Degradation of a Caulobacter soluble cytoplasmic chemoreceptor is ClpX dependent

In order to determine whether ClpXP-mediated proteolysis is a common mechanism used to regulate the chemotaxis machinery during the cell cycle of Caulobacter crescentus, we have characterized a soluble cytoplasmic chemoreceptor, McpB. The mcpB gene lies adjacent to the major chemotaxis operon, which encodes 12 chemotaxis proteins, including the membrane chemoreceptor McpA. Like McpA, McpB possesses a C-terminal CheBR docking motif and three potential methylation sites, which we suggest are methylated. The McpB protein is degraded via a ClpX-dependent pathway during the swarmer-to-stalked cell transition, and a motif, which is 3 amino acids N-terminal to the McpB CheBR docking site, is required for proteolysis. Analysis of the degradation signal in McpB and McpA reveals a common motif present in the other four chemoreceptors that possess CheBR docking sites. A green fluorescent protein (GFP) fusion bearing 58 amino acids from the C terminus of McpA, which contains this motif, is degraded, suggesting that the C-terminal sequence is sufficient to confer ClpXP protease susceptibility.     

Isolation of intact FNR protein (Mr 30 000) of Escherichia coli

FNR, the activator of anaerobic respiratory genes of Escherichia coli, has previously only been isolated as a protein of Mr 29,000, which lacks nine N-terminal amino acid residues. The underlying proteolytic events have been studied with the aim of isolating intact FNR and determining whether cleavage is the result of a physiologically significant intracellular processing mechanism or proteolytic degradation during isolation. The FNR protein was present in aerobically and anaerobically grown bacteria as the intact protein (Mr 30,000). Proteolysis only occurred during and shortly after disruption of the bacteria. The production of FNR (Mr 29,000) must therefore be regarded as an isolation artefact. The proteolysis was caused by a protease which is located outside the cytoplasmic membrane or activated upon disruption of the membrane. Protease inhibitors directed against serine, cysteine or metalloproteases failed to prevent cleavage of FNR. In E. coli strain CAG627, proteolysis was greatly reduced making it possible to isolate FNR of Mr 30,000. The N-terminal sequence of FNR (Mr 30,000) was identical to that predicted from the fnr gene starting with the initiating methionine residue and including a four-cysteine cluster (16)Cys-X3-Cys-X2-Cys-X5-Cys(29).     

Dynamic control of Dps protein levels by ClpXP and ClpAP proteases in Escherichia coli

The Escherichia coli starvation-induced DNA protection protein Dps was observed to be degraded rapidly during exponential growth. This turnover is dependent on the clpP and clpX genes. The clpA gene is not required for Dps proteolysis, suggesting that Dps is a substrate for ClpXP protease but not for ClpAP protease. Dps proteolysis was found to be highly regulated. Upon carbon starvation, Dps is stabilized, which together with increased Dps synthesis allows strong accumulation of Dps in the stationary phase. The addition of glucose to starving cells results in rapid resumption of Dps proteolysis by ClpXP. Oxidative stress also leads to efficient stabilization of Dps. After hyperosmotic shift, however, proteolysis remains unaffected. Thus, regulated proteolysis of Dps strongly contributes to controlling Dps levels under very specific stress conditions. In contrast to the regulated degradation of RpoS by ClpXP, Dps proteolysis is independent of the recognition factor RssB. In addition, during starvation, clpP and, to a somewhat lesser extent, clpA are involved in maintaining ongoing Dps synthesis (acting at the level of Dps translation), which is required for strong Dps accumulation in long-term stationary phase cells. In summary, both ClpXP and ClpAP exert significant control of Dps levels by affecting log phase stability and stationary phase synthesis of Dps respectively.     

Adaptor bypass mutations of Bacillus subtilis spx suggest a mechanism for YjbH‐enhanced proteolysis of the regulator Spx by ClpXP

The global regulator, Spx, is under proteolytic control exerted by the adaptor YjbH and ATP‐dependent protease ClpXP in Bacillus subtilis. While YjbH is observed to bind the Spx C‐terminus, YjbH shows little affinity for ClpXP, indicating adaptor activity that does not operate by tethering. Chimeric proteins derived from B. subtilis AbrB and the Spx C‐terminus showed that a 28‐residue C‐terminal section of Spx (AbrB28), but not the last 12 or 16 residues (AbrB12, AbrB16), was required for YjbH interaction and for ClpXP proteolysis, although the rate of AbrB28 proteolysis was not affected by YjbH addition. The result suggested that the YjbH‐targeted 28 residue segment of the Spx C‐terminus bears a ClpXP‐recognition element(s) that is hidden in the intact Spx protein. Residue substitutions in the conserved helix α6 of the C‐terminal region generated Spx substrates that were degraded by ClpXP at accelerated rates compared to wild‐type Spx, and showed reduced dependency on the YjbH activity. The residue substitutions also weakened the interaction between Spx and YjbH. The results suggest a model in which YjbH, through interaction with residues of helix α6, exposes the C‐terminus of Spx for recognition and proteolysis by ClpXP.     

