Functional analysis of Arabidopsis thaliana isoforms of the Mg-chelatase CHLI subunit

The first step of chlorophyll biosynthesis is catalyzed by a Mg-chelatase composed of the subunits CHLI, CHLD and CHLH. Mg-chelatase requires ATP hydrolysis that can be attributed to CHLI. Arabidopsis has two CHLI isoforms, CHLI1 and CHLI2, that have similar expression profiles, but it has been suggested that CHLI2 has limited function in the Mg-chelatase complex. Recently, we showed that Arabidopsis CHLI1 is an ATPase and a target of chloroplast thioredoxin. Here, we demonstrate that CHLI2 also has ATPase activity but with a lower Vmax and higher Km ATP than CHLI1. We confirmed the thioredoxin-dependent reduction of a disulfide bond in CHLI2 and thiol-modulation of its ATPase activity. We then examined the physiological contribution of CHLI2 using a chli2 T-DNA knockout line. Although visible phenotype of homozygous chli2 mutants was almost comparable to wild type, the mutant accumulated significantly less chlorophyll. Furthermore, cs/cs; chli2/chli2 double mutants were almost albino. There were three phenotypes among progenies segregated from the cs/cs; CHLI2/chli2 parent: cs-like pale green, yellow, and almost albino were obtained in the approximate ratio of 1:2:0.7. PCR analysis confirmed that the chli2 mutation is semidominant on a homozygous cs background. These results reveal that although CHLI2 plays a limited role in chlorophyll biosynthesis, this subunit certainly contributes to the assembly of the Mg-chelatase complex.     

Distinct expression patterns of different subunit isoforms of the V-ATPase in the rat epididymis

In the epididymis and vas deferens, the vacuolar H(+)ATPase (V-ATPase), located in the apical pole of narrow and clear cells, is required to establish an acidic luminal pH. Low pH is important for the maturation of sperm and their storage in a quiescent state. The V-ATPase also participates in the acidification of intracellular organelles. The V-ATPase contains many subunits, and several of these subunits have multiple isoforms. So far, only subunits ATP6V1B1, ATP6V1B2, and ATP6V1E2, previously identified as B1, B2, and E subunits, have been described in the rat epididymis. Here, we report the localization of V-ATPase subunit isoforms ATP6V1A, ATP6V1C1, ATP6V1C2, ATP6V1G1, ATP6V1G3, ATP6V0A1, ATP6V0A2, ATP6V0A4, ATP6V0D1, and ATP6V0D2, previously labeled A, C1, C2, G1, G3, a1, a2, a4, d1, and d2, in epithelial cells of the rat epididymis and vas deferens. Narrow and clear cells showed a strong apical staining for all subunits, except the ATP6V0A2 isoform. Subunits ATP6V0A2 and ATP6V1A were detected in intracellular structures closely associated but not identical to the TGN of principal cells and narrow/clear cells, and subunit ATP6V0D1 was strongly expressed in the apical membrane of principal cells in the apparent absence of other V-ATPase subunits. In conclusion, more than one isoform of subunits ATP6V1C, ATP6V1G, ATP6V0A, and ATP6V0D of the V-ATPase are present in the epididymal and vas deferens epithelium. Our results confirm that narrow and clear cells are well fit for active proton secretion. In addition, the diverse functions of the V-ATPase may be established through the utilization of specific subunit isoforms. In principal cells, the ATP6V0D1 isoform may have a physiological function that is distinct from its role in proton transport via the V-ATPase complex.     

Mutational analysis of the stator subunit E of the yeast V-ATPase

Subunit E is a component of the peripheral stalk(s) that couples membrane and peripheral subunits of the V-ATPase complex. In order to elucidate the function of subunit E, site-directed mutations were performed at the amino terminus and carboxyl terminus. Except for S78A and D233A/T202A, which exhibited V(1)V(o) assembly defects, the function of subunit E was resistant to mutations. Most mutations complemented the growth phenotype of vma4Delta mutants, including T6A and D233A, which only had 25% of the wild-type ATPase activity. Residues Ser-78 and Thr-202 were essential for V(1)V(o) assembly and function. The mutation S78A destabilized subunit E and prevented assembly of V(1) subunits at the membranes. Mutant T202A membranes exhibited 2-fold increased V(max) and about 2-fold less of V(1)V(o) assembly; the mutation increased the specific activity of V(1)V(o) by enhancing the k(cat) of the enzyme 4-fold. Reduced levels of V(1)V(o) and V(o) complexes at T202A membranes suggest that the balance between V(1)V(o) and V(o) was not perturbed; instead, cells adjusted the amount of assembled V-ATPase complexes in order to compensate for the enhanced activity. These results indicated communication between subunit E and the catalytic sites at the A(3)B(3) hexamer and suggest potential regulatory roles for the carboxyl end of subunit E. At the carboxyl end, alanine substitution of Asp-233 significantly reduced ATP hydrolysis, although the truncation 229-233Delta and the point mutation K230A did not affect assembly and activity. The implication of these results for the topology and functions of subunit E within the V-ATPase complex are discussed.     

