The Toxoplasma adhesive protein MIC2 is proteolytically processed at multiple sites by two parasite-derived proteases

MIC2 is an adhesive protein that participates in host cell invasion by the obligate intracellular parasite Toxoplasma gondii. Earlier studies established that MIC2 is secreted into the culture medium by extracellular parasites and that release is coincident with proteolytic modification. Since little is known about proteolytic processing of proteins secreted by T. gondii, we undertook this study to investigate the proteolytic events that accompany secretion of MIC2. We demonstrate that the C-terminal domain of MIC2 is removed by a protease, termed MPP1, when MIC2 is released into the culture supernatant. Additionally, prior to release, a second protease, termed MPP2, trims the N terminus of MIC2, resulting in the release of heterogeneously sized species of MIC2. Although MPP1 activity was unaffected by any of the protease inhibitors tested, MPP2 activity was blocked by a subset of serine and cysteine protease inhibitors. These results establish that MIC2 is proteolytically modified at multiple sites by two distinct enzymes that probably operate on the parasite surface.     

The precursor of sulfatide activator protein is processed to three different proteins

The enzymic degradation of a number of sphingolipids in the lysosomes is stimulated by small acid glycoproteins named activator proteins. We purified and sequenced a new protein, called component C, which seems to be related to sulfatide activator and to a recently described activator of glucosylceramidase (A1 activator) (Kleinschmidt, T., Christomanou, H. & Braunitzer, G. (1987) Biol. Chem. Hoppe-Seyler 368, 1571-1578). It consists of 78 amino acids and carries one carbohydrate chain at aparagine 20. Component C shows 21.5% sequence homology to sulfatide activator and 34.2% homology to A1 activator. Structural similarities between these three proteins have also been detected. Recently the cDNA sequence of the sulfatide activator precursor has been published (Dewji, N.N., Wenger, D.A. & O'Brien, J.S. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8652-8656). We could align the protein sequences of sulfatide activator, A1 activator and component C with that of this large precursor protein. After minor corrections of the DNA sequence we obtained total fit. Thus it seems that three different proteins are derived from the sulfatide activator precursor by proteolytic processing. Possible processing sites were found on the precursor at sites adjacent to the N-termini and C-termini of the mature proteins. The processing of sulfatide activator was studied by Fujibayashi and Wenger (Fujibayashi, S. & Wenger, D.A. (1986) Biochim. Biophys. Acta 875, 554-562). Their data support our assumption that processing occurs by simultaneous cleavage at all possible sites.     

The yeast CLC chloride channel is proteolytically processed by the furin-like protease Kex2p in the first extracellular loop

CLC chloride channels are a family of channel proteins mediating chloride transport across the plasma membrane and intracellular membranes. The single yeast CLC protein Gef1p is localized to the Golgi and endosomal system. Investigating epitope-tagged variants of Gef1p, we found that the channel is proteolytically processed in the secretory pathway. Proteolytic cleavage occurs in the first extracellular loop of the protein at residues KR136/137 and is carried out by the Kex2p protease. Fragments mimicking the N- and C-terminal products of the cleavage reaction are non-functional when expressed alone. However, functional channels can assemble when the two fragments are co-expressed.     

The in-vitro synthesized tomato shikimate kinase precursor is enzymatically active and is imported and processed to the mature enzyme by chloroplasts

Full-length cDNA clones encoding shikimate kinase (EC 2.7.1.71), an enzyme of the central section of the shikimate pathway, have been isolated from tomato (Lycopersicon esculentum L., cv. UC82b). The open reading frame has the capacity to encode a peptide of 300 amino acids. The in-vitro synthesized peptide catalysed the phosphorylation of shikimate thus confirming the identity of the isolated cDNA clones. The N-terminal portion of the deduced amino acid sequence resembles known chloroplast-specific transit peptides. The existence of such a transit peptide was proven by the uptake of the in-vitro synthesized peptide as well as its processing by isolated chloroplasts. Multiple sites of polyadenylation were observed in shikimate kinase mRNAs. The results of Northern and Southern blot analyses are consistent with the existence of only one shikimate kinase gene per haploid genome in tomato. These results are discussed with respect to the dual pathway hypothesis of the shikimate pathway in higher plants.     