Identification of the region of AT2 receptor needed for inhibition of the AT1 receptor-mediated inositol 1,4,5-triphosphate generation

Increase in the intracellular inositol triphosphate (IP3) levels in Xenopus oocytes in response to expression and activation of rat angiotensin II (Ang II) receptor AT1 was inhibited by co-expression of rat AT2 receptor. To identify which region of the AT2 was involved in this inhibition, ability of three AT2 mutants to abolish this inhibition was analyzed. Deletion of the C-terminus of the AT2 did not abolish this inhibition. Replacing Ile249 in the third intracellular loop (3rd ICL) of the AT2 with proline, corresponding amino acid in the AT1, in the mutant M6, resulted in slightly reduced affinity to [125I]Ang II (K(d)=0.259 nM), however, did not abolish the inhibition. In contrast, replacing eight more amino acids in the 3rd ICL of the AT2 (at positions 241-244, 250-251 and 255-256) with that of the AT1 in the mutant M8, not only increased the affinity of the AT2 receptor to [125I]Ang II (K(d)=0.038 nM) but also abolished AT2-mediated inhibition. Interestingly, activation of the M8 by Ang II binding also resulted in increase in the intracellular IP(3) levels in oocytes. These results imply that the region of the 3rd ICL of AT2 spanning amino acids 241-256 is sufficient for the AT2-mediated inhibition of AT1-stimulated IP3 generation. Moreover, these nine mutations are also sufficient to render the AT2 with the ability to activate phospholipase C.     

Identification by mutational analysis of amino acid residues essential in the chaperone function of calreticulin

Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor.     

Location of Dual Sites in E. coli FtsZ Important for Degradation by ClpXP; One at the C-Terminus and One in the Disordered Linker

ClpXP is a two-component ATP-dependent protease that unfolds and degrades proteins bearing specific recognition signals. One substrate degraded by Escherichia coli ClpXP is FtsZ, an essential cell division protein. FtsZ forms polymers that assemble into a large ring-like structure, termed the Z-ring, during cell division at the site of constriction. The FtsZ monomer is composed of an N-terminal polymerization domain, an unstructured linker region and a C-terminal conserved region. To better understand substrate selection by ClpXP, we engineered FtsZ mutant proteins containing amino acid substitutions or deletions near the FtsZ C-terminus. We identified two discrete regions of FtsZ important for degradation of both FtsZ monomers and polymers by ClpXP in vitro. One region is located 30 residues away from the C-terminus in the unstructured linker region that connects the polymerization domain to the C-terminal region. The other region is near the FtsZ C-terminus and partially overlaps the recognition sites for several other FtsZ-interacting proteins, including MinC, ZipA and FtsA. Mutation of either region caused the protein to be more stable and mutation of both caused an additive effect, suggesting that both regions are important. We also observed that in vitro MinC inhibits degradation of FtsZ by ClpXP, suggesting that some of the same residues in the C-terminal site that are important for degradation by ClpXP are important for binding MinC.     

Conserved low-affinity nickel-binding amino acids are essential for the function of the nickel permease NixA of Helicobacter pylori

Nickel acquisition is necessary for urease activity, a major virulence factor of the human gastric pathogen Helicobacter pylori. The nickel permease NixA of H. pylori is a member of the single-component nickel-cobalt transporter family. To identify functionally relevant amino acids of NixA, single-site exchanges were introduced into NixA via PCR-based mutagenesis. This study investigated one of the recognition motifs for this family in transmembrane segment III and other conserved amino acids, mostly with possible nickel-binding capacities. The mutant alleles were expressed in Escherichia coli, and activity of the altered permeases was analyzed by measuring nickel accumulation and urease activity. Expression was checked by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a NixA-specific antibody. Replacement of Phe-75 and His-79-both part of the characteristic sequence motif-and of Asn-127, Thr-195, and Ser-197 with alanine abolished nickel uptake in the E. coli system. The results were unchanged if these amino acids were replaced with residues more similar to the original amino acid. The phenotype of the null mutants was independent of the culture medium. Mutation of Val-82, Tyr-242, Thr-260, His-181, and His-15 strongly affected uptake activity under nickel limitation on complex Luria-Bertani medium but had little effect in minimal medium. Eight other conserved amino acids (Ser-80, Ser-81, Phe-119, Trp-180, Tyr-183, Trp-244, Pro-249, and Asn-256) were found to be dispensable for the function of NixA. These results show that atypical nickel-binding amino acids play an important function in nickel uptake and that most of the essential amino acids are clustered in conserved motifs.     

