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Chloroplasts of maize leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C(4) photosynthesis. Chloroplasts contain thylakoid and envelope membranes that contain the photosynthetic machineries and transporters but also proteins involved in e.g. protein homeostasis. These chloroplast membranes must be specialized within each cell type to accommodate C(4) photosynthesis and regulate metabolic fluxes and activities. This quantitative study determined the differentiated state of BS and M chloroplast thylakoid and envelope membrane proteomes and their oligomeric states using innovative gel-based and mass spectrometry-based protein quantifications. This included native gels, iTRAQ, and label-free quantification using an LTQ-Orbitrap. Subunits of Photosystems I and II, the cytochrome b(6)f, and ATP synthase complexes showed average BS/M accumulation ratios of 1.6, 0.45, 1.0, and 1.33, respectively, whereas ratios for the light-harvesting complex I and II families were 1.72 and 0.68, respectively. A 1000-kDa BS-specific NAD(P)H dehydrogenase complex with associated proteins of unknown function containing more than 15 proteins was observed; we speculate that this novel complex possibly functions in inorganic carbon concentration when carboxylation rates by ribulose-bisphosphate carboxylase/oxygenase are lower than decarboxylation rates by malic enzyme. Differential accumulation of thylakoid proteases (Egy and DegP), state transition kinases (STN7,8), and Photosystem I and II assembly factors was observed, suggesting that cell-specific photosynthetic electron transport depends on post-translational regulatory mechanisms. BS/M ratios for inner envelope transporters phosphoenolpyruvate/P(i) translocator, Dit1, Dit2, and Mex1 were determined and reflect metabolic fluxes in carbon metabolism. A wide variety of hundreds of other proteins showed differential BS/M accumulation. Mass spectral information and functional annotations are available through the Plant Proteome Database. These data are integrated with previous data, resulting in a model for C(4) photosynthesis, thereby providing new rationales for metabolic engineering of C(4) pathways and targeted analysis of genetic networks that coordinate C(4) differentiation.
Mol Cell Proteomics 2008 Sep
PMID:Consequences of C4 differentiation for chloroplast membrane proteomes in maize mesophyll and bundle sheath cells. 1845 40

During cytochrome c maturation (Ccm), the DsbA-dependent thio-oxidative protein-folding pathway is thought to introduce a disulphide bond into the haem-binding motif of apocytochromes c. This disulphide bond is believed to be reduced through a thio-reductive pathway involving the Ccm components CcdA (DsbD), CcmG and CcmH. Here, we show in Rhodobacter capsulatus that in the absence of DsbA cytochrome c levels were decreased and CcdA or CcmG or the putative glutathione transporter CydDC was not needed for Ccm. This decrease was not due to overproduction of the periplasmic protease DegP as a secondary effect of DsbA absence. In contrast, CcmH was absolutely necessary regardless of DsbA, indicating that compensatory thio-redox interactions excluded it. Remarkably, the double (DsbA-CcmG) and triple (DsbA-CcmG-CcdA) mutants produced cytochromes c at lower levels than the DsbA-null mutants, unless they contained a CcmG derivative (CcmG*) lacking its thio-reductive activity. Purified CcmG* can bind apocytochrome c in vitro, revealing for the first time a thiol-independent, direct interaction between apocytochrome c and CcmG. Furthermore, elimination of the thio-redox components does not abolish cytochrome c production, restricting the number of Ccm components essential for haem-apocyt c ligation per se during Ccm.
Mol Microbiol 2008 Nov
PMID:Compensatory thio-redox interactions between DsbA, CcdA and CcmG unveil the apocytochrome c holdase role of CcmG during cytochrome c maturation. 1878 43

Ynm3 is the only budding yeast protein possessing a combination of serine protease and postsynaptic density 95/disc-large/zona occludens domains, a defining feature of the high temperature requirement A (HtrA) protein family. The bacterial HtrA/DegP is involved in protective stress response to aid survival at higher temperatures. The role of mammalian mitochondrial HtrA2/Omi in protein quality control is unclear, although loss of its protease activity results in susceptibility toward Parkinson's disease, in which mitochondrial dysfunction and impairment of protein folding and degradation are key pathogenetic features. We studied the role of the budding yeast HtrA, Ynm3, with respect to unfolding stresses. Similar to Escherichia coli DegP, we find that Ynm3 is a dual chaperone-protease. Its proteolytic activity is crucial for cell survival at higher temperature. Ynm3 also exhibits strong general chaperone activity, a novel finding for a eukaryotic HtrA member. We propose that the chaperone activity of Ynm3 may be important to improve the efficiency of proteolysis of aberrant proteins by averting the formation of nonproductive toxic aggregates and presenting them in a soluble state to its protease domain. Suppression studies with Deltaynm3 led to the discovery of chaperone activity in a nucleolar peptidyl-prolyl cis-trans isomerase, Fpr3, which could partly relieve the heat sensitivity of Deltaynm3.
Mol Biol Cell 2009 Jan
PMID:The yeast HtrA orthologue Ynm3 is a protease with chaperone activity that aids survival under heat stress. 1894 88

