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1.
Proteomic, enzymatic, and mutant analyses revealed that peptidyl-prolyl isomerase (PPIase) activity in the chloroplast thylakoid lumen of Arabidopsis is determined by two immunophilins: AtCYP20-2 and AtFKBP13. These two enzymes are responsible for PPIase activity in both soluble and membrane-associated fractions of thylakoid lumen suggesting that other lumenal immunophilins are not active towards the peptide substrates. In thiol-reducing conditions PPIase activity of the isolated AtFKBP13 and of the total thylakoid lumen is suppressed several fold. Profound redox-dependence of PPIase activity implies oxidative activation of protein folding catalysis under oxidative stress and photosynthetic oxygen production in the thylakoid lumen of plant chloroplasts.  相似文献   

2.
3.
The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates.  相似文献   

4.
The Plasmodium mitochondrial electron transport chain has received considerable attention as a potential target for new antimalarial drugs. Atovaquone, a potent inhibitor of Plasmodium cytochrome bc(1), in combination with proguanil is recommended for chemoprophylaxis and treatment of malaria. The type II NADH:ubiquinone oxidoreductase (NDH2) is considered an attractive drug target, as its inhibition is thought to lead to the arrest of the mitochondrial electron transport chain and, as a consequence, pyrimidine biosynthesis, an essential pathway for the parasite. Using the rodent malaria parasite Plasmodium berghei as an in vivo infection model, we studied the role of NDH2 during Plasmodium life cycle progression. NDH2 can be deleted by targeted gene disruption and, thus, is dispensable for the pathogenic asexual blood stages, disproving the candidacy for an anti-malarial drug target. After transmission to the insect vector, NDH2-deficient ookinetes display an intact mitochondrial membrane potential. However, ndh2(-) parasites fail to develop into mature oocysts in the mosquito midgut. We propose that Plasmodium blood stage parasites rely on glycolysis as the main ATP generating process, whereas in the invertebrate vector, a glucose-deprived environment, the malaria parasite is dependent on an intact mitochondrial respiratory chain.  相似文献   

5.
Michel Havaux  Dominique Rumeau 《BBA》2005,1709(3):203-213
Far-red illumination of plant leaves for a few seconds induces a delayed luminescence rise, or afterglow, that can be measured with the thermoluminescence technique as a sharp band peaking at around 40-45 °C. The afterglow band is attributable to a heat-induced electron flow from the stroma to the plastoquinone pool and the PSII centers. Using various Arabidopsis and tobacco mutants, we show here that the electron fluxes reflected by the afterglow luminescence follow the pathways of cyclic electron transport around PSI. In tobacco, the afterglow signal relied mainly on the ferredoxin-quinone oxidoreductase (FQR) activity while the predominant pathway responsible for the afterglow in Arabidopsis involved the NAD(P)H dehydrogenase (NDH) complex. The peak temperature Tm of the afterglow band varied markedly with the light conditions prevailing before the TL measurements, from around 30 °C to 45 °C in Arabidopsis. These photoinduced changes in Tm followed the same kinetics and responded to the same light stimuli as the state 1-state 2 transitions. PSII-exciting light (leading to state 2) induced a downward shift while preillumination with far-red light (inducing state 1) caused an upward shift. However, the light-induced downshift was strongly inhibited in NDH-deficient Arabidopsis mutants and the upward shift was cancelled in plants durably acclimated to high light, which can perform normal state transitions. Taken together, our results suggest that the peak temperature of the afterglow band is indicative of regulatory processes affecting electron donation to the PQ pool which could involve phosphorylation of NDH. The afterglow thermoluminescence band provides a new and simple tool to investigate the cyclic electron transfer pathways and to study their regulation in vivo.  相似文献   

