the CCA1 gene and is localized to three cellular compartments: mitochondria, nucleus, and cytosol. There are three in-frame ATGs near the 5' end of the CCA1 open reading frame. Primer extension experiments show multiple transcription initiation sites upstream of ATG1 and between ATG1 and ATG2. Fractionation of cells carrying a CCA1-COXIV fusion gene demonstrates that all three in-frame AUGs are used as sites of initiation of translation. Therefore, both transcription of CCA1 mRNA with heterogeneous 5' ends and translation from downstream AUGs in CCA1 mRNAs play a role in the synthesis of three nucleotidyltransferase isozymes. Protein initiating from AUG1 is required for mitochondrial protein synthesis and, like many other proteins targeted to mitochondria, it is processed at the amino terminus upon import into the organelle. The shorter proteins arising from AUG2 and AUG3 provide nuclear/cytosol activity.

Amino Acid Sequence↗
PubMed1994

How single genes provide tRNA processing enzymes to mitochondria, nuclei and the cytosol.

TRM1, MOD5 and CCA1 are yeast genes that provide tRNA processing enzymes to mitochondria and the nuclear/cytosolic compartments. The product of the TRM1 gene is N2,N2 dimethylguanosine tRNA methyltransferase. The product of the MOD5 gene is isopentenyl pyrophosphate: tRNA isopentenyl transferase and the product of the CCA1 gene is ATP (CTP): tRNA nucleotidyltransferase. N2,N2 dimethylguanosine tRNA methyltransferase is found in the mitochondria and the nucleus. The tRNA isopentenyl transferase and tRNA nucleotidyltransferase are found in mitochondria, nuclei and the cytosol. Genes coding for these three enzymes contain more than one in-frame ATG. Where translation begins dictates the efficiency with which these gene products reach mitochondria. Depending on the gene, ATGs choice is by transcription start site selection, by translational selection or by an interplay between these two processes. A short amino acid sequence is necessary and sufficient for the nuclear targeting of the dimethylguanosine transferase. There is a good candidate sequence for a nuclear targeting signal (NTS) for the isopentenyl pyrophosphate: tRNA isopentenyl transferase. There are no obvious candidate sequences for a NTS in the CCA1 sequence.

Cell Nucleus↗
PubMed1994

Subcellular locations of MOD5 proteins: mapping of sequences sufficient for targeting to mitochondria and demonstration that mitochondrial and nuclear isoforms commingle in the cytosol.

MOD5, a gene responsible for the modification of A37 to isopentenyl A37 of both cytosolic and mitochondrial tRNAs, encodes two isozymes. Initiation of translation at the first AUG of the MOD5 open reading frame generates delta 2-isopentenyl pyrophosphate:tRNA isopentanyl transferase I (IPPT-I), which is located predominantly, but not exclusively, in the mitochondria. Initiation of translation at a second AUG generates IPPT-II, which modifies cytoplasmic tRNA. IPPT-II is unable to target to mitochondria. The N-terminal sequence present in IPPT-I and absent in IPPT-II is therefore necessary for mitochondrial targeting. In these studies, we fused MOD5 sequences encoding N-terminal regions to genes encoding passenger proteins, pseudomature COXIV and dihydrofolate reductase, and studied the ability of these chimeric proteins to be imported into mitochondria both in vivo and in vitro. We found that the sequences necessary for mitochondrial import, amino acids 1 to 11, are not sufficient for efficient mitochondrial targeting and that at least some of the amino acids shared by IPPT-I and IPPT-II comprise part of the mitochondrial targeting information. We used indirect immunofluorescence and cell fractionation to locate the MOD5 isozymes in yeast. IPPT-I was found in two subcellular compartments: mitochondria and the cytosol. We also found that IPPT-II had two subcellular locations: nuclei and the cytosol. The nuclear location of this protein is surprising because the A37-->isopentenyl A37 modification had been predicted to occur in the cytoplasm. MOD5 is one of the first genes reported to encode isozymes found in three subcellular compartments.

Alkyl and Aryl Transferases↗
PubMed1994

Identification of two single base substitutions in the UGT1 gene locus which abolish bilirubin uridine diphosphate glucuronosyltransferase activity in vitro.

