A Functional Calvin Cycle Is Not Indispensable for the Light Activation of C4 Phosphoenolpyruvate Carboxylase Kinase and Its Target Enzyme in the Maize Mutant bundle sheath defective2-mutable11

We used a pale-green maize (Zea mays L.) mutant that fails to accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to test the working hypothesis that the regulatory phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) by its Ca2+-insensitive protein-serine/threonine kinase (PEPC kinase) in the C4 mesophyll cytosol depends on cross-talk with a functional Calvin cycle in the bundle sheath. Wild-type (W22) and bundle sheath defective2-mutable1 (bsd2-m1) seeds were grown in a controlled environment chamber at 100 to 130 μmol m−2 s−1 photosynthetic photon flux density, and leaf tissue was harvested 11 d after sowing, following exposure to various light intensities. Immunoblot analysis showed no major difference in the amount of polypeptide present for several mesophyll- and bundle-sheath-specific photosynthetic enzymes apart from Rubisco, which was either completely absent or very much reduced in the mutant. Similarly, leaf net CO2-exchange analysis and in vitro radiometric Rubisco assays showed that no appreciable carbon fixation was occurring in the mutant. In contrast, the sensitivity of PEPC to malate inhibition in bsd2-m1 leaves decreased significantly with an increase in light intensity, and there was a concomitant increase in PEPC kinase activity, similar to that seen in wild-type leaf tissue. Thus, although bsd2-m1 mutant plants lack an operative Calvin cycle, light activation of PEPC kinase and its target enzyme are not grossly perturbed.

The Two Genes Encoding Starch-Branching Enzymes IIa and IIb Are Differentially Expressed in Barley1

The sbeIIa and sbeIIb genes, encoding starch-branching enzyme (SBE) IIa and SBEIIb in barley (Hordeum vulgare L.), have been isolated. The 5′ portions of the two genes are strongly divergent, primarily due to the 2064-nucleotide-long intron 2 in sbeIIb. The sequence of this intron shows that it contains a retro-transposon-like element. Expression of sbeIIb but not sbeIIa was found to be endosperm specific. The temporal expression patterns for sbeIIa and sbeIIb were similar and peaked around 12 d after pollination. DNA gel-blot analysis demonstrated that sbeIIa and sbeIIb are both single-copy genes in the barley genome. By fluorescence in situ hybridization, the sbeIIa and sbeIIb genes were mapped to chromosomes 2 and 5, respectively. The cDNA clones for SBEIIa and SBEIIb were isolated and sequenced. The amino acid sequences of SBEIIa and SBEIIb were almost 80% identical. The major structural difference between the two enzymes was the presence of a 94-amino acid N-terminal extension in the SBEIIb precursor. The (β/α)8-barrel topology of the α-amylase superfamily and the catalytic residues implicated in branching enzymes are conserved in both barley enzymes.

Polypeptides of the Maize Amyloplast Stroma1 : Stromal Localization of Starch-Biosynthetic Enzymes and Identification of an 81-Kilodalton Amyloplast Stromal Heat-Shock Cognate

In the developing endosperm of monocotyledonous plants, starch granules are synthesized and deposited within the amyloplast. A soluble stromal fraction was isolated from amyloplasts of immature maize (Zea mays L.) endosperm and analyzed for enzyme activities and polypeptide content. Specific activities of starch synthase and starch-branching enzyme (SBE), but not the cytosolic marker alcohol dehydrogenase, were strongly enhanced in soluble amyloplast stromal fractions relative to soluble extracts obtained from homogenized kernels or endosperms. Immunoblot analysis demonstrated that starch synthase I, SBEIIb, and sugary1, the putative starch-debranching enzyme, were each highly enriched in the amyloplast stroma, providing direct evidence for the localization of starch-biosynthetic enzymes within this compartment. Analysis of maize mutants shows the deficiency of the 85-kD SBEIIb polypeptide in the stroma of amylose extender cultivars and that the dull mutant lacks a >220-kD stromal polypeptide. The stromal fraction is distinguished by differential enrichment of a characteristic group of previously undocumented polypeptides. N-terminal sequence analysis revealed that an abundant 81-kD stromal polypeptide is a member of the Hsp70 family of stress-related proteins. Moreover, the 81-kD stromal polypeptide is strongly recognized by antibodies specific for an Hsp70 of the chloroplast stroma. These findings are discussed in light of implications for the correct folding and assembly of soluble, partially soluble, and granule-bound starch-biosynthetic enzymes during import into the amyloplast.