6.
The Saccharomyces cerevisiae FAB1 gene encodes a 257-kD protein that contains a cysteine-rich RING-FYVE domain at its NH2-terminus and a kinase domain at its COOH terminus. Based on its sequence, Fab1p was initially proposed to function as a phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinase (Yamamoto et al., 1995). Additional sequence analysis of the Fab1p kinase domain, reveals that Fab1p defines a subfamily of putative PtdInsP kinases that is distinct from the kinases that synthesize PtdIns(4,5)P2. Consistent with this, we find that unlike wild-type cells, fab1Δ, fab1tsf, and fab1 kinase domain point mutants lack detectable levels of PtdIns(3,5)P2, a phosphoinositide recently identified both in yeast and mammalian cells. PtdIns(4,5)P2 synthesis, on the other hand, is only moderately affected even in fab1Δ mutants. The presence of PtdIns(3)P in fab1 mutants, combined with previous data, indicate that PtdIns(3,5)P2 synthesis is a two step process, requiring the production of PtdIns(3)P by the Vps34p PtdIns 3-kinase and the subsequent Fab1p- dependent phosphorylation of PtdIns(3)P yielding PtdIns(3,5)P2. Although Vps34p-mediated synthesis of PtdIns(3)P is required for the proper sorting of hydrolases from the Golgi to the vacuole, the production of PtdIns(3,5)P2 by Fab1p does not directly affect Golgi to vacuole trafficking, suggesting that PtdIns(3,5)P2 has a distinct function. The major phenotypes resulting from Fab1p kinase inactivation include temperature-sensitive growth, vacuolar acidification defects, and dramatic increases in vacuolar size. Based on our studies, we hypothesize that whereas Vps34p is essential for anterograde trafficking of membrane and protein cargoes to the vacuole, Fab1p may play an important compensatory role in the recycling/turnover of membranes deposited at the vacuole. Interestingly, deletion of VAC7 also results in an enlarged vacuole morphology and has no detectable PtdIns(3,5)P2, suggesting that Vac7p functions as an upstream regulator, perhaps in a complex with Fab1p. We propose that Fab1p and Vac7p are components of a signal transduction pathway which functions to regulate the efflux or turnover of vacuolar membranes through the regulated production of PtdIns(3,5)P2.  相似文献   

7.
Alternative splicing is prevalent in plants, but little is known about its regulation in the context of developmental and signaling pathways. We describe here a new factor that influences pre-messengerRNA (mRNA) splicing and is essential for embryonic development in Arabidopsis thaliana. This factor was retrieved in a genetic screen that identified mutants impaired in expression of an alternatively spliced GFP reporter gene. In addition to the known spliceosomal component PRP8, the screen recovered Arabidopsis RTF2 (AtRTF2), a previously uncharacterized, evolutionarily conserved protein containing a replication termination factor 2 (Rtf2) domain. A homozygous null mutation in AtRTF2 is embryo lethal, indicating that AtRTF2 is an essential protein. Quantitative RT-PCR demonstrated that impaired expression of GFP in atrtf2 and prp8 mutants is due to inefficient splicing of the GFP pre-mRNA. A genome-wide analysis using RNA sequencing indicated that 13–16% of total introns are retained to a significant degree in atrtf2 mutants. Considering these results and previous suggestions that Rtf2 represents an ubiquitin-related domain, we discuss the possible role of AtRTF2 in ubiquitin-based regulation of pre-mRNA splicing.  相似文献   

8.
We reported recently that regulation by intracellular pH (pH(i)) of the murine Cl-/HCO(3)(-) exchanger AE2 requires amino acid residues 310-347 of the polypeptide's NH(2)-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl- efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pH(o)) with unclamped or clamped pH(i), or during variation of pH(i) at constant pH(o). Wild-type AE2-mediated 36Cl- efflux was profoundly inhibited by acid pH(o), with a value of pH(o50) = 6.87 +/- 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312-347 identified the greatest acid shift in pH(o(50)) value, approximately 0.8 pH units in the mutant (A)6 342-347, but only a modest acid-shift in the mutant (A)6 336-341. Two of the six (A)6 mutants retained normal pH(i) sensitivity of 36Cl- efflux, whereas the (A)6 mutants 318-323, 336-341, and 342-347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336-347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pH(o) and to pH(i) were found independently and in concert. The E346A mutation acid-shifted the pH(o(0) value to the same extent whether pH(i) was unclamped or held constant during variation of pH(o). Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pH(o) as well as pH(i).  相似文献   