Mutational analysis of the non-homologous region of subunit A of the yeast V-ATPase

Subunit A is the catalytic nucleotide binding subunit of the vacuolar proton-translocating ATPase (or V-ATPase) and is homologous to subunit beta of the F(1)F(0) ATP synthase (or F-ATPase). Amino acid sequence alignment of these subunits reveals a 90-amino acid insert in subunit A (termed the non-homologous region) that is absent from subunit beta. To investigate the functional role of this region, site-directed mutagenesis has been performed on the VMA1 gene that encodes subunit A in yeast. Substitutions were performed on 13 amino acid residues within this region that are conserved in all available A subunit sequences. Most of the 18 mutations introduced showed normal assembly of the V-ATPase. Of these, one (R219K) greatly reduced both proton transport and ATPase activity. By contrast, the P217V mutant showed significantly reduced ATPase activity but higher than normal levels of proton transport, suggesting an increase in coupling efficiency. Two other mutations in the same region (P223V and P233V) showed decreased coupling efficiency, suggesting that changes in the non-homologous region can alter coupling of proton transport and ATP hydrolysis. It was previously shown that the V-ATPase must possess at least 5-10% activity relative to wild type to undergo in vivo dissociation in response to glucose withdrawal. However, four of the mutations studied (G150A, D157E, P177V, and P223V) were partially or completely blocked in dissociation despite having greater than 30% of wild type levels of activity. These results suggest that changes in the non-homologous region can also alter in vivo dissociation of the V-ATPase independent of effects on activity.     

Functional analyses of differentially expressed isoforms of the Arabidopsis inositol phosphorylceramide synthase

Sphingolipids are key components of eukaryotic plasma membranes that are involved in many functions, including the formation signal transduction complexes. In addition, these lipid species and their catabolites function as secondary signalling molecules in, amongst other processes, apoptosis. The biosynthetic pathway for the formation of sphingolipid is largely conserved. However, unlike mammalian cells, fungi, protozoa and plants synthesize inositol phosphorylceramide (IPC) as their primary phosphosphingolipid. This key step involves the transfer of the phosphorylinositol group from phosphatidylinositol (PI) to phytoceramide, a process catalysed by IPC synthase in plants and fungi. This enzyme activity is at least partly encoded by the AUR1 gene in the fungi, and recently the distantly related functional orthologue of this gene has been identified in the model plant Arabidopsis. Here we functionally analysed all three predicted Arabidopsis IPC synthases, confirming them as aureobasidin A resistant AUR1p orthologues. Expression profiling revealed that the genes encoding these orthologues are differentially expressed in various tissue types isolated from Arabidopsis.     

Immunochemical analysis of aquaporin isoforms in Arabidopsis suspension-cultured cells

Aquaporins mediate the movement of water across biomembranes. Arabidopsis thaliana contains 35 aquaporins that belong to four subfamilies (PIP, TIP, SIP, and NIP). We investigated their expression profiles immunochemically in suspension-cultured Arabidopsis thaliana cells during growth and in response to salt and osmotic stresses. Protein amounts of all aquaporins were much lower in cultured cells than in the plant tissues. This is consistent with the low water permeability of protoplasts from cultured cells. After treatment with NaCl, the protein amounts of PIP2;1, PIP2;2, and PIP2;3 in the cells increased several-fold, and those of TIP1;1 and TIP1;2, 15- and 3-fold respectively. PIP1 did not change under the stress. Cell death began after 19 d in culture, accompanied by marked accumulation of PIPs and TIPs and a gradual decrease in SIPs. Our results suggest the followings: (i) Accumulation of aquaporin isoforms was individually regulated at low levels in single cells. (ii) At least PIP2;2, PIP2;3, TIP1;1, and TIP1;2 are stress-responsive aquaporins in suspension cells. (iii) A sudden increment of several members of PIP2 and TIP1 subfamilies might be related to cell death.     