The leucine-rich repeat domain can determine effective interaction between RPS2 and other host factors in arabidopsis RPS2-mediated disease resistance

Like many other plant disease resistance genes, Arabidopsis thaliana RPS2 encodes a product with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains. This study explored the hypothesized interaction of RPS2 with other host factors that may be required for perception of Pseudomonas syringae pathogens that express avrRpt2 and/or for the subsequent induction of plant defense responses. Crosses between Arabidopsis ecotypes Col-0 (resistant) and Po-1 (susceptible) revealed segregation of more than one gene that controls resistance to P. syringae that express avrRpt2. Many F(2) and F(3) progeny exhibited intermediate resistance phenotypes. In addition to RPS2, at least one additional genetic interval associated with this defense response was identified and mapped using quantitative genetic methods. Further genetic and molecular genetic complementation experiments with cloned RPS2 alleles revealed that the Po-1 allele of RPS2 can function in a Col-0 genetic background, but not in a Po-1 background. The other resistance-determining genes of Po-1 can function, however, as they successfully conferred resistance in combination with the Col-0 allele of RPS2. Domain-swap experiments revealed that in RPS2, a polymorphism at six amino acids in the LRR region is responsible for this allele-specific ability to function with other host factors.     

Homology between the ribosomal DNA of Escherichia coli and mitochondrial DNA preparations of maize is principally to sequences other than mitochondrial rRNA genes

E. coli ribosomal DNA has been used to probe maize mitochondrial DNA. It hybridizes primarily with chloroplast ribosomal DNA sequences and with fungal and bacterial sequences which may contaminate the mtDNA preparations. It also hybridizes to the chloroplast 16S ribosomal RNA gene sequence present in the mitochondrial genome (1) as well as to the mitochondrial 18S ribosomal RNA gene sequence. Weak sequence homology was detected between E. coli rDNA and the mitochondrial 26S ribosomal RNA gene.     

The cytosol-synthesized subunit II (Cox2) precursor with the point mutation W56R is correctly processed in yeast mitochondria to rescue cytochrome oxidase

Deletion of the yeast mitochondrial gene COX2 encoding subunit 2 (Cox2) of cytochrome c oxidase (CcO) results in loss of respiration (Δcox2 strain). Supekova et al. (2010) [1] transformed a Δcox2 strain with a vector expressing Cox2 with a mitochondrial targeting sequence (MTS) and the point mutation W56R (Cox2^W56R), restoring respiratory growth. Here, the CcO carrying the allotopically-expressed Cox2^W56R was characterized. Yeast mitochondria from the wild-type (WT) and the Δcox2 + Cox2^W56R strains were subjected to Blue Native electrophoresis. In-gel activity of CcO and spectroscopic quantitation of cytochromes revealed that only 60% of CcO is present in the complemented strain, and that less CcO is found associated in supercomplexes as compared to WT. CcOs from the WT and the mutant exhibited similar subunit composition, although activity was 20–25% lower in the enzyme containing Cox2^W56R than in the one with Cox2^WT. Tandem mass spectrometry confirmed that W₅₆ was substituted by R₅₆ in Cox2^W56R. In addition, Cox2^W56R exhibited the same N-terminus than Cox2^WT, indicating that the MTS of Oxa1 and the leader sequence of 15 residues were removed from Cox2^W56R during maturation. Thus, Cox2^W56R is identical to Cox2^WT except for the point mutation W56R. Mitochondrial Cox1 synthesis is strongly reduced in Δcox2 mutants, but the Cox2^W56R complemented strain led to full restoration of Cox1 synthesis. We conclude that the cytosol-synthesized Cox2^W56R follows a rate-limiting process of import, maturation or assembly that yields lower steady-state levels of CcO. Still, the allotopically-expressed Cox2^W56R restores CcO activity and allows mitochondrial Cox1 synthesis to advance at WT levels.     

P159 is a proteolytically processed, surface adhesin of Mycoplasma hyopneumoniae: defined domains of P159 bind heparin and promote adherence to eukaryote cells

Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, colonizes the respiratory cilia of affected swine causing significant economic losses to swine production worldwide. Heparin is known to inhibit adherence of M. hyopneumoniae to porcine respiratory epithelial cilia. M. hyopneumoniae cells bind heparin but the identity of the heparin-binding proteins is limited. Proteomic analysis of M. hyopneumoniae lysates identified 27 kDa (P27), 110 kDa (P110) and 52 kDa (P52) proteins representing different regions of a 159 kDa (P159) protein derived from mhp494. These cleavage fragments were surface located and present at all growth stages. Following purification of four recombinant proteins spanning P159 (F1P159, F2P159, F3P159 and F4P159), only F3P159 and F4P159 bound heparin in a dose-dependent manner (K(d) values 142.37 +/- 22.01 nM; 75.37 +/- 7.34 nM respectively). Scanning electron microscopic studies showed M. hyopneumoniae bound intimately to porcine kidney epithelial-like cells (PK15 cells) but these processes were inhibited by excess heparin and F4P159. Similarly, latex beads coated with F2P159 and F4P159 adhered to and entered PK15 cells, but heparin, F2P159 and F4P159 was inhibitory. These findings indicate that P159 is a post-translationally cleaved, glycosaminoglycan-binding adhesin of M. hyopneumoniae.     

