Regulation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by Carbamylation and 2-Carboxyarabinitol 1-Phosphate in Tobacco: Insights from Studies of Antisense Plants Containing Reduced Amounts of Rubisco Activase1

The regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity by 2-carboxyarabinitol 1-phosphate (CA1P) was investigated using gas-exchange analysis of antisense tobacco (Nicotiana tabacum) plants containing reduced levels of Rubisco activase. When an increase in light flux from darkness to 1200 μmol quanta m−2 s−1 was followed, the slow increase in CO2 assimilation by antisense leaves contained two phases: one represented the activation of the noncarbamylated form of Rubisco, which was described previously, and the other represented the activation of the CA1P-inhibited form of Rubisco. We present evidence supporting this conclusion, including the observation that this second phase, like CA1P, is only present following darkness or very low light flux. In addition, the second phase of CO2 assimilation was correlated with leaf CA1P content. When this novel phase was resolved from the CO2 assimilation trace, most of it was found to have kinetics similar to the activation of the noncarbamylated form of Rubisco. Additionally, kinetics of the novel phase indicated that the activation of the CA1P-inhibited form of Rubisco proceeds faster than the degradation of CA1P by CA1P phosphatase. These results may be significant with respect to current models of the regulation of Rubisco activity by Rubisco activase.

Rubisco activase constrains the photosynthetic potential of leaves at high temperature and CO2

Net photosynthesis (Pn) is inhibited by moderate heat stress. To elucidate the mechanism of inhibition, we examined the effects of temperature on gas exchange and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation in cotton and tobacco leaves and compared the responses to those of the isolated enzymes. Depending on the CO2 concentration, Pn decreased when temperatures exceeded 35–40°C. This response was inconsistent with the response predicted from the properties of fully activated Rubisco. Rubisco deactivated in leaves when temperature was increased and also in response to high CO2 or low O2. The decrease in Rubisco activation occurred when leaf temperatures exceeded 35°C, whereas the activities of isolated activase and Rubisco were highest at 42°C and >50°C, respectively. In the absence of activase, isolated Rubisco deactivated under catalytic conditions and the rate of deactivation increased with temperature but not with CO2. The ability of activase to maintain or promote Rubisco activation in vitro also decreased with temperature but was not affected by CO2. Increasing the activase/Rubisco ratio reduced Rubisco deactivation at higher temperatures. The results indicate that, as temperature increases, the rate of Rubisco deactivation exceeds the capacity of activase to promote activation. The decrease in Rubisco activation that occurred in leaves at high CO2 was not caused by a faster rate of deactivation, but by reduced activase activity possibly in response to unfavorable ATP/ADP ratios. When adjustments were made for changes in activation state, the kinetic properties of Rubisco predicted the response of Pn at high temperature and CO2.

Moderately High Temperatures Inhibit Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase-Mediated Activation of Rubisco1

We tested the hypothesis that light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inhibited by moderately elevated temperature through an effect on Rubisco activase. When cotton (Gossypium hirsutum L.) or wheat (Triticum aestivum L.) leaf tissue was exposed to increasing temperatures in the light, activation of Rubisco was inhibited above 35 and 30°C, respectively, and the relative inhibition was greater for wheat than for cotton. The temperature-induced inhibition of Rubisco activation was fully reversible at temperatures below 40°C. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45°C. Nonphotochemical fluorescence quenching increased at temperatures that inhibited Rubisco activation, consistent with inhibition of Calvin cycle activity. Initial and maximal chlorophyll fluorescence were not significantly altered until temperatures exceeded 40°C. Thus, electron transport, as measured by Chl fluorescence, appeared to be more stable to moderately elevated temperatures than Rubisco activation. Western-blot analysis revealed the formation of high-molecular-weight aggregates of activase at temperatures above 40°C for both wheat and cotton when inhibition of Rubisco activation was irreversible. Physical perturbation of other soluble stromal enzymes, including Rubisco, phosphoribulokinase, and glutamine synthetase, was not detected at the elevated temperatures. Our evidence indicates that moderately elevated temperatures inhibit light activation of Rubisco via a direct effect on Rubisco activase.

