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L McIntosh

Publications and source records attributed to L McIntosh.

At least 55 records · Page 3Linked to original sources
PubMed1992

Lower growth temperature increases alternative pathway capacity and alternative oxidase protein in tobacco.

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30 degrees C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18 degrees C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30 degrees C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18 degrees C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein.

Journal Article↗
PubMed1992

Coordinate regulation of cytochrome and alternative pathway respiration in tobacco.

In suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow), inhibition of the cytochrome pathway of respiration with antimycin A induced a large increase in the capacity of the alternative pathway over a period of approximately 12 h, as confirmed in both whole cells and isolated mitochondria. The increase in alternative pathway capacity required de novo RNA and protein synthesis and correlated closely with the increase of a 35-kD alternative oxidase protein. When the cytochrome pathway of intact cells was inhibited by antimycin A, respiration proceeded exclusively through the alternative pathway, reached rates significantly higher than before antimycin A addition, and was not stimulated by p-trifluoromethoxycarbonylcyanide (FCCP). When inhibition of the cytochrome pathway was relieved, alternative pathway capacity and the level of the 35-kD alternative oxidase protein declined. Respiration rate also declined and could once again be stimulated by FCCP. These observations show that the capacities of the mitochondrial electron transport pathways can be regulated in a coordinate fashion.

Journal Article↗
PubMed1991

Isolation and characterization of a cDNA clone encoding an alternative oxidase protein of Sauromatum guttatum (Schott).

Polyclonal and monoclonal antibodies that recognize the 35-, 36-, and 37-kDa alternative oxidase proteins of Sauromatum guttatum (Schott) were used to isolate a cDNA clone, pAOSG81, from an S. guttatum cDNA expression library. A fusion protein with an apparent molecular mass of 48 kDa was expressed from a pUC119 derivative of pAOSG81 (pAOSG81-119) in Escherichia coli cells and was recognized by the monoclonal antibodies. When the in vitro translated and immunoprecipitated products made from mRNA hybrid-selected by pAOSG81 were analyzed, a single band corresponding to a protein with an apparent molecular mass of 42 kDa was observed. DNA sequence characterization showed that pAOSG81 contains the entire coding region of a protein with a calculated molecular mass of 38.9 kDa, a putative 63-amino acid transit peptide, and a 9-amino acid match to the authentic N-terminal sequence of the 36-kDa alternative oxidase protein. Analyses of the deduced amino acid sequence indicate: (i) that the transit peptide is predicted to form amphiphilic helices, and (ii) that three regions of the processed protein are likely to form transmembrane alpha-helices. We conclude from these data that pAOSG81 represents a nuclear gene, aox1, encoding a precursor protein of one or more of the alternative oxidase proteins of S. guttatum.

Amino Acid Sequence↗
PubMed1991

Targeted genetic inactivation of the photosystem I reaction center in the cyanobacterium Synechocystis sp. PCC 6803.

We describe the first complete segregation of a targeted inactivation of psaA encoding one of the P700-chlorophyll a apoproteins of photosystem (PS) I. A kanamycin resistance gene was used to interrupt the psaA gene in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Selection of a fully segregated mutant, ADK9, was performed under light-activated heterotrophic growth (LAHG) conditions; complete darkness except for 5 min of light every 24 h and 5 mM glucose. Under these conditions, wild-type cells showed a 4-fold decrease in chlorophyll (chl) per cell, primarily due to a decrease of PS I reaction centers. Evidence for the absence of PS I in ADK9 includes: the lack of EPR (electron paramagnetic resonance) signal I, from P700+; undetectable P700-apoprotein; greatly reduced whole-chain photosynthesis rates; and greatly reduced chl per cell, resulting in a turquoise blue phenotype. The PS I peripheral proteins PSA-C and PSA-D were not detected in this mutant. ADK9 does assemble near wild-type levels of functional PS II per cell, evidenced by: EPR signal II from YD+; high rates of oxygen evolution with 2,6-dichloro-p-benzoquinone (DCBQ), an electron acceptor from PS II; and accumulation of D1, a PS II core polypeptide. The success of this transformation indicates that this cyanobacterium may be utilized for site-directed mutagenesis of the PS I core.

Autoradiography↗
PubMed1991

Partial conservation of the 5' ndhE-psaC-ndhD 3' gene arrangement of chloroplasts in the cyanobacterium Synechocystis sp. PCC 6803: implications for NDH-D function in cyanobacteria and chloroplasts.

