Analysis of the role of trans-translation in the requirement of tmRNA for lambdaimmP22 growth in Escherichia coli.
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The small, stable RNA molecule encoded by ssrA, known as tmRNA or 10Sa RNA, is required for the growth of certain hybrid lambdaimmP22 phages in Escherichia coli. tmRNA has been shown to tag partially synthesized proteins for degradation in vivo by attaching a short peptide sequence, encoded by tmRNA, to the carboxyl termini of these proteins. This tag sequence contains, at its C terminus, an amino acid sequence that is recognized by cellular proteases and leads to degradation of tagged proteins. A model describing this function of tmRNA, the trans-translation model (K. C. Keiler, P. R. Waller, and R. T. Sauer, Science 271:990-993, 1996), proposes that tmRNA acts first as a tRNA and then as a mRNA, resulting in release of the original mRNA template from the ribosome and translocation of the nascent peptide to tmRNA. Previous work from this laboratory suggested that tmRNA may also interact specifically with DNA-binding proteins, modulating their activity. However, more recent results indicate that interactions between tmRNA and DNA-binding proteins are likely nonspecific. In light of this new information, we examine the effects on lambdaimmP22 growth of mutations eliminating activities postulated to be important for two different steps in the trans-translation model, alanine charging of tmRNA and degradation of tagged proteins. This mutational analysis suggests that, while charging of tmRNA with alanine is essential for lambdaimmP22 growth in E. coli, degradation of proteins tagged by tmRNA is required only to achieve optimal levels of phage growth. Based on these results, we propose that trans-translation may have two roles, the primary role being the release of stalled ribosomes from their mRNA template and the secondary role being the tagging of truncated proteins for degradation. (+info)
Chloroplast-targeted ERD1 protein declines but its mRNA increases during senescence in Arabidopsis.
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Arabidopsis ERD1 is a ClpC-like protein that sequence analysis suggests may interact with the chloroplast-localized ClpP protease to facilitate proteolysis. The mRNA encoded by the ERD1 gene has previously been shown to accumulate in response to senescence and to a variety of stresses and hormones. Here we show that the ERD1 protein, in contrast to the ERD1 mRNA, strongly declines in abundance with age, becoming undetectable in fully expanded leaves. Sequence analysis also suggests that ERD1 is chloroplast targeted, and we show in an in vitro system that the native protein is properly imported, processed, and present within the soluble fraction of the chloroplast, presumably the stroma. We show that ClpP protein, which is also present in the stroma, declines with age in parallel with ERD1. These results are consistent with the interaction of ERD1 and ClpP, but they suggest that it is unlikely that either plays a major role during senescence. Certain other chloroplast proteins decline with age coordinately with ERD1 and ClpP, suggesting that these declines are markers of an early age-mediated change that occurs within the chloroplast. (+info)
Recognition, targeting, and hydrolysis of the lambda O replication protein by the ClpP/ClpX protease.
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It has previously been established that sequences at the C termini of polypeptide substrates are critical for efficient hydrolysis by the ClpP/ClpX ATP-dependent protease. We report for the bacteriophage lambda O replication protein, however, that N-terminal sequences play the most critical role in facilitating proteolysis by ClpP/ClpX. The N-terminal portion of lambda O is degraded at a rate comparable with that of wild type O protein, whereas the C-terminal domain of O is hydrolyzed at least 10-fold more slowly. Consistent with these results, deletion of the first 18 amino acids of lambda O blocks degradation of the N-terminal domain, whereas proteolysis of the O C-terminal domain is only slightly diminished as a result of deletion of the C-terminal 15 amino acids. We demonstrate that ClpX retains its capacity to bind to the N-terminal domain following removal of the first 18 amino acids of O. However, ClpX cannot efficiently promote the ATP-dependent binding of this truncated O polypeptide to ClpP, the catalytic subunit of the ClpP/ClpX protease. Based on our results with lambda O protein, we suggest that two distinct structural elements may be required in substrate polypeptides to enable efficient hydrolysis by the ClpP/ClpX protease: (i) a ClpX-binding site, which may be located remotely from substrate termini, and (ii) a proper N- or C-terminal sequence, whose exposure on the substrate surface may be induced by the binding of ClpX. (+info)
Identification and transcriptional control of the genes encoding the Caulobacter crescentus ClpXP protease.
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The region of the Caulobacter crescentus chromosome harboring the genes for the ClpXP protease was isolated and characterized. Comparison of the deduced amino acid sequences of the C. crescentus ClpP and ClpX proteins with those of their homologues from several gram-positive and gram-negative bacteria revealed stronger conservation for the ATPase regulatory subunit (ClpX) than for the peptidase subunit (ClpP). The C. crescentus clpX gene was shown by complementation analysis to be functional in Escherichia coli. However, clpX from E. coli was not able to substitute for the essential nature of the clpX gene in C. crescentus. The clpP and clpX genes are separated on the C. crescentus chromosome by an open reading frame pointing in the opposite direction from the clp genes, and transcription of clpP and clpX was found to be uncoupled. clpP is transcribed as a monocistronic unit with a promoter (PP1) located immediately upstream of the 5' end of the gene and a terminator structure following its 3' end. PP1 is under heat shock control and is induced upon entry of the cells into the stationary phase. At least three promoters for clpX (PX1, PX2, and PX3) were mapped in the clpP-clpX intergenic region. In contrast to PP1, the clpX promoters were found to be downregulated after heat shock but were also subject to growth phase control. In addition, the clpP and clpX promoters showed different activity patterns during the cell cycle. Together, these results demonstrate that the genes coding for the peptidase and the regulatory subunits of the ClpXP protease are under independent transcriptional control in C. crescentus. Determination of the numbers of ClpP and ClpX molecules per cell suggested that ClpX is the limiting component compared with ClpP. (+info)
Molecular cloning and characterization of a mouse homolog of bacterial ClpX, a novel mammalian class II member of the Hsp100/Clp chaperone family.