Nuclear-Gene Mutations Suppress a Defect in the Expression of the Chloroplast-Encoded Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase1

The green alga Chlamydomonas reinhardtii mutant 76–5EN lacks photosynthesis because of a nuclear-gene mutation that specifically inhibits expression of the chloroplast gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39). Photosynthesis-competent revertants were selected from mutant 76–5EN to explore the possibility of increasing Rubisco expression. Genetic analysis of 10 revertants revealed that most arose from suppressor mutations in nuclear genes distinct from the original 76–5EN mutant gene. The revertant strains have regained various levels of Rubisco holoenzyme, but none of the suppressor mutations increased Rubisco expression above the wild-type level in either the presence or absence of the 76–5EN mutation. One suppressor mutation, S107–4B, caused a temperature-conditional, photosynthesis-deficient phenotype in the absence of the original 76–5EN mutation. The S107–4B strain was unable to grow photosynthetically at 35°C, but it expressed a substantial level of Rubisco holoenzyme. Whereas the 76–5EN gene encodes a nuclear factor that appears to be required for the transcription of the Rubisco large-subunit gene, the S107–4B nuclear gene may be required for the expression of other chloroplast genes.

The lethal mutation of the mouse wasted (wst) is a deletion that abolishes expression of a tissue-specific isoform of translation elongation factor 1α, encoded by the Eef1a2 gene

We have identified the mutation responsible for the autosomal recessive wasted (wst) mutation of the mouse. Wasted mice are characterized by wasting and neurological and immunological abnormalities starting at 21 days after birth; they die by 28 days. A deletion of 15.8 kb in wasted mice abolishes expression of a gene called Eef1a2, encoding a protein that is 92% identical at the amino acid level to the translation elongation factor EF1α (locus Eef1a). We have found no evidence for the involvement of another gene in this deletion. Expression of Eef1a2 is reciprocal with that of Eef1a. Expression of Eef1a2 takes over from Eef1a in heart and muscle at precisely the time at which the wasted phenotype becomes manifest. These data suggest that there are tissue-specific forms of the translation elongation apparatus essential for postnatal survival in the mouse.

An isoform of the rod photoreceptor cyclic nucleotide-gated channel β subunit expressed in olfactory neurons

Sensory transduction in olfactory neurons involves the activation of a cyclic nucleotide-gated (CNG) channel by cAMP. Previous studies identified a CNG channel α subunit (CNG2) and a β subunit (CNG5), which when heterologously expressed form a channel with properties similar but not identical to those of native olfactory neurons. We have cloned a new type of CNG channel β subunit (CNG4.3) from rat olfactory epithelium. CNG4.3 derives from the same gene as the rod photoreceptor β subunit (CNG4.1) but lacks the long, glutamic acid-rich domain found in the N terminus of CNG4.1. Northern blot and in situ hybridization revealed that CNG4.3 is expressed specifically in olfactory neurons. Expression of CNG4.3 in human embryonic kidney 293 cells did not lead to detectable currents. Coexpression of CNG4.3 with CNG2 induced a current with significantly increased sensitivity for cAMP whereas cGMP affinity was not altered. Additionally, CNG4.3 weakened the outward rectification of the current in the presence of extracellular Ca2+, decreased the relative permeability for Ca2+, and enhanced the sensitivity for l-cis diltiazem. Upon coexpression of CNG2, CNG4.3, and CNG5, a conductance with a cAMP sensitivity greater than that of either the CNG2/CNG4.3 or the CNG2/CNG5 channel and near that of native olfactory channel was observed. Our data suggest that CNG4.3 forms a subunit of the native olfactory CNG channel. The expression of various CNG4 isoforms in retina and olfactory epithelium indicates that the CNG4 subunit may be necessary for normal function of both photoreceptor and olfactory CNG channels.

Cloning and characterization of a plastidal and a mitochondrial isoform of tobacco protoporphyrinogen IX oxidase

Protoporphyrinogen IX oxidase is the last enzyme in the common pathway of heme and chlorophyll synthesis and provides precursor for the mitochondrial and plastidic heme synthesis and the predominant chlorophyll synthesis in plastids. We cloned two different, full-length tobacco cDNA sequences by complementation of the protoporphyrin-IX-accumulating Escherichia coli hemG mutant from heme auxotrophy. The two sequences show similarity to the recently published Arabidopsis PPOX, Bacillus subtilis hemY, and to mammalian sequences encoding protoporphyrinogen IX oxidase. One cDNA sequence encodes a 548-amino acid residues protein with a putative transit sequence of 50 amino acid residues, and the second cDNA encodes a protein of 504 amino acid residues. Both deduced protein sequences share 27.2% identical amino acid residues. The first in vitro translated protoporphyrinogen IX oxidase could be translocated to plastids, and the approximately 53-kDa mature protein was detected in stroma and membrane fraction. The second enzyme was targeted to mitochondria without any detectable reduction in size. Localization of both enzymes in subcellular fractions was immunologically confirmed. Steady-state RNA analysis indicates an almost synchronous expression of both genes during tobacco plant development, greening of young seedlings, and diurnal and circadian growth. The mature plastidal and the mitochondrial isoenzyme were overexpressed in E. coli. Bacterial extracts containing the recombinant mitochondrial enzyme exhibit high protoporphyrinogen IX oxidase activity relative to control strains, whereas the plastidal enzyme could only be expressed as an inactive peptide. The data presented confirm a compartmentalized pathway of tetrapyrrole synthesis with protoporphyrinogen IX oxidase in plastids and mitochondria.