The psaC gene, which encodes the 8.9 kDa iron-sulfur containing subunit of Photosystem I, has been sequenced from Synechocystis sp. PCC 6803 and shows greater similarity to reported plant sequences than other cyanobacterial psaC sequences. The deduced amino acid sequence of the protein encoded by the Synechocystis psaC gene is identical to the tobacco PSA-C sequence. In plants psaC is located in the small single-copy region of the chloroplast genome between two genes (designated ndhE and ndhD) with similarity to genes encoding subunits of the mitochondrial NADH Dehydrogenase Complex I. The 5' ndhE-psaC-ndhD3' gene arrangement of higher plants is only partially conserved in Synechocystis. An open reading frame (ORF) upstream of the Synechocystis psaC gene has 85% identity to the tobacco ndhE gene. Downstream of psaC there is a 273 bp ORF with 48% identity to the 5' portion of the tobacco ndhD gene (1527 bp). psaC, ndhE and the region of similarity to ndhD are present in a single copy in the Synechocystis genome. Part of the wheat ndhD gene was sequenced and used as a probe for the presence of the 3' portion of the ndhD gene. The wheat ndhD probe did not hybridize to Synechocystis or Anabaena sp. PCC 7120 genomic DNA, but did hybridize to Oenothera chloroplast DNA. These results indicate the complete ndhD gene is absent in two cyanobacteria, and raises the question of what role, if any, the ndhD gene product plays in the facultative heterotroph Synechocystis sp. PCC 6803.

Amino Acid Sequence↗
PubMed1991

Expression of photosynthesis genes in the cyanobacterium Synechocystis sp. PCC 6803: psaA-psaB and psbA transcripts accumulate in dark-grown cells.

We have cloned and sequenced the psaA and psaB genes from the unicellular cyanobacterium Synechocystis sp. PCC 6803. These genes are arranged in tandem, are co-transcribed, and are highly homologous to the psaA and psaB genes previously characterized. RNA was isolated from light-grown cells, from cells put in total darkness with and without glucose, and from cells grown under light-activated heterotrophic growth (LAHG) conditions. Quantitation of hybridization to northern blots revealed only a slight decrease in the accumulation of the psaA-psaB transcript in cells grown in complete darkness with glucose and in LAHG cells, relative to light-grown cells. Accumulation of the psbA transcript steadily declines through dark incubation, with a steady-state level in LAHG cells 28% of that in light-grown cells. Transcripts from psbD, psaD, and rbcLS accumulate in cells grown in complete darkness and in LAHG cells to approximately the same levels as in light-grown cells. Photosynthesis gene transcripts in cells grown in the dark without glucose were detected, but were highly degraded. Our data prove that transcripts from photosynthesis genes do accumulate in dark-grown Synechocystis 6803, which may allow for synthesis and assembly of photosystem (PS) I and PS II in the dark.

Amino Acid Sequence↗
PubMed1991

The cytochrome oxidase II gene in mitochondria of the sugar-beet Beta vulgaris L.

We have cloned and analyzed the sugar-beet mitochondrial gene for cytochrome oxidase subunit II (coxII). The sugar-beet and its deduced amino acid sequence were compared to its homologous coxII gene sequences from both monocot and dicot plants. It was found to be highly conserved (89-95%) compared to homologue in other plant species. The 780 bp coding sequence of the sugar beet coxII gene is interrupted at position 383 by a 1463 bp intron. This intron contains an additional 107 bp sequence that is not found in any of the plant coxII genes studied thus far. The structure of the intron suggests that a large intron existed in an ancestral coxII gene before monocots and dicots diverged in evolution. Three CGG codons in the sugar-beet coxII coding sequence align with conserved tryptophan residues in the homologous gene of other species, suggesting that RNA editing takes place also in sugar-beet mitochondria. In 13 out of 24 codons of coxII mRNA that were found to be edited in four other plants, the sugar-beet gene already utilizes the edited codons. This phenomenon may indicate that the mitochondrial genome in sugar-beet is phylogenetically more archaic relative to these plants. An additional sequence of 279 bp that is identical to the first exon of coxII was identified in the mtDNA of the sugar-beet. This 'pseudo-gene' is transcribed and its existence in the mitochondrial genome is unexplained.

Amino Acid Sequence↗
PubMed1991

Light-activated heterotrophic growth of the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring process.

