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M G Douglas

Publications and source records attributed to M G Douglas.

At least 55 records · Page 3Linked to original sources
PubMed1987

The role of protein structure in the mitochondrial import pathway. Unfolding of mitochondrially bound precursors is required for membrane translocation.

In order to examine the influence of protein structure on the post-translational import of a protein into mitochondria, the carboxyl-terminal 129 residues of F1-ATPase beta-subunit precursor (511aa) have been replaced with 61 residues of yeast copper metallothionein. Import of the F1 beta-copper metallothionein (beta CuMT) hybrid into mitochondria was as efficient as that of the F1 beta precursor in the absence of copper. Addition of copper to mitochondrial import reactions, which had no significant effect on import of the F1 beta-subunit precursor, blocked import of the beta CuMT protein. This copper-dependent transport block for the beta CuMT precursor occurred after the precursor was bound to mitochondria. Expression of the beta CuMT protein in vivo revealed that beta CuMT would bind copper and allow growth of a copper-sensitive yeast host on an otherwise inhibitory level of the cation as long as it was localized in the cytoplasm. These data indicate that the binding of copper by beta CuMT renders it refractile for partial unfolding which is necessary for its translocation into mitochondria. These observations provide an alternative scheme for the selection of mutants defective in mitochondrial import.

Amino Acid Sequence↗
PubMed1987

Phosphodiester bond cleavage outside mitochondria is required for the completion of protein import into the mitochondrial matrix.

The present studies show that hydrolysis of a phosphodiester bond, most likely ATP, is a distinct, second step required to complete import of the F1-ATPase beta-subunit into the mitochondria. This step follows a membrane potential-dependent first step. We show, using an inhibitor of adenine nucleotide transport and the analogue beta,gamma-AMP-PCP, that the activity required for this phosphodiester hydrolysis-dependent completion of protein import resides outside the mitochondrial inner membrane. This activity is proposed to act on the precursor at the site of translocation either to render it competent or to catalyze its vectorial movement directly through the import apparatus. This activity shares properties ascribed to proteins of the heat-shock family, which are proposed to participate in the ATP-dependent refolding of partially denatured proteins and nascent peptides.

Adenosine Triphosphate↗
PubMed1987

Sequences distal to the mitochondrial targeting sequences are necessary for the maturation of the F1-ATPase beta-subunit precursor in mitochondria.

The beta-subunit of the mitochondrial F1-ATPase is synthesized as a precursor in the cytoplasm which is delivered through two bilayers bounding the mitochondria prior to its assembly with other proteins into a functional complex. In order to determine the role of the amino-terminal 50 residues of the precursor on its localization, maturation, and assembly, a set of deletions within this region of the ATP2 gene encoding the beta-subunit has been analyzed. These studies reveal that deletions between residue 10 of the F1 beta-presequence and residue 36 can still direct in vivo mitochondrial import and assembly of the mutant subunit into a functional complex. Deletions within ATP2 which contain less than the first 10 residues of the precursor are not imported. Thus, the extreme amino terminus (about half of the transient presequence) of the F1 beta-subunit can direct its mitochondrial import. The wild-type F1 beta-subunit precursor is matured by the matrix-located metalloprotease at Lys19-Gln20; however, small in-frame deletions up to 17 residues distal to this site fail to be matured either in vitro or in vivo. This nonmatured F1 beta-subunit is also assembled into a functional enzyme and supports growth of its host on a nonfermentable carbon source. These data indicate that maturation of the F1 beta-subunit precursor is dependent on a protein sequence located distal to the proteolytic maturation site which is distinct from the mitochondrial targeting sequence.

Amino Acid Sequence↗
PubMed1986

Selection by genetic complementation and characterization of the gene coding for the yeast porphobilinogen deaminase.