NADH-Monodehydroascorbate Oxidoreductase Is One of the Redox Enzymes in Spinach Leaf Plasma Membranes1

Amino acid analysis of internal sequences of purified NADH-hexacyanoferrate(III) oxidoreductase (NFORase), obtained from highly purified plasma membranes (PM) of spinach (Spinacia oleracea L.) leaves, showed 90 to 100% homology to internal amino acid sequences of monodehydroascorbate (MDA) reductases (EC 1.6.5.4) from three different plant species. Specificity, kinetics, inhibitor sensitivity, and cross-reactivity with anti-MDA reductase antibodies were all consistent with this identification. The right-side-out PM vesicles were subjected to consecutive salt washing and detergent (polyoxyethylene 20 dodecylether and 3-[(3-cholamido-propyl)-dimethylammonio]-1-propane sulfonate [CHAPS]) treatments, and the fractions were analyzed for NFORase and MDA reductase activities. Similar results were obtained when the 300 mm sucrose in the homogenization buffer and in all steps of the salt-washing and detergent treatments had been replaced by 150 mm KCl to mimic the conditions in the cytoplasm. We conclude that (a) MDA reductase is strongly associated with the inner (cytoplasmic) surface of the PM under in vivo conditions and requires washing with 1.0 m KCl or CHAPS treatment for removal, (b) the PM-bound MDA reductase activity is responsible for the majority of PM NFORase activity, and (c) there is another redox enzyme(s) in the spinach leaf PM that cannot be released from the PM by salt-washing and/or CHAPS treatment. The PM-associated MDA reductase may have a role in reduction of ascorbate in both the cytosol and the apoplast.

4-Coumarate:Coenzyme A Ligase in Hybrid Poplar1 : Properties of Native Enzymes, cDNA Cloning, and Analysis of Recombinant Enzymes

The enzyme 4-coumarate:coenzyme A ligase (4CL) is important in providing activated thioester substrates for phenylpropanoid natural product biosynthesis. We tested different hybrid poplar (Populus trichocarpa × Populus deltoides) tissues for the presence of 4CL isoforms by fast-protein liquid chromatography and detected a minimum of three 4CL isoforms. These isoforms shared similar hydroxycinnamic acid substrate-utilization profiles and were all inactive against sinapic acid, but instability of the native forms precluded extensive further analysis. 4CL cDNA clones were isolated and grouped into two major classes, the predicted amino acid sequences of which were 86% identical. Genomic Southern blots showed that the cDNA classes represent two poplar 4CL genes, and northern blots provided evidence for their differential expression. Recombinant enzymes corresponding to the two genes were expressed using a baculovirus system. The two recombinant proteins had substrate utilization profiles similar to each other and to the native poplar 4CL isoforms (4-coumaric acid > ferulic acid > caffeic acid; there was no conversion of sinapic acid), except that both had relatively high activity toward cinnamic acid. These results are discussed with respect to the role of 4CL in the partitioning of carbon in phenylpropanoid metabolism.

Identification and Stereospecificity of the First Three Enzymes of 3-Dimethylsulfoniopropionate Biosynthesis in a Chlorophyte Alga1