A glucose-tolerant strain of Synechocystis sp. strain 6803 will not grow on glucose under complete darkness unless given a daily pulse of white light, typically 5 min of 40 mumol m-2 s-1 (light-pulsed conditions). The light pulse is insufficient for photoautotrophy, as glucose is required and growth yield is dependent on glucose concentration. Growth rate is independent of fluence, but growth yield is dependent on fluence, saturating at 40 to 75 mumol m-2 s-1. A Synechocystis strain 6803 psbA mutant strain grows under light-pulsed conditions at rates similar to those for the glucose-tolerant strain, indicating that photosystem II is not required for growth. The relative spectral sensitivity of the growth of light-pulsed cultures (growth only in blue light, 400 to 500 nm, maximum at 450 nm) precludes energetic contribution from cyclic electron transport around photosystem I. Pulses of long-wavelength light (i.e., 550 and 650 nm) did not support the growth of Synechocystis strain 6803 and, when supplied before or after a blue-light pulse, did not inhibit blue-light-stimulated growth of Synechocystis strain 6803. We conclude that the required blue-light pulse does not support growth via photosynthetic electron transport but appears instead to function as an environmental signal regulating heterotrophic metabolism, cell division, or other photomorphogenic processes. We have termed the growth of Synechocystis strain 6803 pulsed with light and kept otherwise in complete darkness light-activated heterotrophic growth. This observation of a blue-light requirement for the growth of Synechocystis strain 6803 represents a novel blue light effect on the growth of a cyanobacterium.

Cyanobacteria↗
PubMed1990

Copper-induced expression, cloning, and regulatory studies of the plastocyanin gene from the cyanobacterium Synechocystis sp. PCC 6803.

Plastocyanin can be detected in Synechocystis sp. PCC 6803 when 3 microM copper is added to the growth medium, BG-11. The plastocyanin gene (petE) was cloned from a genomic lambda EMBL 3 library by screening with the petE gene from Anabaena sp. PCC 7937. The Synechocystis 6803 petE gene is present as a single copy and, as deduced from the DNA sequence, encodes a precursor protein of 126 amino acids. The predicted 29 amino acid transit peptide shows substantial homology to the Anabaena 7937 transit peptide, thought to direct the plastocyanin precursor to the thylakoid lumen. Putative promoter sites -16 and -38 base pairs from the start of the petE gene have been identified. The deduced amino acid sequence has the greatest homology (61%) to the green alga Scenedemus obliquus plastocyanin. Despite the lower homology, the copper binding residues and certain aromatic residues remain highly conserved. Northern hybridization analysis indicates that the Synechocystis sp. PCC 6803 petE gene is not transcriptionally regulated since the accumulation of petE mRNA appears to be independent of the copper concentration in the growth media. The possibility of an additional polypeptide needed to facilitate the electron transfer from plastocyanin to P700+ is also discussed.

Amino Acid Sequence↗
PubMed1990

Residues in three conserved regions of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase are required for quaternary structure.

To explore the role of individual residues in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), small subunits with single amino acid substitutions in three regions of relative sequence conservation were produced by directed mutagenesis of the rbcS gene from Anabaena 7120. These altered small subunits were cosynthesized with large subunits (from an expressed Anabaena rbcL gene) in Escherichia coli. Mutants were analyzed for effects on quaternary structure and catalytic activity. Changing Glu-13S (numbering used is that of the spinach enzyme) to Val, Trp-67S to Arg, Pro-73S to His, or Tyr-98S to Asn prevented accumulation of stable holoenzyme. Interpretation of these results using a model for the three-dimensional structure of the spinach enzyme based on x-ray crystallographic data suggests that our small subunit mutants containing substitutions at positions 13S and 67S probably do not assemble because of mispairing or nonpairing of charged residues on the interfacing surfaces of the large and small subunits. The failure of small subunits substituted at positions 73S or 98S to assemble correctly may result from disruption of intersubunit or intrasubunit hydrophobic pockets, respectively.

Amino Acid Sequence↗
PubMed1990

Alternative Oxidase of Potato Is an Integral Membrane Protein Synthesized de Novo during Aging of Tuber Slices.

The rise in alternative respiratory capacity upon aging of potato (Solanum tuberosum) tuber slices is correlated with changes in mitochondrial membrane protein composition and a requirement for cytoplasmic protein synthesis. However, the lack of an antibody specific to the alternative oxidase has, until recently, prevented examination of the alternative oxidase protein(s) itself. We have employed a monoclonal antibody raised against the Sauromatum guttatum alternative oxidase to investigate developmental changes in the alternative pathway of aging potato slice mitochondria and to characterize the potato alternative oxidase by one- and two-dimensional gel electrophoresis. The relative levels of a 36 kilodalton protein parallel the rise in alternative path capacity. A plausible interpretation is that this alternative oxidase protein is synthesized de novo during aging of potato slices.

