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R J Duronio

Publications and source records attributed to R J Duronio.

28 records · Page 2Linked to original sources
PubMed1992

Comparative analysis of the beta transducin family with identification of several new members including PWP1, a nonessential gene of Saccharomyces cerevisiae that is divergently transcribed from NMT1.

While investigating the expression of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene (NMT: E.C. 2.3.1.97) by Northern blot analysis, we observed another RNA transcript whose expression resembled that of NMT1 during meiosis and was derived from a gene located less than 1 kb immediately upstream of NMT1. This new gene, designated PWP1 (for periodic tryptophan protein), is divergently transcribed from NMT1 and encodes a 576-residue protein. Null mutants of PWP1 are viable, but their growth is severely retarded and steady-state levels of several cellular proteins (including at least two proteins that label with exogenous [3H]myristic acid) are drastically reduced. New methods for database searching and assessing the statistical significance of sequence similarities identify PWP1 as a member of the beta-transducin protein superfamily. Two other previously unrecognized beta-transducin-like proteins (S. cerevisiae MAK11 and D. discoideum AAC3) were also identified, and an unexpectedly high degree of sequence homology was found between a Chlamydomonas beta-like polypeptide and the C12.3 gene of chickens. A systematic and quantitative comparative analysis resulted in classifying all beta-transducin-like sequences into 11 nonorthologous families. Based on specific sequence attributes, however, not all beta-transducin-like sequences are expected to be functionally similar, and quantitative criteria for inferring functional analogies are discussed. Possible roles of repetitive tryptophan residues in proteins are also considered.

Acyltransferases↗
PubMed1992

Isolation of a Saccharomyces cerevisiae long chain fatty acyl:CoA synthetase gene (FAA1) and assessment of its role in protein N-myristoylation.

Regulation of myristoylCoA pools in Saccharomyces cerevisiae plays an important role in modulating the activity of myristoylCoA:protein N-myristoyltransferase (NMT), an essential enzyme with an ordered Bi Bi reaction that catalyzes the transfer of myristate from myristoylCoA to greater than or equal to 12 cellular proteins. At least two pathways are available for generating myristoylCoA: de novo synthesis by the multifunctional, multisubunit fatty acid synthetase complex (FAS) and activation of exogenous myristate by acylCoA synthetase. The FAA1 (fatty acid activation) gene has been isolated by genetic complementation of a faal mutant. This single copy gene, which maps to the right arm of chromosome XV, specifies a long chain acylCoA synthetase of 700 amino acids. Analyses of strains containing NMT1 and a faal null mutation indicated that FAA1 is not essential for vegetative growth when an active de novo pathway for fatty acid synthesis is present. The role of FAA1 in cellular lipid metabolism and protein N-myristoylation was therefore assessed in strains subjected to biochemical or genetic blockade of FAS. At 36 degrees C, FAA1 is required for the utilization of exogenous myristate by NMT and for the synthesis of several phospholipid species. This requirement is not apparent at 24 or 30 degrees C, suggesting that S. cerevisiae contains another acylCoA synthetase activity whose chain length and/or temperature optima may differ from Faalp.

Acyl Coenzyme A↗
PubMed1991

Analyzing the substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase by co-expressing it with mammalian G protein alpha subunits in Escherichia coli.

A dual plasmid system was used to examine the protein and acyl-CoA specificities of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) by co-expressing it in Escherichia coli with each of four homologous alpha subunits of the signal-transducing, heterotrimeric G proteins. Exogenous [3H]myristate was incorporated into rat Gi alpha 1 and rat Go alpha but not into bovine Gs alpha or human Gz alpha. Oxygen for methylene group substitutions in myristate result in analogs with comparable chain length and stereochemistry but marked reductions in hydrophobicity. Metabolic labeling studies with 6-, 11-, or 13-[3H]oxatetradecanoic acid indicated that they were incorporated into rat Gi alpha 1 and Go alpha with an efficiency that could be correlated with their accumulation into E. coli and their interactions with purified NMT in vitro. Octapeptides derived from the NH2-terminal sequences of these four G alpha polypeptides were tested as substrates for purified S. cerevisiae NMT. None were bound by the enzyme. Acidic residues at positions 7 and 8 appear to contribute to this effect; deletion of these two amino acids or addition of the next 9 residues of rat Go alpha produced active substrates. These results imply that productive interactions between NMT and G alpha protein substrates in vivo require structural features that are not fully represented within their NH2-terminal 8 residues.

Acyltransferases↗
PubMed1991

Incorporation of 12-methoxydodecanoate into the human immunodeficiency virus 1 gag polyprotein precursor inhibits its proteolytic processing and virus production in a chronically infected human lymphoid cell line.