Disparate roles for the regulatory A subunit isoforms in Arabidopsis protein phosphatase 2A

The heterotrimeric protein phosphatase 2A (PP2A) complex comprises a catalytic subunit and regulatory A and B subunits that modulate enzyme activity and mediate interactions with other proteins. We report here the results of a systematic analysis of the Arabidopsis (Arabidopsis thaliana) regulatory A subunit gene family, which includes the ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1), PP2AA2, and PP2AA3 genes. All three A subunit isoforms accumulate in the organs of seedlings and adult plants, suggesting extensive overlap in expression domains. We have isolated pp2aa2 and pp2aa3 mutants and found that their phenotypes are largely normal and do not resemble that of rcn1. Whereas rcn1 pp2aa2 and rcn1 pp2aa3 double mutants exhibit striking abnormalities in all stages of development, the pp2aa2 pp2aa3 double mutant shows only modest defects. Together, these data suggest that RCN1 performs a cardinal role in regulation of phosphatase activity and that PP2AA2 and PP2AA3 functions are unmasked only when RCN1 is absent.     

Dimer formation of subunit G of the yeast V-ATPase

The G subunit of the vacuolar ATPase (V-ATPase) is a component of the stalk connecting the V(1) and V(O) sectors of the enzyme and is essential for normal assembly and function. Subunit G (Vma10p) of the yeast V-ATPase was expressed in Escherichia coli as a soluble protein and was purified to homogeneity. The molecular mass of subunit G, determined by Native-polyacrylamide gel electrophoresis, gel filtration analysis and small-angle X-ray scattering, was approximately 28+/-2 kDa, indicating that this protein is dimeric. With a radius of gyration (R(g)) and a maximum size (D(max)) of 2.7+/-0.2 nm and 8.0+/-0.3 nm, respectively, the G-dimer is rather elongated. To understand which region of subunit G is required to mediate dimerization, a G(38-144) form (the carboxyl-terminus) was expressed and purified. G(38-144) is homogeneous, with a molecular mass of approximately 12+/-3 kDa, indicating a monomeric form in solution.     

The reconstructed ancestral subunit a functions as both V-ATPase isoforms Vph1p and Stv1p in Saccharomyces cerevisiae

The vacuolar-type, proton-translocating ATPase (V-ATPase) is a multisubunit enzyme responsible for organelle acidification in eukaryotic cells. Many organisms have evolved V-ATPase subunit isoforms that allow for increased specialization of this critical enzyme. Differential targeting of the V-ATPase to specific subcellular organelles occurs in eukaryotes from humans to budding yeast. In Saccharomyces cerevisiae, the two subunit a isoforms are the only difference between the two V-ATPase populations. Incorporation of Vph1p or Stv1p into the V-ATPase dictates the localization of the V-ATPase to the vacuole or late Golgi/endosome, respectively. A duplication event within fungi gave rise to two subunit a genes. We used ancestral gene reconstruction to generate the most recent common ancestor of Vph1p and Stv1p (Anc.a) and tested its function in yeast. Anc.a localized to both the Golgi/endosomal network and vacuolar membrane and acidified these compartments as part of a hybrid V-ATPase complex. Trafficking of Anc.a did not require retrograde transport from the late endosome to the Golgi that has evolved for retrieval of the Stv1p isoform. Rather, Anc.a localized to both structures through slowed anterograde transport en route to the vacuole. Our results suggest an evolutionary model that describes the differential localization of the two yeast V-ATPase isoforms.     

Structural and functional features of yeast V-ATPase subunit C

V-ATPase is a multi-subunit membrane protein complex, it translocates protons across biological membranes, generating electrical and pH gradients which are used for varieties of cellular processes. V-ATPase is composed of two distinct sub-complexes: a membrane bound V0 sub-complex, composed of 6 different subunits, which is responsible for proton transport and a soluble cytosolic facing V1 sub-complex, composed of 8 different subunits which hydrolyse ATP. The two sub-complexes are held together via a flexible stator. One of the main features of eukaryotic V-ATPase is its ability to reversibly dissociate to its sub-complexes in response to changing cellular conditions, which arrest both proton translocation and ATP hydrolysis, suggesting a regulation function. Subunit C (vma5p in yeast) was shown by several biochemical, genetic and recent structural data to function as a flexible stator holding the two sectors of the complex together and regulating the reversible association/dissociation of the complex, partly via association with F-actin filaments. Structural features of subunit C that allow smooth energy conversion and interaction with actin and nucleotides are discussed.     