Many marine algae produce 3-dimethylsulfoniopropionate (DMSP), a potent osmoprotective compound whose degradation product dimethylsulfide plays a central role in the biogeochemical S cycle. Algae are known to synthesize DMSP via the four-step pathway, l-Met → 4-methylthio-2-oxobutyrate → 4-methylthio-2-hydroxybutyrate → 4-dimethylsulfonio-2-hydroxy-butyrate (DMSHB) → DMSP. Substrate-specific enzymes catalyzing the first three steps in this pathway were detected and partially characterized in cell-free extracts of the chlorophyte alga Enteromorpha intestinalis. The first is a 2-oxoglutarate-dependent aminotransferase, the second an NADPH-linked reductase, and the third an S-adenosylmethionine-dependent methyltransferase. Sensitive radiometric assays were developed for these enzymes, and used to show that their activities are high enough to account for the estimated in vivo flux from Met to DMSP. The activities of these enzymes in other DMSP-rich chlorophyte algae were at least as high as those in E. intestinalis, but were ≥20-fold lower in algae without DMSP. The reductase and methyltransferase were specific for the d-enantiomer of 4-methylthio-2-hydroxybutyrate in vitro, and both the methyltransferase step and the step(s) converting DMSHB to DMSP were shown to prefer d-enantiomers in vivo. The intermediate DMSHB was shown to act as an osmoprotectant, which indicates that the first three steps of the DMSP synthesis pathway may be sufficient to confer osmotolerance.

Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55Gag

Ubiquitination appears to be involved in virus particle release from infected cells. Free ubiquitin (Ub), as well as Ub covalently bound to a small fraction of p6 Gag, is detected in mature HIV particles. Here we report that the p6 region in the Pr55Gag structural precursor polyprotein binds to Tsg101, a putative Ub regulator that is involved in trafficking of plasma membrane-associated proteins. Tsg101 was found to interact with Gag in (i) a yeast two-hybrid assay, (ii) in vitro coimmunoprecipitation by using purified Pr55Gag and rabbit reticulocyte lysate-synthesized Tsg101, and (iii) in vivo in the cytoplasm of COS cells transfected with gag. The PTAPP motif [or late (L) domain] within p6, which is required for release of mature virus from the plasma membrane, was the determinant for binding Pr55Gag. The N-terminal region in Tsg101, which is homologous to the Ubc4 class of Ub-conjugating (E2) enzymes, was the determinant of interaction with p6. Mutation of Tyr-110 in Tsg101, present in place of the active-site Cys that binds Ub in E2 enzymes, and other residues unique to Tsg101, impaired p6 interaction, indicating that features that distinguish Tsg101 from active E2 enzymes were important for binding the viral protein. The results link L-domain function in HIV to the Ub machinery and a specific component of the cellular trafficking apparatus.

Newly identified stress-responsive protein kinases, Krs-1 and Krs-2.

The activation of protein kinases is a frequent response of cells to treatment with growth factors, chemicals, heat shock, or apoptosis-inducing agents. However, when several agents result in the activation of the same enzymes, it is unclear how specific biological responses are generated. We describe here two protein kinases that are activated by a subset of stress conditions or apoptotic agents but are not activated by commonly used mitogenic stimuli. Purification and cloning demonstrate that these protein kinases are members of a subfamily of kinases related to Ste20p, a serine/threonine kinase that functions early in a pheromone responsive signal transduction cascade in yeast. The specificity of Krs-1 and Krs-2 activation and their similarity to Ste20p suggest that they may function at an early step in phosphorylation events that are specific responses to some forms of chemical stress or extreme heat shock.

A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes.

The crystal structure of the xanthine oxidase-related molybdenum-iron protein aldehyde oxido-reductase from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas (Mop) was analyzed in its desulfo-, sulfo-, oxidized, reduced, and alcohol-bound forms at 1.8-A resolution. In the sulfo-form the molybdenum molybdopterin cytosine dinucleotide cofactor has a dithiolene-bound fac-[Mo, = O, = S, ---(OH2)] substructure. Bound inhibitory isopropanol in the inner compartment of the substrate binding tunnel is a model for the Michaelis complex of the reaction with aldehydes (H-C = O,-R). The reaction is proposed to proceed by transfer of the molybdenum-bound water molecule as OH- after proton transfer to Glu-869 to the carbonyl carbon of the substrate in concert with hydride transfer to the sulfido group to generate [MoIV, = O, -SH, ---(O-C = O, -R)). Dissociation of the carboxylic acid product may be facilitated by transient binding of Glu-869 to the molybdenum. The metal-bound water is replenished from a chain of internal water molecules. A second alcohol binding site in the spacious outer compartment may cause the strong substrate inhibition observed. This compartment is the putative binding site of large inhibitors of xanthine oxidase.

Phosphorylation by protein kinase C of serine-23 of the alpha-1 subunit of rat Na+,K(+)-ATPase affects its conformational equilibrium.