MucA sequesters extracytoplasmic function (ECF) sigma(22) (algT/U encoded) from target promoters including PalgD for alginate biosynthesis. We have shown that cell wall stress (e.g. d-cycloserine) is a potent inducer of the algD operon. Here we showed that MucB, encoded by the algT-mucABCD operon, interacts with MucA in the sigma-sequestration complex. We hypothesized that AlgW protease (a DegS homologue) is activated by cell wall stress to cleave MucA and release sigma(22). When strain PAO1 was exposed to d-cycloserine, MucA was degraded within just 10 min, and sigma(22) was activated. However, in an algW mutant, MucA was stable with no increased sigma(22) activity. Studies on a yaeL mutant, defective in an RseP/YaeL homologue, suggest that YaeL protease cleaves MucA only after cleavage by AlgW. A defect in mucD, encoding a periplasmic HtrA/DegP homologue, caused MucA instability, suggesting MucD degrades cell wall stress signals. Overall, these data indicate that cell wall stress signals release sigma(22) by regulated intramembrane proteolysis (RIP). Microarray analyses identified genes of the early and late cell wall stress stimulon, which included genes for alginate production. The subset of genes in the sigma(22) regulon was then determined, which included gene products predicted to contribute to recovery from cell wall stress.
Mol Microbiol 2009 Apr
PMID:Use of cell wall stress to characterize sigma 22 (AlgT/U) activation by regulated proteolysis and its regulon in Pseudomonas aeruginosa. 1922 27

HtrA1 belongs to a family of serine proteases found in organisms ranging from bacteria to humans. Bacterial HtrA1 (DegP) is a heat shock-induced protein that behaves as a chaperone at low temperature and as a protease at high temperature to help remove unfolded proteins during heat shock. In contrast to bacterial HtrA1, little is known about the function of human HtrA1. Here, we report the first evidence that human HtrA1 is a microtubule-associated protein and modulates microtubule stability and cell motility. Intracellular HtrA1 is localized to microtubules in a PDZ (PSD95, Dlg, ZO1) domain-dependent, nocodazole-sensitive manner. During microtubule assembly, intracellular HtrA associates with centrosomes and newly polymerized microtubules. In vitro, purified HtrA1 promotes microtubule assembly. Moreover, HtrA1 cosediments and copurifies with microtubules. Purified HtrA1 associates with purified alpha- and beta-tubulins, and immunoprecipitation of endogenous HtrA1 results in coprecipitation of alpha-, beta-, and gamma-tubulins. Finally, downregulation of HtrA1 promotes cell motility, whereas enhanced expression of HtrA1 attenuates cell motility. These results offer an original identification of HtrA1 as a microtubule-associated protein and provide initial mechanistic insights into the role of HtrA1 in the regulation of cell motility by modulating microtubule stability.
Mol Cell Biol 2009 Aug
PMID:Serine protease HtrA1 associates with microtubules and inhibits cell migration. 1947 Jul 53

Sorting of proteins destined to the surface or the extracellular milieu is mediated by specific machineries, which guide the protein substrates towards the proper route of secretion and determine the compartment in which folding occurs. In gram-negative bacteria, the two-partner secretion (TPS) pathway is dedicated to the secretion of large proteins rich in beta-helical structure. The secretion of the filamentous haemagglutinin (FHA), a 230 kDa adhesin of Bordetella pertussis, represents a model TPS system. FHA is exported by the Sec machinery and transits through the periplasm in an extended conformation. From there it is translocated across the outer membrane by its dedicated transporter FhaC to finally fold into a long beta-helix at the cell surface in a progressive manner. In this work, we show that B. pertussis lacking the periplasmic chaperone/protease DegP has a strong growth defect at 37 degrees C, and the integrity of its outer membrane is compromised. While both phenotypes are significantly aggravated by the presence of FHA, the chaperone activity of DegP markedly alleviates the periplasmic stress. In vitro, DegP binds to non-native FHA with high affinity. We propose that DegP chaperones the extended FHA polypeptide in the periplasm and is thus involved in the TPS pathway.
Mol Microbiol 2009 Oct
PMID:Role of DegP for two-partner secretion in Bordetella. 1970 6