Covalent linkage of myristate (tetradecanoate; 14:0) to the NH2-terminal glycine residue of the human immunodeficiency virus 1 (HIV-1) 55-kDa gag polyprotein precursor (Pr55gag) is necessary for its proteolytic processing and viral assembly. We have shown recently that several analogs of myristate in which a methylene group is replaced by a single oxygen or sulfur atom are substrates for Saccharomyces cerevisiae and mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97; NMT) despite their reduced hydrophobicity. Some inhibit HIV-1 replication in acutely infected CD4+H9 cells without accompanying cellular toxicity. To examine the mechanism of their antiviral effects, we performed labeling studies with two analogs, 12-methoxydodecanoate (13-oxamyristate; 13-OxaMyr) and 5-octyloxypentanoate (6-oxamyristate; 6-OxaMyr), the former being much more effective than the latter in blocking virus production. [3H]Myristate and [3H]13-OxaMyr were incorporated into Pr55gag with comparable efficiency when it was coexpressed with S. cerevisiae NMT in Escherichia coli. [3H]6-OxaMyr was not incorporated, even though its substrate properties in vitro were similar to those of 13-OxaMyr and myristate. [3H]13-OxaMyr, but not [3H]6-OxaMyr, was also efficiently incorporated into HIV-1 Pr55gag and nef (negative factor) in chronically infected H9 cells. Analog incorporation produced a redistribution of Pr55gag from membrane to cytosolic fractions and markedly decreased its proteolytic processing by viral protease. 13-OxaMyr and 3'-azido-3'-deoxythymidine (AZT) act synergistically to reduce virus production in acutely infected H9 cells. Unlike AZT, the analog is able to inhibit virus production (up to 70%) in chronically infected H9 cells. Moreover, the inhibitory effect lasts 6-8 days. These results suggest that (i) its mechanism of action is distinct from that of AZT and involves a late step in virus assembly; (ii) the analog may allow reduction in the dose of AZT required to affect viral replication; and (iii) combinations of analog and HIV-1 protease inhibitors may have synergistic effects on the processing of Pr55gag.

Antiviral Agents↗
PubMed1991

Lipid modifications of G protein subunits. Myristoylation of Go alpha increases its affinity for beta gamma.

Myristoylated recombinant proteins can be synthesized in Escherichia coli by concurrent expression of the enzyme myristoyl-CoA:protein N-myristoyl-transferase with its protein substrates (Duronio, R.J., Jackson-Machelski, E., Heuckeroth, R.O., Olins, P. O., Devine, C.S., Yonemoto, W., Slice, L. W., Taylor, S. S., and Gordon, J. I. (1990) Proc. Natl. Acad. Sci. U. S.A. 87, 1506-1510). Expression of the G protein subunit Go alpha in this system results in the synthesis of two forms of the protein; these were separated on a column of heptylamine-Sepharose. Purification of the more abundant form of Go alpha yielded a product that has a blocked amino terminus. Chemical analysis of the fatty acids released by acid hydrolysis of the protein revealed myristic acid. The second form of the protein was not myristoylated. Myristoylated and nonmyristoylated recombinant Go alpha were compared with brain Go alpha (which is myristoylated) for their ability to interact with G protein beta gamma subunits. The nonmyristoylated recombinant protein clearly had a reduced affinity for beta gamma, while the myristoylated recombinant protein was indistinguishable from native Go alpha in its subunit interactions. Thus, myristoylation increases the affinity of alpha subunits for beta gamma. We propose that the function of myristoylation of G protein alpha subunits is, at least in part, to facilitate formation of the heterotrimer and the localization of alpha to the plasma membrane.

Acylation↗
PubMed1991

Myristic acid auxotrophy caused by mutation of S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase.

The S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene (NMT1) is essential for vegetative growth. NMT1 was found to be allelic with a previously described, but unmapped and unidentified mutation that causes myristic acid (C14:0) auxotrophy. The mutant (nmt1-181) is temperature sensitive, but growth at the restrictive temperature (36 degrees C) is rescued with exogenous C14:0. Several analogues of myristate with single oxygen or sulfur for methylene group substitutions partially complement the phenotype, while others inhibit growth even at the permissive temperature (24 degrees C). Cerulenin, a fatty acid synthetase inhibitor, also prevents growth of the mutant at 24 degrees C. Complementation of growth at 36 degrees C by exogenous fatty acids is blocked by a mutation affecting the acyl:CoA synthetase gene. The nmt1-181 allele contains a single missense mutation of the 455 residue acyltransferase that results in a Gly451----Asp substitution. Analyses of several intragenic suppressors suggest that Gly451 is critically involved in NMT catalysis. In vitro kinetic studies with purified mutant enzyme revealed a 10-fold increase in the apparent Km for myristoyl-CoA at 36 degrees C, relative to wild-type, that contributes to an observed 200-fold reduction in catalytic efficiency. Together, the data indicate that nmt-181 represents a sensitive reporter of the myristoyl-CoA pools utilized by NMT.