9.
AtCYP38 is a thylakoid lumen protein comprising the immunophilin domain and the phosphatase inhibitor module. Here we show the association of AtCYP38 with the photosystem II (PSII) monomer complex and address its functional role using AtCYP38-deficient mutants. The dynamic greening process of etiolated leaves failed in the absence of AtCYP38, due to specific problems in the biogenesis of PSII complexes. Also the development of leaves under short-day conditions was severely disturbed. Detailed biophysical and biochemical analysis of mature AtCYP38-deficient plants from favorable growth conditions (long photoperiod) revealed: (i) intrinsic malfunction of PSII, which (ii) occurred on the donor side of PSII and (iii) was dependent on growing light intensity. AtCYP38 mutant plants also showed decreased accumulation of PSII, which was shown not to originate from impaired D1 synthesis or assembly of PSII monomers, dimers and supercomplexes as such but rather from the incorrect fine-tuning of the oxygen-evolving side of PSII. This, in turn, rendered PSII centers extremely susceptible to photoinhibition. AtCYP38 deficiency also drastically decreased the in vivo phosphorylation of PSII core proteins, probably related to the absence of the AtCYP38 phosphatase inhibitor domain. It is proposed that during PSII assembly AtCYP38 protein guides the proper folding of D1 (and CP43) into PSII, thereby enabling the correct assembly of the water-splitting Mn4–Ca cluster even with high turnover of PSII.  相似文献   

10.
We have successfully prepared E- and Z- isomers of 17-20 dehydro analogs of 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 (2MD). Both isomers bind to the recombinant rat vitamin D receptor (VDR) with high affinity. The Z-isomer (Vit-III 17-20Z) displays activity in vivo and in vitro that is similar to 2MD. The in vitro activity of the E-isomer (Vit-III 17-20E) is comparable to the natural hormone, though in vivo this analog is significantly less calcemic. Crystal structures of the rat VDR ligand binding domain complexed with the analogs demonstrate that the Vit-III 17-20Z analog is oriented almost identically to 2MD, with only minor differences induced by the planar configuration around the C17-C20 double bond. The Vit-III 17-20E analog is oriented in a conformation distinct from both 2MD and the natural hormone. The structural comparisons suggest that the position of C21 in the ligand binding site may be an important determinant of biological activity.  相似文献   

11.
Chloroplastic NAD(P)H dehydrogenase (NDH) plays a role in cyclic electron flow around photosystem I to produce ATP, especially in adaptation to environmental changes. Although the NDH complex contains 11 subunits that are homologous to NADH:ubiquinone oxidoreductase (complex I; EC 1.6.5.3), recent genetic and biological studies have indicated that NDH also comprises unique subunits. We describe here an in silico approach based on co-expression analysis and phylogenetic profiling that was used to identify 65 genes as potential candidates for NDH subunits. Characterization of 21 Arabidopsis T-DNA insertion mutants among these ndh gene candidates indicated that three novel ndf (NDH-dependent cyclic electron flow) mutants ( ndf1 , ndf2 and ndf4 ) had impaired NDH activity as determined by measurement of chlorophyll fluorescence. The amount of NdhH subunit was greatly decreased in these mutants, suggesting that the loss of NDH activity was caused by a defect in accumulation of the NDH complex. In addition, NDF1, NDF2 and NDF4 proteins co-migrated with the NdhH subunit, as shown by blue native electrophoresis. These results strongly suggest that NDF proteins are novel subunits of the NDH complex. Further analysis revealed that the NDF1 and NDF2 proteins were unstable in the mutants lacking hydrophobic subunits of the NDH complex, but were stable in mutants lacking the hydrophilic subunits, suggesting that NDF1 and NDF2 interact with a hydrophobic sub-complex. NDF4 protein was predicted to possess a redox-active iron–sulfur cluster domain that may be involved in the electron transfer.  相似文献   

