N-myristoylation and S-acylation are common modifications of Ca2+ -regulated Arabidopsis kinases and are required for activation of the SLAC1 anion channel.

N-myristoylation and S-acylation promote protein membrane association, allowing regulation of membrane proteins. However, how widespread this targeting mechanism is in plant signaling processes remains unknown. Through bioinformatics analyses, we determined that among plant protein kinase families, the occurrence of motifs indicative for dual lipidation by N-myristoylation and S-acylation is restricted to only five kinase families, including the Ca2+ -regulated CDPK-SnRK and CBL protein families. We demonstrated N-myristoylation of CDPK-SnRKs and CBLs by incorporation of radiolabeled myristic acid. We focused on CPK6 and CBL5 as model cases and examined the impact of dual lipidation on their function by fluorescence microscopy, electrophysiology and functional complementation of Arabidopsis mutants. We found that both lipid modifications were required for proper targeting of CBL5 and CPK6 to the plasma membrane. Moreover, we identified CBL5-CIPK11 complexes as phosphorylating and activating the guard cell anion channel SLAC1. SLAC1 activation by CPK6 or CBL5-CIPK11 was strictly dependent on dual lipid modification, and loss of CPK6 lipid modification prevented functional complementation of cpk3 cpk6 guard cell mutant phenotypes. Our findings establish the general importance of dual lipid modification for Ca2+ signaling processes, and demonstrate their requirement for guard cell anion channel regulation.

Identification and characterization of trans-3-hydroxy-l-proline dehydratase and Δ(1)-pyrroline-2-carboxylate reductase involved in trans-3-hydroxy-l-proline metabolism of bacteria.

trans-4-Hydroxy-l-proline (T4LHyp) and trans-3-hydroxy-l-proline (T3LHyp) occur mainly in collagen. A few bacteria can convert T4LHyp to α-ketoglutarate, and we previously revealed a hypothetical pathway consisting of four enzymes at the molecular level (J Biol Chem (2007) 282, 6685-6695; J Biol Chem (2012) 287, 32674-32688). Here, we first found that Azospirillum brasilense has the ability to grow not only on T4LHyp but also T3LHyp as a sole carbon source. In A. brasilense cells, T3LHyp dehydratase and NAD(P)H-dependent Δ(1)-pyrroline-2-carboxylate (Pyr2C) reductase activities were induced by T3LHyp (and d-proline and d-lysine) but not T4LHyp, and no effect of T3LHyp was observed on the expression of T4LHyp metabolizing enzymes: a hypothetical pathway of T3LHyp â†’ Pyr2C â†’ l-proline was proposed. Bacterial T3LHyp dehydratase, encoded to LhpH gene, was homologous with the mammalian enzyme. On the other hand, Pyr2C reductase encoded to LhpI gene was a novel member of ornithine cyclodeaminase/µ-crystallin superfamily, differing from known bacterial protein. Furthermore, the LhpI enzymes of A. brasilense and another bacterium showed several different properties, including substrate and coenzyme specificities. T3LHyp was converted to proline by the purified LhpH and LhpI proteins. Furthermore, disruption of LhpI gene from A. brasilense led to loss of growth on T3LHyp, d-proline and d-lysine, indicating that this gene has dual metabolic functions as a reductase for Pyr2C and Δ(1)-piperidine-2-carboxylate in these pathways, and that the T3LHyp pathway is not linked to T4LHyp and l-proline metabolism.

Distinct features of protein folding by the GroEL system from a psychrophilic bacterium, Colwellia psychrerythraea 34H.

We investigated the protein folding mechanism of the GroEL system of a psychrophilic bacterium, Colwellia psychrerythraea 34H. The amount of mRNA of the groESL operon of C. psychrerythraea was increased about 6-fold after a temperature upshift from 8 to 18 °C for 30 min, suggesting that this temperature causes heat stress in this bacterium. A σ(32)-type promoter was found upstream of the groESL, suggesting that the C. psychrerythraea groESL is regulated by the σ(32) system, like the groESL in E. coli. The maximum ATPase and CTPase activities of CpGroEL were observed at 45 and 35 °C, respectively, which are much higher than the growth temperatures of C. psychrerythraea. We found that the refolding activity of the CpGroEL system in the presence of ATP is lower than that in the presence of CTP. This suggests that ATP is not the optimum energy source of the CpGroEL system. Analyses for the interaction of CpGroEL-CpGroES revealed that CTP could weaken this interaction, resulting in effective refolding function of the CpGroEL system. From these findings, we consider that the CpGroEL system possesses an energy-saving mechanism for avoiding excess consumption of ATP to ensure growth in a low-temperature environment.