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In this paper, we present the molecular cloning and characterization of a murine homolog of the Escherichia coli chaperone ClpX. Murine ClpX shares 38% amino acid sequence identity with the E. coli homolog and is a novel member of the Hsp100/Clp family of molecular chaperones. ClpX localizes to human chromosome 15q22.2-22.3 and in mouse is expressed tissue-specifically as one transcript of approximately 2.9 kilobases (kb) predominantly within the liver and as two isoforms of approximately 2.6 and approximately 2.9 kb within the testes. Purified recombinant ClpX displays intrinsic ATPase activity, with a Km of approximately 25 microM and a Vmax of approximately 660 pmol min-1 microgram-1, which is active over a broad range of pH, temperature, ethanol, and salt parameters. Substitution of lysine 300 with alanine in the ATPase domain P-loop abolishes both ATP hydrolysis and binding. Recombinant ClpX can also interact with its putative partner protease subunit ClpP in overexpression experiments in 293T cells. Subcellular studies by confocal laser scanning microscopy localized murine ClpX green fluorescent protein fusions to the mitochondria. Deletion of the N-terminal mitochondrial targeting sequence abolished mitochondrial compartmentalization. Our results thus suggest that murine ClpX acts as a tissue-specific mammalian mitochondrial chaperone that may play a role in mitochondrial protein homeostasis. (+info)
Lon and Clp family proteases and chaperones share homologous substrate-recognition domains.
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Lon protease and members of the Clp family of molecular chaperones and protease regulatory subunits contain homologous regions with properties expected for substrate-binding domains. Fragments corresponding to these sequences are stably and independently folded for Lon, ClpA, and ClpY. The corresponding regions from ClpB and ClpX are unstable. All five fragments exhibit distinct patterns of binding to three proteins that are protease substrates in vivo: the heat shock transcription factor sigma32, the SOS mutagenesis protein UmuD, and Arc repressor bearing the SsrA degradation tag. Recognition of UmuD is mediated through peptide sequences within a 24-residue N-terminal region whereas recognition of both sigma32 and SsrA-tagged Arc requires sequences at the C terminus. These results indicate that the Lon and Clp proteases use the same mechanism of substrate discrimination and suggest that these related ATP-dependent bacterial proteases scrutinize accessible or disordered regions of potential substrates for the presence of specific targeting sequences. (+info)
Redundant in vivo proteolytic activities of Escherichia coli Lon and the ClpYQ (HslUV) protease.
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The ClpYQ (HslUV) ATP-dependent protease of Escherichia coli consists of an ATPase subunit closely related to the Clp ATPases and a protease component related to those found in the eukaryotic proteasome. We found that this protease has a substrate specificity overlapping that of the Lon protease, another ATP-dependent protease in which a single subunit contains both the proteolytic active site and the ATPase. Lon is responsible for the degradation of the cell division inhibitor SulA; lon mutants are UV sensitive, due to the stabilization of SulA. lon mutants are also mucoid, due to the stabilization of another Lon substrate, the positive regulator of capsule transcription, RcsA. The overproduction of ClpYQ suppresses both of these phenotypes, and the suppression of UV sensitivity is accompanied by a restoration of the rapid degradation of SulA. Inactivation of the chromosomal copy of clpY or clpQ leads to further stabilization of SulA in a lon mutant but not in lon+ cells. While either lon, lon clpY, or lon clpQ mutants are UV sensitive at low temperatures, at elevated temperatures the lon mutant loses its UV sensitivity, while the double mutants do not. Therefore, the degradation of SulA by ClpYQ at elevated temperatures is sufficient to lead to UV resistance. Thus, a protease with a structure and an active site different from those of Lon is capable of recognizing and degrading two different Lon substrates and appears to act as a backup for Lon under certain conditions. (+info)
Concurrent chaperone and protease activities of ClpAP and the requirement for the N-terminal ClpA ATP binding site for chaperone activity.
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ClpA, a member of the Clp/Hsp100 family of ATPases, is both an ATP-dependent molecular chaperone and the regulatory component of ClpAP protease. We demonstrate that chaperone and protease activities occur concurrently in ClpAP complexes during a single round of RepA binding to ClpAP and ATP-dependent release. This result was substantiated with a ClpA mutant, ClpA(K220V), carrying an amino acid substitution in the N-terminal ATP binding site. ClpA(K220V) is unable to activate RepA, but the presence of ClpP or chemically inactivated ClpP restores its ability to activate RepA. The presence of ClpP simultaneously facilitates degradation of RepA. ClpP must remain bound to ClpA(K220V) for these effects, indicating that both chaperone and proteolytic activities of the mutant complex occur concurrently. ClpA(K220V) itself is able to form stable complexes with RepA in the presence of a poorly hydrolyzed ATP analog, adenosine 5'-O-(thiotriphosphate), and to release RepA upon exchange of adenosine 5'-O-(thiotriphosphate) with ATP. However, the released RepA is inactive in DNA binding, indicating that the N-terminal ATP binding site is essential for the chaperone activity of ClpA. Taken together, these results suggest that substrates bound to the complex of the proteolytic and ATPase components can be partitioned between release/reactivation and translocation/degradation. (+info)