The porphobilinogen deaminase (PBG-D) gene of Saccharomyces cerevisiae has been isolated by genetic complementation of a mutant GL7 (alpha hem 3) strain, previously shown to be defective in this haembiosynthetic enzyme [Gollub, Liu, Dayan, Adlersberg & Sprinson (1977) J. Biol. Chem. 252, 2846-2854]. The gene was selected from a yeast wild-type genomic DNA library ligated into the shuttle vector YEp13. The complementing gene restored growth of the hem 3 (PBG-D) mutant strain on media in the absence of exogeneous haem or fatty acid and sterol supplements. The recombinant plasmid was retained in the Hem+ transformant provided that selective pressure for plasmid-dependent growth was maintained. Transformation of the mutant strain (hem 3) restored the PBG-D activity to levels up to 10-fold those of the parental strain. The mutant strain GL7 does not show any measurable enzymic activity. Analysis of the plasmid designated YEpPBG-D (containing the PBG-D gene) by hybrid-selected translation revealed that it contained the coding information for a single protein of apparent Mr 43,000. The coding region was localized on an 1.5 kb endonuclease-EcoRI fragment (E4), within the 5.5 kb genomic insert in YEpPBG-D.

Ammonia-Lyases↗
PubMed1986

Nuclear genes encoding the yeast mitochondrial ATPase complex. Analysis of ATP1 coding the F1-ATPase alpha-subunit and its assembly.

Mitochondria prepared from the yeast nuclear pet mutant N9-84 lack a detectable F1-ATPase activity. Genetic complementation of this mutant with a pool of yeast genomic DNA in the yeast Escherichia coli shuttle vector YEp13 restored its growth on a nonfermentable carbon source. Mitochondria prepared from the transformed host contained an 8-fold higher than normal level of the F1 alpha-subunit and restored ATPase activity to 50% that of the wild-type strain. Deletion and nucleotide sequence analysis of the complementing DNA on the plasmid revealed a coding sequence designated ATP1 for a protein of 544 amino acids which exhibits 60 and 54% direct protein sequence homology with the proton-translocating ATPase alpha-subunits from tobacco chloroplast and E. coli, respectively. In vitro expression and mitochondrial import experiments using this ATP1 sequence showed that additional amino-terminal sequences not present in the comparable plant and bacterial subunits function as transient sequences for import.

Amino Acid Sequence↗
PubMed1986

The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal.

The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase. To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S. cerevisiae SUC2 gene (which codes for invertase). The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo. The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle. Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle. Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells.

Amino Acid Sequence↗
PubMed1986

Sequences required for delivery and localization of the ADP/ATP translocator to the mitochondrial inner membrane.

The ADP/ATP translocator, a transmembrane protein of the mitochondrial inner membrane, is coded in Saccharomyces cerevisiae by the nuclear gene PET9. DNA sequence analysis of the PET9 gene showed that it encoded a protein of 309 amino acids which exhibited a high degree of homology with mitochondrial translocator proteins from other sources. This mitochondrial precursor, in contrast to many others, does not contain a transient presequence which has been shown to direct the posttranslational localization of proteins in the organelle. Gene fusions between the PET9 gene and the gene encoding beta-galactosidase (lacZ) were constructed to define the location of sequences necessary for the mitochondrial delivery of the ADP/ATP translocator protein in vivo. These studies reveal that the information to target the hybrid molecule to the mitochondria is present within the first 115 residues of the protein. In addition, these studies suggest that the "import information" of the amino-terminal region of the ADP/ATP translocator precursor is twofold. In addition to providing targeting function of the precursor to the organelle, these amino-terminal sequences act to prevent membrane-anchoring sequences located between residues 78 and 98 from stopping import at the outer mitochondrial membrane. These results are discussed in light of the function of distinct protein elements at the amino terminus of mitochondrially destined precursors in both organelle delivery and correct membrane localization.

Amino Acid Sequence↗
PubMed1985

Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Primary sequence analysis of ATP2 encoding the F1-ATPase beta-subunit precursor.

The yeast nuclear gene ATP2 encodes a F1-ATPase beta-subunit protein of 509 amino acids with a predicted mass of 54,575 daltons. In contrast to the ATPase beta-subunit proteins determined previously from Escherichia coli and various plant sources, the yeast mitochondrial precursor peptide contains a unique cysteine residue within its immediate amino terminus. Expression of an in-frame deletion in ATP2 between residues 28 and 34 to eliminate this single cysteine residue located near the processing site of the matrix protease does not prevent the in vivo delivery of the subunit to mitochondria or its assembly into a functional ATPase complex. Thus, the import F1 beta-subunit into mitochondria does not require a covalent modification of the type utilized for the secretion of the major lipoprotein from E. coli. In addition, analysis of the level of the major F1-ATPase subunits in mitochondria prepared from an atp2- disruption mutant demonstrates that the in vivo import of these catalytic subunits is not dependent on each other. These data and additional studies, therefore, suggest that the determinants for mitochondrial delivery reside within the amino terminus of the individual precursors.