Cloning of the cDNA encoding the large subunit of human RNase HI, a homologue of the prokaryotic RNase HII

Two RNases H of mammalian tissues have been described: RNase HI, the activity of which was found to rise during DNA replication, and RNase HII, which may be involved in transcription. RNase HI is the major mammalian enzyme representing around 85% of the total RNase H activity in the cell. By using highly purified calf thymus RNase HI we identified the sequences of several tryptic peptides. This information enabled us to determine the sequence of the cDNA coding for the large subunit of human RNase HI. The corresponding ORF of 897 nt defines a polypeptide of relative molecular mass of 33,367, which is in agreement with the molecular mass obtained earlier by SDS/PAGE. Expression of the cloned ORF in Escherichia coli leads to a polypeptide, which is specifically recognized by an antiserum raised against calf thymus RNase HI. Interestingly, the deduced amino acid sequence of this subunit of human RNase HI displays significant homology to RNase HII from E. coli, an enzyme of unknown function and previously judged as a minor activity. This finding suggests an evolutionary link between the mammalian RNases HI and the prokaryotic RNases HII. The idea of a mammalian RNase HI large subunit being a strongly conserved protein is substantiated by the existence of homologous ORFs in the genomes of other eukaryotes and of all eubacteria and archaebacteria that have been completely sequenced.

Preferential activation of the p46 isoform of JNK/SAPK in mouse macrophages by TNFα

A pleiotropic cytokine, tumor necrosis factor-α (TNFα), regulates the expression of multiple macrophage gene products and thus contributes a key role in host defense. In this study, we have investigated the specificity and mechanism of activation of members of the c-Jun-NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of mitogen-activated protein kinases (MAPKs) in mouse macrophages in response to stimulation with TNFα. Exposure of macrophages to TNFα stimulated a preferential increase in catalytic activity of the p46 JNK/SAPK isoform compared with the p54 JNK/SAPK isoform as determined by: (i) separation of p46 and p54 JNK/SAPKs by anion exchange liquid chromatography and (ii) selective immunodepletion of the p46 JNK/SAPK from macrophage lysates. To investigate the level of regulation of p46 JNK/SAPK activation, we determined the ability of MKK4/SEK1/JNKK, an upstream regulator of JNK/SAPKs, to phosphorylate recombinant kinase-inactive p46 and p54 JNK/SAPKs. Endogenous MKK4 was able to transphosphorylate both isoforms. In addition, both the p46 and p54 JNK/SAPK isoforms were phosphorylated on their TPY motif in response to TNFα stimulation as reflected by immunoblotting with a phospho-specific antibody that recognizes both kinases. Collectively, these results suggest that the level of control of p46 JNK/SAPK activation is distal not only to MKK4 but also to the p54 JNK/SAPK. Preferential isoform activation within the JNK/SAPK subfamily of MAPKs may be an important mechanism through which TNFα regulates macrophage phenotypic heterogeneity and differentiation.

A bcr-3 isoform of RARα-PML potentiates the development of PML-RARα-driven acute promyelocytic leukemia

Acute promyelocytic leukemia (APML) most often is associated with the balanced reciprocal translocation t(15;17) (q22;q11.2) and the expression of both the PML-RARα and RARα-PML fusion cDNAs that are formed by this translocation. In this report, we investigated the biological role of a bcr-3 isoform of RARα-PML for the development of APML in a transgenic mouse model. Expression of RARα-PML alone in the early myeloid cells of transgenic mice did not alter myeloid development or cause APML, but its expression significantly increased the penetrance of APML in mice expressing a bcr-1 isoform of PML-RARα (15% of animals developed APML with PML-RARα alone vs. 57% with both transgenes, P < 0.001). The latency of APML development was not altered substantially by the expression of RARα-PML, suggesting that it does not behave as a classical “second hit” for development of the disease. Leukemias that arose from doubly transgenic mice were less mature than those from PML-RARα transgenic mice, but they both responded to all-trans retinoic acid in vitro. These findings suggest that PML-RARα drives the development of APML and defines its basic phenotype, whereas RARα-PML potentiates this phenotype via mechanisms that are not yet understood.