The consensus motif for N-myristoylation of plant proteins in a wheat germ cell-free translation system.

Protein N-myristoylation plays key roles in various cellular functions in eukaryotic organisms. To clarify the relationship between the efficiency of protein N-myristoylation and the amino acid sequence of the substrate in plants, we have applied a wheat germ cell-free translation system with high protein productivity to examine the N-myristoylation of various wild-type and mutant forms of Arabidopsis thaliana proteins. Evaluation of the relationship between removal of the initiating Met and subsequent N-myristoylation revealed that constructs containing Pro at position 3 do not undergo N-myristoylation, primarily because of an inhibitory effect of this amino acid on elimination of the initiating Met by methionyl aminopeptidase. Our analysis of the consensus sequence for N-myristoylation in plants focused on the variability of amino acids at positions 3, 6 and 7 of the motif. We found that not only Ser at position 6 but also Lys at position 7 affects the selectivity for the amino acid at position 3. The results of our analyses allowed us to identify several A. thaliana proteins as substrates for N-myristoylation that had previously been predicted not to be candidates for such modification with a prediction program. We have thus shown that a wheat germ cell-free system is a useful tool for plant N-myristoylome analysis. This in vitro approach will facilitate comprehensive determination of N-myristoylated proteins in plants.

Involvement of HMG-12 and CAR-1 in the cdc-48.1 expression of Caenorhabditis elegans.

Caenorhabditis elegans possesses two p97/VCP/Cdc48p homologues, named CDC-48.1 (C06A1.1) and CDC-48.2 (C41C4.8), and their expression patterns and levels are differently regulated. To clarify the regulatory mechanisms of differential expression of two p97 proteins of C. elegans, we performed detailed deletion analysis of their promoter regions. We found that the promoter of cdc-48.1 contains two regions necessary for embryonic and for post-embryonic expression, while the promoter of cdc-48.2 contains the single region necessary for embryonic expression. In particular, two elements (Element A and Element B) and three conserved boxes (Box a, Box b and Box c) were essential for cdc-48.1 expression in embryos and at post-embryonic stages, respectively. By using South-Western blotting and MALDI-TOF MS analysis, we identified HMG-12 and CAR-1 as proteins that bind to Element A and Element B, respectively, from the embryonic nuclear extract. Importantly, we found the decreased expression of p97 in embryos prepared from hmg-12(RNAi) or car-1(RNAi) worms. These results indicate that both HMG-12 and CAR-1 play important roles in embryonic expression of cdc-48.1.

Differential expression pattern of UBX family genes in Caenorhabditis elegans.

UBX (ubiquitin regulatory X)-containing proteins belong to an evolutionary conserved protein family and determine the specificity of p97/VCP/Cdc48p function by binding as its adaptors. Caenorhabditis elegans was found to possess six UBX-containing proteins, named UBXN-1 to -6. However, no general or specific function of them has been revealed. During the course of understanding not only their function but also specified function of p97, we investigated spatial and temporal expression patterns of six ubxn genes in this study. Transcript analyses showed that the expression pattern of each ubxn gene was different throughout worm's development and may show potential developmental dynamics in their function, especially ubxn-5 was expressed specifically in the spermatogenic germline, suggesting a crucial role in spermatogenesis. In addition, as ubxn-4 expression was induced by ER stress, it would function as an ERAD factor in C. elegans. In vivo expression analysis by using GFP translational fusion constructs revealed that six ubxn genes show distinct expression patterns. These results altogether demonstrate that the expression of all six ubxn genes of C. elegans is differently regulated.

Comparative analysis of expression of two p97 homologues in Caenorhabditis elegans.