Amino Acid Sequence↗
PubMed1984

Intracellular targeting and import of an F1-ATPase beta-subunit-beta-galactosidase hybrid protein into yeast mitochondria.

The gene coding for the yeast mitochondrial F1-ATPase beta subunit (ATP2) has been fused to the Escherichia coli lacZ gene. The chimeric ATP2-lacZ gene codes for a hybrid protein consisting of some 350 amino acids of the F1-ATPase beta subunit at its amino terminus and a large enzymatically active portion of the lacZ gene product, beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), at its carboxyl terminus. The beta-subunit-beta-galactosidase hybrid protein is expressed in both E. coli and yeast. In yeast, this hybrid molecule is targeted to the mitochondrion and is protected in isolated mitochondria from added protease under conditions in which an outer membrane enzymatic marker is digested. Yeast cells carrying the ATP2-lacZ gene fusion on plasmid p beta Z1 are unable to grow on a nonfermentable carbon source. Upon loss of the p beta Z1 plasmid, growth of the cured host strain on the nonfermentable substrate is restored. In the presence of the beta-subunit-beta-galactosidase hybrid protein, the energy-transducing capacity of the mitochondrial membrane as measured by the 32Pi-ATP exchange reaction is only 9% of that measured in the absence of the gene fusion product. The results indicate that it is the presence of the beta-subunit-beta-galactosidase hybrid protein within mitochondria that interferes with function(s) essential for respiratory growth. These observations open up the prospect of genetic characterization of the signals and cellular machinery responsible for mitochondrial protein delivery.

Amino Acid Sequence↗
PubMed1983

Complementation of a Schizosaccharomyces pombe mutant lacking the beta subunit of the mitochondrial ATPase by the ATP2 gene of Saccharomyces cerevisiae.

A chimeric plasmid carrying the structural gene (ATP2) for the mitochondrial ATPase beta subunit of Saccharomyces cerevisiae has been used to complement a mutant of Schizosaccharomyces pombe lacking the beta subunit (Boutry, M., and Goffeau, A. (1982) Eur. J. Biochem. 125, 471-477). Transformation with ATP2 restored the growth rate of S. pombe mutant on glycerol as well as the mitochondrial ATPase and 32Pi-ATP exchange activities to approximately 20% of the parental strain. Mitochondria prepared from the transformant contained a normal amount of a hybrid F1-ATPase consisting of the S. cerevisiae beta subunit assembled with the remaining subunits of the S. pombe ATPase complex. The presence of the S. cerevisiae beta subunit in the S. pombe ATPase complex conferred a sensitivity to the energy transfer inhibitors citreoviridin and oligomycin which was like that of the intact S. cerevisiae enzyme. The S. cerevisiae beta subunit assembled into the hybrid ATPase complex was the same size as the mature subunit in S. cerevisiae. These data indicate that the mechanism of mitochondrial import and the assembly of the cytoplasmically synthesized subunits is similar or identical in these evolutionary divergent yeasts. In addition, this study provides a new approach for the construction of hybrid mitochondrial ATPase complexes which can be used to examine the function of selected subunits in energy transduction.

Adenosine Triphosphatases↗
PubMed1983

Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Isolation of ATP2 coding the F1-ATPase beta subunit.

A yeast nuclear pet mutant of Saccharomyces cerevisiae lacking any detectable mitochondrial F1-ATPase activity was genetically complemented upon transformation with a pool of wild type genomic DNA fragments carried in the yeast Escherchia coli shuttle vector YEp 13. Plasmid-dependent complementation restored both growth of the pet mutant on a nonfermentable carbon source as well as functional mitochondrial ATPase activity. Characterization of the complementing plasmid by plasmid deletion analysis indicated that the complementing gene was contained on adjoining BamH1 fragments with a combined length of 3.05 kilobases. Gel analysis of the product of this DNA by in vitro translation in a rabbit reticulocyte lysate programmed with yeast mRNA hybrid selected by the plasmid revealed a product which could be immunoprecipitated by antisera against the beta subunit of the yeast mitochondrial ATPase complex. A comparison of the protein sequence derived from partial DNA sequence analysis indicated that the beta subunit of the yeast mitochondrial ATPase complex exhibits greater than 70% conservation of protein sequence when compared to the same subunit from the ATPase of E. coli, beef heart, and chloroplast. The gene coding the beta subunit (subunit 2) of yeast mitochondrial adenosine triphosphatase is designated ATP2. The utilization of cloned nuclear structural genes of mitochondrial proteins for the analysis of the post-translational targeting and import events in organelle assembly is discussed.