12.
13.
This paper presents domain complementation studies in the mannitol transporter, EIImtl, from Escherichia coli. EIImtl is responsible for the transport and concomitant phosphorylation of mannitol over the cytoplasmic membrane. By using tryptophan-less EIImtl as a basis, each of the four phenylalanines located in the cytoplasmic loop between putative transmembrane helices II and III in the membrane-embedded C domain were replaced by tryptophan, yielding the mutants W97, W114, W126, and W133. Except for W97, these single-tryptophan mutants exhibited a high, wild-type-like, binding affinity for mannitol. Of the four mutants, only W114 showed a high mannitol phosphorylation activity. EIImtl is functional as a dimer and the effect of these mutations on the oligomeric activity was investigated via heterodimer formation (C/C domain complementation studies). The low phosphorylation activities of W126 and W133 could be increased 7-28 fold by forming heterodimers with either the C domain of W97 (IICmtlW97) or the inactive EIImtl mutant G196D. W126 and W133, on the other hand, did not complement each other. This study points towards a role of positions 97, 126 and 133 in the oligomeric activation of EIImtl. The involvement of specific residue positions in the oligomeric functioning of a sugar-translocating EII protein has not been presented before.  相似文献   

14.

Background

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). Studies in the yeast Saccharomyces cerevisiae have provided valuable insights into the mechanisms of cellular dysfunction associated with the expression of faulty PD genes.

Methods

We developed a yeast model for full-length LRRK2 studies. We expressed wild-type (wt) LRRK2 and mutations and evaluated their role during oxidative stress conditions. The involvement of mitochondria was assessed by using rho-zero mutants and by evaluating reactive oxygen species (ROS) production and mitochondrial membrane potential by flow cytometry. The involvement of endocytosis was also studied by testing several endocytic mutants and by following the vacuolar delivery of the probe FM4-64.

Results

Expression of LRRK2 in yeast was associated to increased hydrogen peroxide resistance. This phenotype, which was dependent on mitochondrial function, was not observed for PD-mutants G2019S and R1441C or in the absence of the kinase activity and the WD40 repeat domain. Expression of the pathogenic mutants stimulated ROS production and increased mitochondrial membrane potential. For the PD-mutants, but not for wild-type LRRK2, endocytic defects were also observed. Additionally, several endocytic proteins were required for LRRK2-mediated protection against hydrogen peroxide.

Conclusions

Our results indicate that LRRK2 confers cellular protection during oxidative stress depending on mitochondrial function and endocytosis.

General significance

Both the loss of capacity of LRRK2 pathogenic mutants to protect against oxidative stress and their enhancement of dysfunction may be important for the development of PD during the aging process.  相似文献   

15.
The yeast α-1,3-mannosyltransferase (Mnn1p) is localized to the Golgi by independent transmembrane and lumenal domain signals. The lumenal domain is localized to the Golgi complex when expressed as a soluble form (Mnn1-s) by exchange of its transmembrane domain for a cleavable signal sequence (Graham, T. R., and V. A. Krasnov. 1995. Mol. Biol. Cell. 6:809–824). Mutants that failed to retain the lumenal domain in the Golgi complex, called lumenal domain retention (ldr) mutants, were isolated by screening mutagenized yeast colonies for those that secreted Mnn1-s. Two genes were identified by this screen, HOG1, a gene encoding a mitogen-activated protein kinase (MAPK) that functions in the high osmolarity glycerol (HOG) pathway, and LDR1. We have found that basal signaling through the HOG pathway is required to localize Mnn1-s to the Golgi in standard osmotic conditions. Mutations in HOG1 and LDR1 also perturb localization of intact Mnn1p, resulting in its loss from early Golgi compartments and a concomitant increase of Mnn1p in later Golgi compartments.  相似文献   