Caenorhabditis elegans possesses two p97/VCP/Cdc48p homologues, named CDC-48.1 (C06A1.1) and CDC-48.2 (C41C4.8), although their expression regulation and functional diversity have not yet been studied. We therefore investigated spatial and temporal expression patterns of two p97 homologues in this study. RT-PCR and Western blot analysis showed that the amount of cdc-48.1 was about twofold of that of cdc-48.2 in adults and that two p97 homologues were induced by ER stress. The amount of cdc-48.1 mRNA did not increase in the cdc-48.2 deletion mutant and vice versa. In situ hybridization showed that two p97 homologues are mainly expressed in germ cells. In vivo expression analysis by using GFP translational fusion constructs revealed that CDC-48.1::GFP was expressed from embryos through to adult worms, while CDC-48.2::GFP was expressed mainly in embryos. These results suggest that the expression of two p97 homologues of C. elegans is differently regulated and independent of each other.

The rpoH gene encoding heat shock sigma factor sigma32 of psychrophilic bacterium Colwellia maris.

The rpoH gene encoding a heat shock sigma factor, sigma(32), was cloned from the psychrophilic bacterium Colwellia maris. The deduced amino acid sequence of sigma(32) from C. maris is more than 60% homologous to that of sigma(32) from mesophilic bacteria. The RpoH box, a 9-amino-acid sequence region (QRKLFFNLR) specific to sigma(32), and two downstream box sequences complementary to a part of 16S rRNA were identified. Primer extension analysis showed that the C. maris rpoH is expressed from only one sigma(70)-type promoter. Northern blot analysis showed that the level of rpoH mRNA was clearly increased at 20 degrees C, a temperature that induces heat shock in this organism. In the presence of an inhibitor of transcriptional initiation, the degradation of rpoH mRNA was much slower at 20 degrees C than at 10 degrees C. Thus, increased stability of the rpoH mRNA might be responsible for the rpoH mRNA accumulation. The predicted secondary structure of the 5'-region of C. maris rpoH mRNA was different from the conserved patterns reported for most mesophilic bacteria, and the base pairing of the downstream boxes appeared to be less stable than that of Escherichia coli rpoH mRNA. Thus, essential features that ensure the HSP expression at a relatively low temperature are embedded in the rpoH gene of psychrophiles.

Gene structure and transcriptional regulation of dnaK and dnaJ genes from a psychrophilic bacterium, Colwellia maris.

The dnaK and dnaJ genes, encoding heat shock proteins, were cloned from a psychrophilic bacterium, Colwellia maris. Significant homology was evident comparing DnaK and DnaJ of the psychrophilile with the counterparts of mesophilic and thermophilic bacteria. In the DnaJ protein, three conserved regions of the Hsp40 family were observed. A putative promoter similar to the sigma32 consensus sequence was found upstream of the dnaK gene. The G+C content in the 5'-untranslated region of the dnaK gene was much lower than that in the corresponding region of mesophilic bacteria. Northern-blot analysis and primer-extension analysis showed that both genes were transcribed separately as monocistronic mRNAs. Following several temperature upshifts from 10 to 26 degrees C, maximum induction of the dnaK and dnaJ mRNAs was detected at 20 degrees C, suggesting that this temperature induces the heat shock response in this bacterium. In addition, the level of the induction of the dnaJ gene was much lower than that of the dnaK gene. These findings together revealed several specific features of the heat shock response at a relatively low temperature in psychrophiles.

Gene structure and transcriptional regulation specific to the groESL operon from the psychrophilic bacterium Colwellia maris.

The groESL operon of a psychrophilic bacterium, Colwellia maris, was cloned and sequenced. The operon contains two ORFs of 291 bp and 1,650 bp separated by 210 bp. Northern blot analysis suggested that the groESL operon was transcribed as a bicistronic mRNA, and that the amount of mRNA markedly increased after the temperature was raised from 10 degrees C to 20 degrees C. Although the optimum temperatures for GroESL function are different in psychrophilic, mesophilic, and thermophilic bacteria, the deduced amino acid sequences of C. maris GroES and GroEL showed remarkably high similarity with those of GroES and GroEL from mesophilic and thermophilic bacteria. A putative promoter similar to the Escherichia coli sigma(32) consensus sequence was identified. One specific feature of C. maris groESL was that in the putative untranslated region the G+C content was about 24 mol%, which is much lower than that of mesophilic bacteria such as E. coli. The low G+C content may be important for maintaining transcription at low temperatures.