SLO-2 is cytoprotective and contributes to mitochondrial potassium transport

Mitochondrial potassium channels are important mediators of cell protection against stress. The mitochondrial large-conductance "big" K(+) channel (mBK) mediates the evolutionarily-conserved process of anesthetic preconditioning (APC), wherein exposure to volatile anesthetics initiates protection against ischemic injury. Despite the role of the mBK in cardioprotection, the molecular identity of the channel remains unknown. We investigated the attributes of the mBK using C. elegans and mouse genetic models coupled with measurements of mitochondrial K(+) transport and APC. The canonical Ca(2+)-activated BK (or "maxi-K") channel SLO1 was dispensable for both mitochondrial K(+) transport and APC in both organisms. Instead, we found that the related but physiologically-distinct K(+) channel SLO2 was required, and that SLO2-dependent mitochondrial K(+) transport was triggered directly by volatile anesthetics. In addition, a SLO2 channel activator mimicked the protective effects of volatile anesthetics. These findings suggest that SLO2 contributes to protection from hypoxic injury by increasing the permeability of the mitochondrial inner membrane to K(+).     

Crimean-congo hemorrhagic fever virus glycoprotein precursor is cleaved by Furin-like and SKI-1 proteases to generate a novel 38-kilodalton glycoprotein

Crimean-Congo hemorrhagic fever virus (genus Nairovirus, family Bunyaviridae) genome M segment encodes an unusually large (in comparison to members of other genera) polyprotein (1,684 amino acids in length) containing the two major structural glycoproteins, Gn and Gc, that are posttranslationally processed from precursors PreGn and PreGc by SKI-1 and SKI-1-like proteases, respectively. The characteristics of the N-terminal 519 amino acids located upstream of the mature Gn are unknown. A highly conserved furin/proprotein convertase (PC) cleavage site motif (RSKR247) is located between the variable N-terminal region that is predicted to have mucin-like properties and the rest of PreGn. Mutational analysis of the RSKR247 motif and use of a specific furin/PC inhibitor and brefeldin A demonstrate that furin/PC cleavage occurs at the RSKR247 motif of PreGn as the protein transits the trans Golgi network and generates a novel glycoprotein designated GP38. Immunoprecipitation analysis identified two additional proteins, GP85 and GP160, which contain both mucin and GP38 domain regions, and whose generation does not involve furin/PC cleavage. Consistent with glycosylation predictions, heavy O-linked glycosylation and moderate levels of N-glycans were detected in the GP85 and GP160 proteins, both of which contain the mucin domain. GP38, GP85, and GP160 are likely soluble proteins based on the lack of predicted transmembrane domains, their detection in virus-infected cell supernatants, and the apparent absence from virions. Analogy with soluble glycoproteins and mucin-like proteins encoded by other hemorrhagic fever-associated RNA viruses suggests these proteins could play an important role in viral pathogenesis.     

The Erwinia amylovora avrRpt2EA gene contributes to virulence on pear and AvrRpt2EA is recognized by Arabidopsis RPS2 when expressed in pseudomonas syringae

The enterobacterium Erwinia amylovora is a devastating plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. In an attempt to identify genes induced during infection of host plants, we identified and cloned a putative effector gene, avrRpt2EA. The deduced amino-acid sequence of the translated AvrRpt2EA protein is homologous to the effector protein AvrRpt2 previously reported in Pseudomonas syringae pv. tomato. These two proteins share 58% identity (70% similarity) in the functional domain; however, the secretion and translocation signal domain varied. The avrRpt2EA promoter region contains a typical 'hrp box,' which suggests that avrRpt2EA is regulated by the alternative sigma factor, HrpL. avrRpt2EA was detected in all E. amylovora strains tested but not in other closely related Erwinia species. An avrRpt2EA deletion mutant was reduced in its ability to cause systemic infection on immature pear fruits as compared with the wild-type strain, indicating that avrRpt2EA acts as a virulence factor on its native host. Growth of P. syringae pv. tomato DC3000 expressing avrRpt2EA was 10-fold higher than that of P. syringae pv. tomato DC3000 in an Arabidopsis rps2 mutant, indicating that avrRpt2EA promotes virulence of P. syringae pv. tomato DC3000 on Arabidopsis similar to P. syringae pv. tomato avrRpt2. When avrRpt2EA was expressed in P. syringae pv. tomato DC3000 in its native form, a weak hypersensitive response (HR) was induced in Arabidopsis; however, a hybrid protein containing the P. syringae pv. tomato avrRpt2 signal sequence, when expressed from the P syringae pv. tomato avrRpt2 promoter, caused a strong HR. Thus, the signal sequence and promoter of avrRpt2EA may affect its expression, secretion, or translocation, singly or in combination, in P. syringae pv. tomato DC3000. These results indicated that avrRpt2EA is genetically recognized by the RPS2 disease resistance gene in Arabidopsis when expressed in P. syringae pv. tomato DC3000. The results also suggested that although distinct pathogens such as E. amylovora and P. syringae may contain similar effector genes, expression and secretion of these effectors can be under specific regulation by the native pathogen.     