Amino Acid Sequence↗
PubMed1983

Molecular cloning of fatty acid synthetase genes from Saccharomyces cerevisiae.

Fatty acid synthetase from Saccharomyces cerevisiae is a multifunctional enzyme which catalyzes the synthesis of long chain fatty acids from acetyl- and malonyl-CoA. The enzyme is composed of two nonidentical subunits, alpha (Mr = 212,000) and beta (Mr = 203,000), which are coded for by two unlinked genes FAS2 and FAS1, respectively. Individual yeast strains containing mutations in either of the FAS genes were transformed with a bank of yeast DNA sequences in the vector YEp13. Plasmids YEpFAS1 and YEpFAS2 were selected by their ability to complement the fas1 or fas2 mutations, respectively. Additionally, we utilized an immunologic screening of a second yeast DNA bank and selected two clones 33F1 and 102B5 which produce antigenically reactive material to anti-yeast fatty acid synthetase antibodies. Through Southern hybridization experiments and restriction endonuclease mapping, a region of 5.3 kilobase pairs of 33F1 was shown to be homologous with YEpFAS1, and a span of 3.4 kilobase pairs of 102B5 was homologous with YEpFAS2. These experiments identify the yeast DNA sequences cloned into 33F1 as originating from the FAS1 gene and those DNA sequences in 102B5, from the FAS2 gene.

Base Composition↗
PubMed1983

Presence and stoichiometry of two forms of subunit 6 of the mitochondrial ATPase complex of yeast.

One of the mitochondrically coded components of the yeast mitochondrial ATPase complex (subunit 6) can be resolved into two components on certain polyacrylamide gels in the presence of sodium dodecyl sulfate. Purification of the ATPase complex from commercially processed yeast as well as immunoprecipitation of the holo-enzyme from cells labeled in vivo with 14C-labeled amino acids demonstrate that both forms of subunit 6 are physically associated with the assembled enzyme and present in two copies each per complex. One-dimensional papain-generated peptide maps of the two components are identical except for the mobility of a single fragment. It is concluded that the two components of subunit 6 are different forms of a single protein and are present on an average of two copies each per complex.

Adenosine Triphosphatases↗
PubMed1983

A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast.

Polyethylene glycol (PEG) can induce genetic transformation in both bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) without cell wall removal. PEG-mediated transformation of E. coli is technically simple and yields transformants with an efficiency of 10(6)-10(7) transformants/microgram DNA. Detailed analysis of the parameters involved in PEG-mediated transformation of E. coli reveals basic differences between the PEG and standard CaCl2 methods for transformation of E. coli. PEG-mediated transformation of yeast is far simpler than existing protoplast methods and is comparable in efficiency. The new methods described here for PEG-mediated genetic transformation may prove to be of general utility in performing genetic transformation in a wide variety of organisms.

Cations↗
PubMed1982

A neutral metallo endoprotease involved in the processing of an F1-ATPase subunit precursor in mitochondria.

A post-translational processing assay of the precursor to the yeast F1-ATPase subunit has been utilized to examine a mitochondrial endoprotease which cleaves this subunit precursor to the size of a mature subunit. The endoprotease is extracted from purified mitochondria as a soluble complex of Mr = 115,000 which is composed of subunits of lower molecular weight when examined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It exhibits a pH optimum of between pH 7 and 8 and is inactive at pH 6.5 and below. The mitochondrial endoprotease is insensitive to serine esterase inhibitors, but is inhibited by EDTA and o-phenanthroline. Restoration of precursor subunit processing activity in the presence of metal chelators is strictly dependent on excess Co2+ and Mn2+ over other heavy metals examined. These and additional data indicate that this soluble metallo endoprotease is involved in the processing of other cytoplasmically synthesized precursor subunits of the ATPase complex in addition to the subunit 2 precursor. The role of this processing enzyme in the assembly of mitochondrial inner membrane complexes is discussed in light of the current model of mitochondrial biogenesis.

Adenosine Triphosphatases↗