Functional analysis of isoforms of NADPH: protochlorophyllide oxidoreductase (POR), PORB and PORC, in Arabidopsis thaliana

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide. To elucidate the physiological function of three differentially regulated POR isoforms (PORA, PORB and PORC) in Arabidopsis thaliana, we isolated T-DNA tagged null mutants of porB and porC. The mature seedlings of the mutants had normal photosynthetic competencies, showing that PORB and PORC are interchangeable and functionally redundant in developed plants. In etiolated seedlings, only porB showed a reduction in the photoactive protochlorophyllide and the size of prolamellar bodies (PLBs), indicating that PORB, as well as PORA, functioned in PLB assembly and photoactive protochlorophyllide formation in etiolated seedlings. When illuminated, the etiolated porB seedling was able to green to a similar extent as the wild type, whereas the greening was significantly reduced under low light conditions. During greening, high light irradiation increased the level of PORC protein, and the greening of porC was repressed under high light conditions. The porB, but not porC, etiolated seedling was more sensitive to the far-red block of greening than the wild type, which is caused by depletion of endogenous POR proteins resulting in photo-oxidative damage. These results suggest that, at the onset of greening, PLBs are important for efficient capture of light energy for photoconversion under various light conditions, and PORC, which is induced by high light irradiation, contributes to photoprotection during greening of the etiolated seedlings.     

Functional analysis of two isoforms of leaf-type ferredoxin-NADP(+)-oxidoreductase in rice using the heterologous expression system of Arabidopsis

Ferredoxin-NADP(+)-oxidoreductase (FNR) mediates electron transfer between ferredoxin (Fd) and NADP(+); therefore, it is a key enzyme that provides the reducing power used in the Calvin cycle. Other than FNR, nitrite reductase, sulfite reductase, glutamate synthase, and Fd-thioredoxin reductase also accept electrons from Fd, an electron carrier protein in the stroma. Therefore, the regulation of electron partitioning in the chloroplast is important for photosynthesis and other metabolic pathways. The regulatory mechanism of electron partitioning, however, remains to be elucidated. We found, by taking advantage of a gain-of-function approach, that expression of two rice (Oryza sativa) full-length cDNAs of leaf-type FNRs (OsLFNR1 and OsLFNR2) led to altered chlorophyll fluorescence and growth in Arabidopsis (Arabidopsis thaliana) and rice. We revealed that overexpression of the OsLFNR1 and OsLFNR2 full-length cDNAs resulted in distinct phenotypes despite the high sequence similarity between them. Expression of OsLFNR1 affected the nitrogen assimilation pathway without inhibition of photosynthesis under normal conditions. On the other hand, OsLFNR2 expression led to the impairment of photosynthetic linear electron transport as well as Fd-dependent cyclic electron flow around photosystem I. The endogenous protein level of OsLFNR was found to be suppressed in both OsLFNR1- and OsLFNR2-overexpressing rice plants, leading to changes in the stoichiometry of the two LFNR isoforms within the thylakoid and soluble fractions. Thus, we propose that the stoichiometry of two LFNR isoforms plays an important role in electron partitioning between carbon fixation and nitrogen assimilation.     

Structural analysis of the stalk subunit Vma5p of the yeast V-ATPase in solution

Vma5p (subunit C) of the yeast V-ATPase was produced in Escherichia coli and purified to homogeneity. Analysis of secondary structure by circular dichroism spectroscopy showed that Vma5p comprises 64% -helix and 17% β-sheet content. The molecular mass of this subunit, determined by gel filtration analysis and small angle X-ray scattering (SAXS), was approximately 51 ± 4 kDa, indicating a high hydration level of the protein in solution. The radius of gyration and the maximum size of Vma5p were determined to be 3.74 ± 0.03 and 12.5 ± 0.1 nm, respectively. Using two independent ab initio approaches, the first low-resolution shape of the protein was determined. Vma5p is an elongated boot-shaped particle consisting of two distinct domains. Co-reconstitution of Vma5p to V₁ without C from Manduca sexta resulted in a V₁–Vma5p hybrid complex and a 20% increase in ATPase hydrolysis activity.     

A WNK kinase binds and phosphorylates V-ATPase subunit C

WNK (with no lysine (K)) protein kinases are found in many eukaryotes and share a unique active site. Here, we report that a member of the Arabidopsis WNK family (AtWNK8) interacts with subunit C of the vacuolar H+-ATPase (V-ATPase) via a short C-terminal domain. AtWNK8 is shown to autophosphorylate intermolecularly and to phosphorylate Arabidopsis subunit C (AtVHA-C) at multiple sites as determined by MALDI-TOF MS analysis. Furthermore, we show that AtVHA-C and other V-ATPase subunits are phosphorylated when V1-complexes are used as substrates for AtWNK8. Taken together, our results provide evidence that V-ATPases are potential targets of WNK kinases and their associated signaling pathways.