16.
17.
The multifunctional cytochrome P450 monooxygenases P450-1 and P450-2 from Fusarium fujikuroi catalyze the formation of GA14 and GA4, respectively, in the gibberellin (GA)-biosynthetic pathway. However, the activity of these enzymes is qualitatively and quantitatively different in mutants lacking the NADPH:cytochrome P450 oxidoreductase (CPR) compared to CPR-containing strains. 3beta-Hydroxylation, a major P450-1 activity in wild-type strains, was strongly decreased in the mutants relative to oxidation at C-6 and C-7, while synthesis of C19-GAs as a result of oxidative cleavage of C-20 by P450-2 was almost absent whereas the C-20 alcohol, aldehyde and carboxylic acid derivatives accumulated. Interaction of the monooxygenases with alternative electron transport proteins could account for these different product distributions. In the absence of CPR, P450-1 activities were NADH-dependent, and stimulated by cytochrome b5 or by added FAD. These properties as well as the decreased efficiency of P450-1 and P450-2 in the mutants are consistent with the participation of cytochrome b5:NADH cytochrome b5 reductase as redox partner of the gibberellin monooxygenases in the absence of CPR. We provide evidence, from either incubations of GA12 (C-20 methyl) with cultures of the mutant suspended in [18O]H2O or maintained under an atmosphere of [18O]O2:N2 (20:80), that GA15 (C-20 alcohol) and GA24 (C-20 aldehyde) are formed directly from dioxygen and not from hydrolysis of covalently enzyme-bound intermediates. Thus these partially oxidized GAs correspond to intermediates of the sequential oxidation of C-20 catalyzed by P450-2.  相似文献   

18.
Yeast flavocytochrome b2 (Fcb2) is an l-lactate:cytochrome c oxidoreductase in the mitochondrial intermembrane space participating in cellular respiration. Each enzyme subunit consists of a cytochrome b5-like heme domain and a flavodehydrogenase (FDH) domain. In the Fcb2 crystal structure, the heme domain is mobile relative to the tetrameric FDH core in one out of two subunits. The monoclonal antibody B2B4, elicited against the holoenzyme, recognizes only the native heme domain in the holoenzyme. When bound, it suppresses the intramolecular electron transfer from flavin to heme b2, hence cytochrome c reduction. We report here the crystal structure of the heme domain in complex with the Fab at 2.7 Å resolution. The Fab epitope on the heme domain includes the two exposed propionate groups of the heme, which are hidden in the interface between the domains in the complete subunit. The structure discloses an unexpected plasticity of Fcb2 in the neighborhood of the heme cavity, in which the heme has rotated. The epitope overlaps with the docking area of the FDH domain onto the heme domain, indicating that the antibody displaces the heme domain in a movement of large amplitude. We suggest that the binding sites on the heme domain of cytochrome c and of the FDH domain also overlap and therefore that cytochrome c binding also requires the heme domain to move away from the FDH domain, so as to allow electron transfer between the two hemes. Based on this hypothesis, we propose a possible model of the Fcb2·cytochrome c complex. Interestingly, this model shares similarity with that of the cytochrome b5·cytochrome c complex, in which cytochrome c binds to the surface around the exposed heme edge of cytochrome b5. The present results therefore support the idea that the heme domain mobility is an inherent component of the Fcb2 functioning.  相似文献   

19.
20.
The activities of electron transport are compared between wild-type Arabidopsis and two Arabidopsis mutants deficient for the chloroplastic NAD(P)H dehydrogenase (NDH) which catalyzes cyclic electron transport around photosystem I. The quantum yield of photosystem II and the degree of non-photochemical quenching of chlorophyll fluorescence were of similar levels in the two NDH-deficient mutants and the wild type under non-stressed standard growth conditions. Stromal over-reduction was induced in Arabidopsis NDH mutants with high light treatment, as is the case in tobacco NDH mutants. However, unlike tobacco mutants, photoinhibition was not observed in the Arabidopsis NDH mutants.  相似文献   

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