Transport of proteins into chloroplasts. The precursor of small subunit of ribulose bisphosphate carboxylase is processed to the mature size in two steps

The precursor of the small subunit of ribulose bisphosphate carboxylase in Pisum sativum (relative molecular mass 20 000) is processed to the mature size (relative molecular mass 14 000) by the purified processing enzyme in two steps. The maturation proceeds via an intermediate of Mr 18 000. Both processing reactions may be carried out by the same enzyme although different residues are involved in the two cleavage sites. The second cleavage is inhibited if the precursor is pre-incubated with iodoacetate. The processing intermediate cannot be detected during the uptake of the precursor by intact isolated chloroplasts but iodoacetate-treated precursor is taken up and converted to a number of polypeptides of Mr 18 000 and below.     

The interplay between SUCLA2, SUCLG2, and mitochondrial DNA depletion

SUCLA2-related mitochondrial DNA (mtDNA) depletion syndrome is a result of mutations in the β subunit of the ADP-dependent isoform of the Krebs cycle succinyl-CoA synthase (SCS). The mechanism of tissue specificity and mtDNA depletion is elusive but complementation by the GDP-dependent isoform encoded by SUCLG2, and the association with mitochondrial nucleoside diphosphate kinase (NDPK), is a plausible link. We have investigated this relationship by studying SUCLA2 deficient fibroblasts derived from patients and detected normal mtDNA content and normal NDPK activity. However, knockdown of SUCLG2 by shRNA in both patient and control fibroblasts resulted in a significant decrease in mtDNA amount, decreased NDPK and cytochrome c oxidase activities, and a marked growth impairment. This suggests that, SUCLG2, to a higher degree than SUCLA2, is crucial for mtDNA maintenance and that mitochondrial NDPK is involved. Although results pertain to a cell culture system, the findings might explain the pathomechanism and tissue specificity in mtDNA depletion caused by defective SUCLA2.     

The ABC transporter AtABCB14 is a malate importer and modulates stomatal response to CO2

Carbon dioxide uptake and water vapour release in plants occur through stomata, which are formed by guard cells. These cells respond to light intensity, CO2 and water availability, and plant hormones. The predicted increase in the atmospheric concentration of CO2 is expected to have a profound effect on our ecosystem. However, many aspects of CO2-dependent stomatal movements are still not understood. Here we show that the ABC transporter AtABCB14 modulates stomatal closure on transition to elevated CO2. Stomatal closure induced by high CO2 levels was accelerated in plants lacking AtABCB14. Apoplastic malate has been suggested to be one of the factors mediating the stomatal response to CO2 (Refs 4,5) and indeed, exogenously applied malate induced a similar AtABCB14-dependent response as high CO2 levels. In isolated epidermal strips that contained only guard cells, malate-dependent stomatal closure was faster in plants lacking the AtABCB14 and slower in AtABCB14-overexpressing plants, than in wild-type plants, indicating that AtABCB14 catalyses the transport of malate from the apoplast into guard cells. Indeed, when AtABCB14 was heterologously expressed in Escherichia coli and HeLa cells, increases in malate transport activity were observed. We therefore suggest that AtABCB14 modulates stomatal movement by transporting malate from the apoplast into guard cells, thereby increasing their osmotic pressure.     

5-(phenylthiomethyl)-2'-deoxyuridine as an efficient photoreactive precursor to generate single and multiple lesions within DNA fragments

5-(Phenylthiomethyl)-2'-deoxyuridine was successfully incorporated into DNA oligomers by automated DNA synthesis using phosphoramidite chemistry. UV exposure of the latter thionucleoside containing oligonucleotides under anaerobic and aerobic conditions gives rise to specific base lesions. The photoproducts have been isolated and further characterized on the basis of NMR and mass spectrometric analyses.     

The DEAD-box RNA helicase ZmRH48 is required for the splicing of multiple mitochondrial introns,mitochondrial complex biosynthesis,and seed development in maize

RNA helicases participate in nearly all aspects of RNA metabolism by rearranging RNAs or RNA–protein complexes in an adenosine triphosphatedependent manner. Due to the large RNA helicase families in plants, the precise roles of many RNA helicases in plant physiology and development remain to be clarified. Here, we show that mutations in maize(Zea mays) DEAD-box RNA helicase48(Zm RH48) impair the splicing of mitochondrial introns, mitochondrial complex biosynthesis,and seed development. Loss of Z...