Accumulating evidence indicates that mutations in the human UGT1 gene locus abolish hepatic bilirubin UDP-glucuronosyltransferase activity and cause the subsequent accumulation of bilirubin to toxic levels in patients with Crigler-Najjar type 1 (CN-I). Genetic and biochemical criteria are required to link CN-I with mutations in UGT1. Here we present analysis of mutations at the UGT1 locus in three individuals that were clinically diagnosed with CN-I (two related and one unrelated). Each patient carries a single base substitution that alters conserved residues in the transferase enzyme molecule, serine to phenylalanine at codon 376 and glycine to glutamic acid at codon 309. Each was homozygous for the defect as demonstrated by sequencing and RFLPs. Mutant cDNAs, constructed by site-directed mutagenesis, inserted into expression vectors, and transfected into COS-1 cells, supported the synthesis of the bilirubin transferase protein but only cells transfected with the wild-type cDNA expressed bilirubin UDP-glucuronosyltransferase activity. The data provide conclusive evidence that alterations at Gly 309 and Ser 376 are the genetic basis for CN-I in these families. These results suggest that the two codons, located in conserved regions of the molecule, are part of the active site of the bilirubin enzyme.

Adolescent↗
PubMed1994

maf1 mutation alters the subcellular localization of the Mod5 protein in yeast.

Two forms of Mod5p, a tRNA modification enzyme, are found in three intracellular compartments, mitochondria, cytoplasm and nucleus, but are encoded by a single MOD5 gene. The two forms of the enzyme, Mod5p-I and Mod5p-II differ at the N-termini and are produced by initiation of translation at different start codons. Mod5p-I does contain a mitochondrial targeting signal and is distributed between mitochondria and cytoplasm, whereas Mod5p-II is found in the cytosol and nucleus (Boguta, M., et al. 1994, Mol. Cell. Biol. 14, 2298-2306). In the present work mutants which mislocalize the Mod5p-I enzyme were isolated. The screen was based on a correlation between the amount of cytosolic protein and the efficiency of tRNA mediated suppression. Identification of mutants is possible because a red pigment accumulates in the cells unable to suppress an ade2-1 nonsense allele. The maf1 mutant, with an altered intracellular localization of the Mod5p-I protein, was isolated. Immunofluorescence data suggest that the mutation causes mislocalization of the Mod5p-I to the nucleus.

Antimicrobial Cationic Peptides↗
PubMed1993

Yeast mitochondrial RNase P. Sequence of the RPM2 gene and demonstration that its product is a protein subunit of the enzyme.

We report here the sequence of the RPM2 gene which codes for the 105-kDa protein previously purified from the mitochondria of Saccharomyces cerevisiae and shown by genetic techniques to be required for mitochondrial RNase P activity. The sequence predicts a primary translation product of 1202 residues with a molecular mass of 139 kDa and no obvious sequence similarity to any known protein in the data bases. There are 122 amino-terminal amino acids predicted by the gene that are not found in the purified protein, some of which may play a role in mitochondrial targeting of the protein. Antibodies raised against a trpE-105-kDa fusion protein recognize a 105-kDa protein in wild-type cells but not in cells carrying a disruption of the RMP2 gene. Immune, but not preimmune serum, immunoprecipitates the RNase P RNA and the mitochondrial RNase P activity. Thus, the 105-kDa protein forms a complex with RNase P RNA and is required for RNase P activity as predicted for a bona fide subunit of the enzyme.

Amino Acid Sequence↗
PubMed1993

Isolation and characterization of LIP5. A lipoate biosynthetic locus of Saccharomyces cerevisiae.

A number of mutants with pleiotropic effects on mitochondrial metabolism have been isolated in yeast Saccharomyces cerevisiae, and for many the biochemical function that is impaired is not yet known. We report here the isolation and characterization of the LIP5 gene involved in lipoic acid metabolism which complements the g189 mutant (Tzagoloff, A., and Dieckmann, C. L. (1990) Microbiol. Rev. 54, 211-225). DNA sequence analysis of complementing yeast genomic DNA revealed an open reading frame predicting a protein of 414 amino acids. The protein sequence deduced from the gene shares 43% identical residues with the product of the Escherichia coli lip gene, which codes an enzyme involved in lipoic acid synthesis. The LIP5 mutant is not capable of synthesizing lipoic acid but still possesses the activity necessary for attachment of lipoic acid to protein. Relative to the E. coli lip gene product, the LIP5 protein has an amino-terminal extension with characteristics of mitochondrial targeting signals. Cells carrying a disrupted copy of the LIP5 gene show slow growth on ethanol-rich media and barely detectable growth on glycerol-rich media. Unlike other strains with defects in the tricarboxylic acid cycle, LIP5 mutants undergo a high frequency of mitochondrial DNA deletions.