Journal Article↗
PubMed1989

Monoclonal antibodies to the alternative oxidase of higher plant mitochondria.

The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain which terminates with cytochrome oxidase, an alternative pathway that terminates with an alternative oxidase. The alternative oxidase of Sauromatum guttatum Schott has recently been identified as a cluster of proteins with apparent M(r) of 37, 36, and 35 kilodaltons (kD). Monoclonal antibodies have now been prepared to these proteins and designated as AOA (binding all three proteins of the alternative oxidase cluster), AOU (binding the upper or 37 kD protein), and AOL (binding the lower or 36 and 35 kD proteins). All three antibodies bind to their respective alternative oxidase proteins whether the proteins are in their native or denatured states (as on protein blots). AOA and AOU inhibit alternative oxidase activity around 49%, whereas AOL inhibits activity only 14%. When coupled individually to Sepharose 4B, all three monoclonal resins were capable of retaining the entire cluster of alternative oxidase proteins, suggesting that these proteins are physically associated in some manner. The monoclonals were capable of binding similar mitochondrial proteins in a number of thermogenic and nonthermogenic species, indicating that they will be useful in characterizing and purifying the alternative oxidase of different systems. The ability of the monoclonal-Sepharose 4B resins to retain the cluster of previously identified alternative oxidase proteins, along with the inhibition of alternative oxidase activity by these monoclonals, supports the role of these proteins in constituting the alternative oxidase.

Journal Article↗
PubMed1989

Immunological identification of the alternative oxidase of Neurospora crassa mitochondria.

Neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. We used a monoclonal antibody to the alternative oxidase of the higher plant Sauromatum guttatum to identify a similar set of related polypeptides (Mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of N. crassa mitochondria. These polypeptides were not present constitutively in the mitochondria of a wild-type N. crassa strain, but were produced in high amounts under conditions that induced alternative oxidase activity. Under the same conditions, mutants in the aod-1 gene, with one exception, produced apparently inactive alternative oxidase polypeptides, whereas mutants in the aod-2 gene failed to produce these polypeptides. The latter findings support the hypothesis that aod-1 is a structural gene for the alternative oxidase and that the aod-2 gene encodes a component that is required for induction of alternative oxidase activity. Finally, our results indicate that the alternative oxidase is highly conserved, even between plant and fungal species.

Antibodies, Monoclonal↗
PubMed1988

Directed mutagenesis indicates that the donor to P+680 in photosystem II is tyrosine-161 of the D1 polypeptide.

Photosystem II contains two redox-active tyrosines. One of these, YZ, reduces the reaction center chlorophyll, P680, and transfers the oxidizing equivalent to the oxygen-evolving complex. The second, YD, has a long-lived free radical state of unknown function. We recently established that YD is Tyr-160 of the D2 polypeptide by site-directed mutagenesis of a psbD gene in the unicellular cyanobacterium Synechocystis 6803 [Debus, R. J., Barry, B. A., Babcock, G. T., & McIntosh, L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 427-430]. YZ is most likely the symmetry-related Tyr-161 of the D1 polypeptide. To test this hypothesis, we have changed Tyr-161 to phenylalanine by site-directed mutagenesis of a psbA gene in Synechocystis. The resulting mutant assembles PSII, as judged by its ability to produce the stable Y+D radical, but is unable to grow photosynthetically and exhibits altered fluorescence properties. The nature of the fluorescence change indicates that forward electron transfer to P+680 is disrupted in the mutant. These results provide strong support for our identification of Tyr-161 in the D1 polypeptide with YZ.

Chlorophyll↗
PubMed1988

Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthetic oxygen-evolving system.

Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P+.680, while D+. gives rise to the dark-stable EPR spectrum known as signal II. D+. has recently been shown to be a tyrosine radical. Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and D2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, we have changed Tyr-160 of the D2 polypeptide to phenylalanine by site-directed mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D+.. We conclude that D is Tyr-160 of the D2 polypeptide. We suggest that the C2 symmetry in photosystem II extends beyond P680 to its immediate electron donor and conclude that Z is Tyr-161 of the D1 polypeptide.

Chlorophyll↗