A dynamically localized protease complex and a polar specificity factor control a cell cycle master regulator

Regulated proteolysis is essential for cell cycle progression in both prokaryotes and eukaryotes. We show here that the ClpXP protease, responsible for the degradation of multiple bacterial proteins, is dynamically localized to specific cellular positions in Caulobacter where it degrades colocalized proteins. The CtrA cell cycle master regulator, that must be cleared from the Caulobacter cell to allow the initiation of chromosome replication, interacts with the ClpXP protease at the cell pole where it is degraded. We have identified a novel, conserved protein, RcdA, that forms a complex with CtrA and ClpX in the cell. RcdA is required for CtrA polar localization and degradation by ClpXP. The localization pattern of RcdA is coincident with and dependent upon ClpX localization. Thus, a dynamically localized ClpXP proteolysis complex in concert with a cytoplasmic factor provides temporal and spatial specificity to protein degradation during a bacterial cell cycle.     

Spirulina ferredoxin-NADP+ reductase. Further characterization with an improved preparation

The preparation procedure for Spirulina ferredoxin-NADP+ reductase (ferredoxin: NADP+ oxidoreductase, EC 1.18.1.2, FNR) was improved by adding protease inhibitors, phenylmethylsulfonylfluoride (PMSF) and EDTA, through the whole process of preparation and by introducing an affinity chromatography step on Blue Sepharose CL-6B. The addition of the inhibitors largely prevented the formation of the minor component (FNR I), and the affinity gel chromatography simplified the preparation process, shortening the exposure period of FNR to proteolysis. However, complete removal of the heterogeneity of FNR found at the amino (N)-terminal region was not achieved even by applying the new method. The affinity chromatography on the Blue Sepharose gel was also effective in purifying spinach FNR. The affinity of this gel for Spirulina FNR was compared with that for the enzyme derived from spinach leaves. The spinach enzyme had a higher affinity than the Spirulina one. Both enzymes showed the highest affinities to Blue Sepharose at 20--30 mM NaCl concentration. The N-terminal sequence analysis revealed that there was 4 forms, which were probably modifications produced by exopeptidase action during the preparation, or even in the living cells. The longest component gave the N-terminal sequence Ala-Lys-Thr-Asp-Ile-Pro-Val-Asn-Ile-Tyr-. The others lacked amino acids successively one by one from the N-terminus. In contrast, the carboxyl(C)-terminal residues of all 4 FNR forms were tyrosine. The probable C-terminal sequence was predicted to be -Trp-His-Val-Gln-Thr-Tyr based on a study of a cyanogen bromide peptide.     

The impact of O(2) on the Fe-S cluster biogenesis requirements of Escherichia coli FNR

In this study, the functions of two established Fe-S cluster biogenesis pathways, Isc (iron-sulfur cluster) and Suf (sulfur mobilization), under aerobic and anaerobic growth conditions were compared by measuring the activity of the Escherichia coli global anaerobic regulator FNR. A [4Fe-4S] cluster is required for FNR activity under anaerobic conditions. An assay of the expression of FNR-dependent promoters in strains containing various deletions of the iscSUAhscBAfdx operon revealed that, under anaerobic conditions, FNR activity was reduced by 60% in the absence of the Isc pathway. In contrast, a mutant lacking the entire Suf pathway had normal FNR activity, although overexpression of the suf operon fully rescued the anaerobic defect in FNR activity in strains lacking the Isc pathway. Expression of the sufA promoter and levels of SufD protein were upregulated by twofold to threefold in Isc ⁻ strains under anaerobic conditions, suggesting that increased expression of the Suf pathway may be partially responsible for the FNR activity remaining in strains lacking the Isc pathway. In contrast, use of the O₂-stable [4Fe-4S] cluster FNR variant FNR-L28H showed that overexpression of the suf operon did not restore FNR activity to strains lacking the Isc pathway under aerobic conditions. In addition, FNR-L28H activity was more impaired under aerobic conditions than under anaerobic conditions. The greater requirement for the Isc pathway under aerobic conditions was not due to a change in the rate of Fe-S cluster acquisition by FNR-L28H under aerobic and anaerobic conditions, as shown by ⁵⁵Fe-labeling experiments. Using [³⁵S]methionine pulse-chase assays, we observed that the Isc pathway, but not the Suf pathway, is the major pathway required for conversion of O₂-inactivated apo-FNR into [4Fe-4S]FNR upon the onset of anaerobic growth conditions. Taken together, these findings indicate a major role for the Isc pathway in FNR Fe-S cluster biogenesis under both aerobic and anaerobic conditions.