Amino Acid Sequence↗
PubMed1992

A 105-kDa protein is required for yeast mitochondrial RNase P activity.

RNase P from the mitochondria of Saccharomyces cerevisiae was purified to near homogeneity > 1800-fold with a yield of 1.6% from mitochondrial extracts. The most abundant protein in the purified fractions is, at 105 kDa, considerably larger than the 14-kDa bacterial RNase P protein subunits. Oligonucleotides designed from the amino-terminal sequence of the 105-kDa protein were used to identify and isolate the 105-kDa protein-encoding gene. Strains carrying a disruption of the gene for the 105-kDa protein are viable but respiratory deficient and accumulate mitochondrial tRNA precursors with 5' extensions. As this is the second gene known to be necessary for yeast mitochondrial RNase P activity, we have named it RPM2 (for RNase P mitochondrial).

Amino Acid Sequence↗
PubMed1992

Cytoplasmic and mitochondrial tRNA nucleotidyltransferase activities are derived from the same gene in the yeast Saccharomyces cerevisiae.

ATP (CTP):tRNA-specific tRNA nucleotidyltransferase is an enzyme required for the synthesis of functional tRNAs in eukaryotic cells. Neither the tRNA genes in the nucleus nor in organelles encode the CCA end, so it must be added post-transcriptionally. The gene that codes for the enzyme that adds the CCA end to nuclear coded tRNAs in Saccharomyces cerevisiae has been isolated (Aebi, M., Kirchner, G., Chen, J.-Y., Vijayraghavan, U., Jacobson, A., Martin, N. C., and Abelson, J. (1990) J. Biol. Chem. 265, 16216-16220). We now demonstrate that there is a mitochondrial tRNA nucleotidyltransferase activity in yeast and that it is a matrix enzyme. A comparison of purified mitochondrial enzyme with its cytoplasmic counterpart revealed no differences. These results suggest that proteins responsible for this step in the maturation of tRNAs in the nucleus and mitochondria might be identical and coded by the same nuclear gene. Accumulation of shortened mitochondrial as well as cytoplasmic tRNAs in a strain with a temperature-sensitive tRNA nucleotidyltransferase is consistent with this hypothesis. Alteration of the wild type gene such that amino-terminal truncated proteins are produced leads to a defect in mitochondrial function and a decrease in mitochondrial nucleotidyltransferase activity. This provides a direct demonstration that one gene provides this enzyme activity for the biosynthesis of tRNAs in both the nuclear/cytoplasmic and mitochondrial compartments in yeast.

Amino Acid Sequence↗
PubMed1992

Separate information required for nuclear and subnuclear localization: additional complexity in localizing an enzyme shared by mitochondria and nuclei.

The TRM1 gene of Saccharomyces cerevisiae codes for a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase (m2(2)Gtase), shared by mitochondria and nuclei. Immunofluorescent staining at the nuclear periphery demonstrates that m2(2)Gtase localizes at or near the nuclear membrane. In determining sequences necessary for targeting the enzyme to nuclei and mitochondria, we found that information required to deliver the enzyme to the nucleus is not sufficient for its correct subnuclear localization. We also determined that mislocalizing the enzyme from the nucleus to the cytoplasm does not destroy its biological function. This change in location was caused by altering a sequence similar to other known nuclear targeting signals (KKSKKKRC), suggesting that shared enzymes are likely to use the same import pathway as proteins that localize only to the nucleus. As with other well-characterized mitochondrial proteins, the mitochondrial import of the shared methyltransferase depends on amino-terminal amino acids, and removal of the first 48 amino acids prevents its import into mitochondria. While this truncated protein is still imported into nuclei, the immunofluorescent staining is uniform throughout rather than at the nuclear periphery, a staining pattern identical to that described for a fusion protein consisting of the first 213 amino acids of m2(2)Gtase in frame with beta-galactosidase. As both of these proteins together contain the entire m2(2)Gtase coding region, the information necessary for association with the nuclear periphery must be more complex than the short linear sequence necessary for nuclear localization.

Amino Acid Sequence↗