Acyltransferases↗
PubMed1990

Structural and functional studies of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase produced in Escherichia coli. Evidence for an acyl-enzyme intermediate.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase has been efficiently expressed in Escherichia coli and subsequently purified to homogeneity using phosphocellulose chromatography. The interactions between apoenzyme and its acyl-CoA and peptide ligands were examined by an isoelectric focusing gel shift assay, circular dichroism, and fluorescence spectroscopy, and a continuous assay of enzyme activity which measures the release of CoA from acyl-CoA using the thiol-specific reagent 5-5'-dithiobis-2-nitrobenzoate. Addition of myristoyl-CoA (without a substrate peptide) results in the formation of a high affinity reaction intermediate which can be operationally defined by the appearance of a more acidic enzyme isoform and by quenching of the tryptophan emission with a maximal difference at 340 nm. Circular dichroism spectroscopy indicates that these changes are accompanied by minimal changes in the enzyme's secondary structure. Incubation of purified NMT with [1-14C] myristoyl-CoA, followed by chymotryptic digestion, denaturing polyacrylamide gel electrophoresis, and treatment with hydroxylamine yielded results that are highly suggestive of a covalent ester-linked acyl-enzyme complex. Edman degradation of chymotryptic peptides has narrowed the site of interaction to a domain spanning Arg42 to Thr220 of the 455 amino acid acyltransferase. An octapeptide containing Gly but not Ala at position 1 is able to reverse the change in pI and reduce the quenching almost entirely. These data suggest a preferred order or ping-pong reaction mechanism with the acyl-CoA substrate binding event occurring first. They also indicate that Gly1 is absolutely necessary for the reaction to proceed forward from the acyl-enzyme reaction intermediate.

Acyltransferases↗
PubMed1990

Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria.

Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH2-terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. We have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activity relationships. Expression of the yeast NMT1 gene in Escherichia coli, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH2-terminal 39 amino acids. Each E. coli-synthesized NMT species has fatty acid and peptide substrate specificities that are indistinguishable from those of NMT recovered from Saccharomyces cerevisiae, suggesting that the NH2-terminal domain of this enzyme is not required for its catalytic activity. By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted in E. coli by simultaneous expression of the yeast NMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A). The fatty acid specificity of N-myristoylation was preserved in this system: [9,10(n)-3H]myristate but not [9,10(n)3H]palmitate was efficiently linked to Gly-1 of the C subunit. [13,14(n)-3H]10-Propoxydecanoic acid, a heteroatom-containing analog of myristic acid with reduced hydrophobicity but similar chain length, was an effective alternative substrate for NMT that also could be incorporated into the C subunit of PK-A. Such analogs have recently been shown to inhibit replication of certain retroviruses that depend upon linkage of a myristoyl group to their gag polyprotein precursors (e.g., the Pr55gag of human immunodeficiency virus type 1). A major advantage of the bacterial system over eukaryotic systems is the absence of endogenous NMT and substrates, providing a more straightforward way of preparing myristoylated, analog-substituted, and nonmyristoylated forms of a given protein for comparison of their structural and functional properties. The system should facilitate screening of enzyme inhibitors as well as alternative NMT fatty acid substrates for their ability to be incorporated into a specific target protein. Our experimental system may prove useful for recapitulating other eukaryotic protein modifications in E. coli so that structure-activity relationships of modifying enzymes and their substrates can be more readily assessed.

Acyltransferases↗
PubMed1989

Disruption of the yeast N-myristoyl transferase gene causes recessive lethality.

The structural gene for N-myristoyl transferase (NMT1) has been cloned from the budding yeast Saccharomyces cerevisiae. The gene encodes a polypeptide of 455 amino acids (Mr = 52,837) that has no identifiable significant primary sequence homology with any protein in currently available databases. Overexpression of NMT activity was achieved by means of the yeast episomal plasmid YEp24 without obvious effects on growth kinetics, cell morphology, or acylprotein metabolic labeling patterns. Insertional mutagenesis of the NMT1 locus on yeast chromosome XII caused recessive lethality, indicating that this protein acyltransferase activity is necessary for vegetative cell growth.

Acyltransferases↗