To upgrade the proteome reference map of Shigella flexneri 2a 2457T, the protein expression profiles of log phase and stationary phase cells grown at 30 and 37 degrees C were thoroughly analyzed using multiple overlapping narrow pH range (between pH 4.0 and 11.0) two-dimensional gel electrophoresis. A total of 723 spots representing 574 protein entries were identified by MALDI-TOF/TOF MS, including the majority of known key virulence factors. 64 hypothetical proteins and six misannotated proteins were also experimentally identified. A comparison between the four proteome maps showed that most of the virulence-related proteins were up-regulated at 37 degrees C, and the differences were more notable in stationary phase cells, suggesting that the expressions of these virulence factors were not only controlled by temperature but also controlled by the nutrients available in the environment. The expression patterns of some virulence-related genes under the four different conditions suggested that they might also be regulated at the post-transcriptional level. A further significant finding was that the expression of the protein ArgT was dramatically up-regulated at 30 degrees C. The results of semiquantitative RT-PCR analysis showed that expression of argT was not regulated at the transcriptional level. Therefore, we carried out a series of experiments to uncover the mechanism regulating ArgT levels and found that the differential expression of ArgT was due to its degradation by a periplasmic protease, HtrA, whose activity, but not its synthesis, was affected by temperature. The cleavage site in ArgT was between position 160 (Val) and position 161 (Ala). These results may provide useful insights for understanding the physiology and pathogenesis of S. flexneri.
Mol Cell Proteomics 2010 Jun
PMID:The proteome of Shigella flexneri 2a 2457T grown at 30 and 37 degrees C. 2016 57

The PDZ protease DegS senses mislocalized outer membrane proteins and initiates the sigmaE pathway in the bacterial periplasm. This unfolded protein response pathway is activated by processing of the anti-sigma factor RseA by DegS and other proteases acting downstream of DegS. DegS mediates the rate-limiting step of sigma E induction and its activity must be highly specific and tightly regulated. While DegS is structurally and biochemically well studied, the determinants of its pronounced substrate specificity are unknown. We therefore performed swapping experiments by introducing elements of the homologous but unspecific PDZ protease DegP. Introduction of loop L2 of DegP into DegS converted the enzyme into a non-specific protease, while swapping of PDZ domains did not. Therefore, loop L2 of the protease domain is a key determinant of substrate specificity. Interestingly, swapping of loop L2 did not affect the tight regulation of DegS. In addition, the combined introduction of loop L2 and PDZ domain 1 of DegP into DegS converted DegS even further into a DegP-like protease. These and other data suggest that homologous enzymes with distinct activities and regulatory features can be converted by simple genetic modifications.
J Mol Biol 2010 Apr 09
PMID:Conversion of a regulatory into a degradative protease. 2018 96

When proteins are damaged under stresses conditions, these proteins are either refolded or degraded by quality control system of molecular chaperones and protease. High-temperature requirement A (htrA) is of particular interest because it can perform the roles of both protease and a chaperone. HtrA plays an important role in maintaining the physiological homeostasis of bacteria against environmental stress such as elevated temperature, oxidative and osmotic stress. Inactivation of htrA genes can thus restrict the survival ability of bacteria. These observations suggested that htrA might be responsible for acid tolerance of Streptococcus mutans. In this study, we have generated an htrA mutant and an htrA-complemented strain of S. mutans K7 isolated from a Korean in order to investigate the role of htrA in growth under acidic conditions. In terms of growth under cidic conditions, the htrA mutant exhibited 20% to 23% lower growth than the control group. In addition, glucosyltransferase B and glucosyltransferase C expression levels significantly decreased. When the htrA expression level was restored by adding the htrA gene to the htrA mutant strain, the normal growth phenotype was restored under acid stress. Further, similar results were obtained for S. mutans UA159. Thus, htrA in S. mutans K7, as well as S. mutans UA159, can be concluded to play an important role during acid stress.
Mol Cells 2010 Mar
PMID:The influence of HtrA expression on the growth of Streptococcus mutans during acid stress. 2038 36

The Cpx envelope stress response is induced by the misfolding of periplasmic proteins and restores envelope homeostasis by upregulating several periplasmic protein folding and degrading factors. The Cpx response also regulates the expression of a variety of envelope-spanning protein complexes, including flagella, secretion systems and pili, which play an important role in pathogenesis. In a previous study, we inactivated the Cpx response in enteropathogenic Escherichia coli (EPEC), a causative agent of infant diarrhoea, and observed decreased expression of its major adhesin, the bundle-forming pilus (BFP). Here, we examined the mechanism underlying this BFP expression defect, and found that this phenotype can be attributed to insufficient expression of periplasmic folding factors, such as DsbA, DegP and CpxP. Hence, a low level of Cpx pathway activity promotes BFP synthesis by upregulating factors important for folding of BFP component proteins. Conversely, we found that full induction of the Cpx response inhibits BFP expression, mainly by repressing transcription of the bfp gene cluster. In combination with a previous report examining EPEC type III secretion, our results demonstrate that the Cpx response co-ordinates the repression of cell-surface structures during periods of envelope stress.
Mol Microbiol 2010 Jun 01
PMID:The Cpx envelope stress response both facilitates and inhibits elaboration of the enteropathogenic Escherichia coli bundle-forming pilus. 2044 97


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