A Splice-Isoform of Vesicle-associated Membrane Protein-1 (VAMP-1) Contains a Mitochondrial Targeting Signal

Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell.

Overexpression of the large subunit of the protein Ku suppresses metallothionein-I induction by heavy metals

Metallothioneins (MT) are involved in the scavenging of the toxic heavy metals and protection of cells from reactive oxygen intermediates. To investigate the potential role of the protein Ku in the expression of MT, we measured the level of MT-I mRNA in the parental rat fibroblast cell line (Rat 1) and the cell lines that stably and constitutively overexpress the small subunit, the large subunit, and the heterodimer of Ku. Treatment with CdS04 or ZnS04 elevated the MT-I mRNA level 20- to 30-fold in the parental cells and the cells (Ku-70) that overproduce the small subunit or those (Ku-7080) overexpressing the heterodimer. By contrast, the cells (Ku-80) overexpressing the large subunit of Ku failed to induce MT-I. In vitro transcription assay showed that the MT-I promoter activity was suppressed selectively in the nuclear extracts from Ku-80 cells. The specificity of the repressor function was shown by the induction of hsp 70, another Cd-inducible gene, in Ku-80 cells. Addition of the nuclear extract from Ku-80 cells at the start of the transcription reaction abolished the MT-l promoter activity in the Rat 1 cell extract. The transcript once formed in Rat 1 nuclear extract was not degraded by further incubation with the extract from Ku-80 cells. The repressor was sensitive to heat. The DNA-binding activities of at least four transcription factors that control the MT-I promoter activity were not affected in Ku-80 cells. These observations have set the stage for further exploration of the mechanisms by which the Ku subunit mediates suppression of MT induction.

Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase

γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, is synthesized by two glutamate decarboxylase isoforms, GAD65 and GAD67. The separate role of the two isoforms is unknown, but differences in saturation with cofactor and subcellular localization suggest that GAD65 may provide reserve pools of GABA for regulation of inhibitory neurotransmission. We have disrupted the gene encoding GAD65 and backcrossed the mutation into the C57BL/6 strain of mice. In contrast to GAD67−/− animals, which are born with developmental abnormalities and die shortly after birth, GAD65−/− mice appear normal at birth. Basal GABA levels and holo-GAD activity are normal, but the pyridoxal 5′ phosphate-inducible apo-enzyme reservoir is significantly decreased. GAD65−/− mice develop spontaneous seizures that result in increased mortality. Seizures can be precipitated by fear or mild stress. Seizure susceptibility is dramatically increased in GAD65−/− mice backcrossed into a second genetic background, the nonobese diabetic (NOD/LtJ) strain of mice enabling electroencephalogram analysis of the seizures. The generally higher basal brain GABA levels in this backcross are significantly decreased by the GAD65−/− mutation, suggesting that the relative contribution of GABA synthesized by GAD65 to total brain GABA levels is genetically determined. Seizure-associated c-fos-like immunoreactivity reveals the involvement of limbic regions of the brain. These data suggest that GABA synthesized by GAD65 is important in the dynamic regulation of neural network excitability, implicate at least one modifier locus in the NOD/LtJ strain, and present GAD65−/− animals as a model of epilepsy involving GABA-ergic pathways.

Identification of a Novel Isoform of the Chloroplast-Coupling Factor α-Subunit 1

Studies were conducted to identify a 64-kD thylakoid membrane protein of unknown function. The protein was extracted from chloroplast thylakoids under low ionic strength conditions and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four peptides generated from the proteolytic cleavage of the wheat 64-kD protein were sequenced and found to be identical to internal sequences of the chloroplast-coupling factor (CF1) α-subunit. Antibodies for the 64-kD protein also recognized the α-subunit of CF1. Both the 64-kD protein and the 61-kD CF1 α-subunit were present in the monocots barley (Hordeum vulgare), maize (Zea mays), oat (Avena sativa), and wheat (Triticum aestivum); but the dicots pea (Pisum sativum), soybean (Glycine max Merr.), and spinach (Spinacia oleracea) contained only a single polypeptide corresponding to the CF1 α-subunit. The 64-kD protein accumulated in response to high irradiance (1000 μmol photons m−2 s−1) and declined in response to low irradiance (80 μmol photons m−2 s−1) treatments. Thus, the 64-kD protein was identified as an irradiance-dependent isoform of the CF1 α-subunit found only in monocots. Analysis of purified CF1 complexes showed that the 64-kD protein represented up to 15% of the total CF1 α-subunit.