Phosphorylation of the alpha-1 subunit of rat Na+,K(+)-ATPase by protein kinase C has been shown previously to decrease the activity of the enzyme in vitro. We have now undertaken an investigation of the mechanism by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains of the protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosphorylated the alpha-1 subunit of the rat Na+,K(+)-ATPase within the extreme NH2-terminal domain, on serine-23. The phosphorylation of this residue resulted in a shift in the equilibrium toward the E1 form, as measured by eosin fluorescence studies, and this was associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays. The rate of transition from E2 to E1 was apparently unaffected by phosphorylation by protein kinase C. These results, together with previous studies that examined the effects of tryptic digestion of Na+,K(+)-ATPase, suggest that the NH2-terminal domain of the alpha-1 subunit, including serine-23, is involved in regulating the activity of the enzyme.

The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins.

Although transcription and pre-mRNA processing are colocalized in eukaryotic nuclei, molecules linking these processes have not previously been described. We have identified four novel rat proteins by their ability to interact with the repetitive C-terminal domain (CTD) of RNA polymerase II in a yeast two-hybrid assay. A yeast homolog of one of the rat proteins has also been shown to interact with the CTD. These CTD-binding proteins are all similar to the SR (serine/arginine-rich) family of proteins that have been shown to be involved in constitutive and regulated splicing. In addition to alternating Ser-Arg domains, these proteins each contain discrete N-terminal or C-terminal CTD-binding domains. We have identified SR-related proteins in a complex that can be immunoprecipitated from nuclear extracts with antibodies directed against RNA polymerase II. In addition, in vitro splicing is inhibited either by an antibody directed against the CTD or by wild-type but not mutant CTD peptides. Thus, these results suggest that the CTD and a set of CTD-binding proteins may act to physically and functionally link transcription and pre-mRNA processing.

Mutation of a conserved serine in TM4 of opioid receptors confers full agonistic properties to classical antagonists.

The involvement of a conserved serine (Ser196 at the mu-, Ser177 at the delta-, and Ser187 at the kappa-opioid receptor) in receptor activation is demonstrated by site-directed mutagenesis. It was initially observed during our functional screening of a mu/delta-opioid chimeric receptor, mu delta2, that classical opioid antagonists such as naloxone, naltrexone, naltriben, and H-Tyr-Tic[psi,CH2NH]Phe-Phe-OH (TIPPpsi; Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing the chimeric receptor. Antagonists also activated the G protein-coupled inward rectifying potassium channel (GIRK1) in Xenopus oocytes coexpressing the mu delta2 opioid receptor and the GIRK1 channel. By sequence analysis and back mutation, it was determined that the observed antagonist activity was due to the mutation of a conserved serine to leucine in the fourth transmembrane domain (S196L). The importance of this serine was further demonstrated by analogous mutations created in the mu-opioid receptor (MORS196L) and delta-opioid receptor (DORS177L), in which classical opioid antagonists could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing either MORS196L or DORS177L. Again, antagonists could activate the GIRK1 channel coexpressed with either MORS196L or DORS177L in Xenopus oocytes. These data taken together suggest a crucial role for this serine residue in opioid receptor activation.

A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain.

Phosphoprotein phosphatase 2A (PP2A) is one of the four major protein serine/threonine phosphatases found in all eukaryotic cells. We have shown that the 36-kDa catalytic subunit of PP2A is carboxyl methylated in eukaryotic cells, and we have previously identified and purified a novel methyltransferase (MTase) that is responsible for this modification. Here, we describe a novel protein carboxyl methyl-esterase (MEase) from bovine brain that demethylates PP2A. The enzyme has been purified to homogeneity as a monomeric 46-kDa soluble protein. The MEase is highly specific for PP2A. It does not catalyze the demethylation of other protein or peptide methylesters. Moreover, MEase activity is dramatically inhibited by nanomolar concentrations of okadaic acid, a specific inhibitor of PP2A. From these results, we conclude that PP2A methylation is controlled by two specific enzymes, a MTase and a MEase. Since PP2A methylation is highly conserved in eukaryotes ranging from human to yeast, it is likely that this system plays an important role in phosphatase regulation.