Subunit Composition, Structure, and Distribution of Bacterial V-Type ATPases

The overall structure of V-ATPase complexes resembles that of F-type ATPases, but the stalk region is different and more complex. Database searches followed by sequence analysis of the five water-soluble stalk region subunits C–G revealed that (i) to date V-ATPases are found in 16 bacterial species, (ii) bacterial V-ATPases are closer to archaeal A-ATPases than to eukaryotic V-ATPases, and (iii) different groups of bacterial V-ATPases exist. Inconsistencies in the nomenclature of types and subunits are addressed. Attempts to assign subunit positions in V-ATPases based on biochemical experiments, chemical cross-linking, and electron microscopy are discussed. A structural model for prokaryotic and eukaryotic V-ATPases is proposed. The prokaryotic V-ATPase is considered to have a central stalk between headpiece and membrane flanked by two peripheral stalks. The eukaryotic V-ATPases have one additional peripheral stalk.     

过表达ThVHAc1对拟南芥V-ATPase各亚基表达的影响

V-ATPase是多亚基复合蛋白,其c亚基负责V-ATPase的组装及质子通道的形成。本研究拟分析盐胁迫下过表达ThVHAc1基因拟南芥V-ATPase各亚基的表达,探讨过表达外源c亚基对拟南芥V-ATPase全酶响应盐胁迫表达模式的影响。实时荧光定量PCR结果显示,盐胁迫下,过表达外源ThVHAc1拟南芥V-ATPase 28个亚基的表达发生了明显改变,且拟南芥5个c亚基的表达均不同程度的被抑制。表明外源ThVHAc1基因能影响拟南芥V-ATPase各亚基的表达以调节V-ATPase全酶的活性,但各亚基的表达模式与V-ATPase活性非简单对应关系,各亚基互相协调决定V-ATPase活性。     

Initial Steps in the Assembly of the Vacuole-Type H+-ATPase

The plant vacuole is acidified by a complex multimeric enzyme, the vacuole-type H⁺-ATPase (V-ATPase). The initial association of ATPase subunits on membranes was studied using an in vitro assembly assay. The V-ATPase assembled onto microsomes when V-ATPase subunits were supplied. However, when the A or B subunit or the proteolipid were supplied individually, only the proteolipid associated with membranes. By using poly(A⁺) RNA depleted in the B subunit and proteolipid subunit mRNA, we demonstrated A subunit association with membranes at substoichiometric amounts of the B subunit or the 16-kD proteolipid. These data suggest that poly(A⁺) RNA-encoded proteins are required to catalyze the A subunit membrane assembly. Initial events were further studied by in vivo protein labeling. Consistent with a temporal ordering of V-ATPase assembly, membranes contained only the A subunit at early times; at later times both the A and B subunits were found on the membranes. A large-mass ATPase complex was not efficiently formed in the absence of membranes. Together, these data support a model whereby the A subunit is first assembled onto the membrane, followed by the B subunit.     

Primary structure of V-ATPase subunit B from Manduca sexta midgut.

The amino acid sequence of a vacuolar-type ATPase (V-ATPase) subunit B has been deduced from a cDNA clone isolated from a Manduca sexta larval midgut library. The library was screened by hybridization with a labeled cDNA encoding subunit B of Arabidopsis thaliana tonoplast V-ATPase. The M. sexta V-ATPase subunit B consists of 494 amino acids with a calculated M(r) of 54,902. The amino acid sequence deduced for V-ATPase subunit B of M. sexta is between 98% and 76% identical with that of seven other V-ATPase subunits B and greater than 52% identical with three archaebacterial ATPase subunits B.     

Molecular cloning of cDNA encoding the C subunit of H(+)-ATPase from bovine chromaffin granules

A cDNA encoding subunit C of the V-ATPase from bovine chromaffin granules was cloned and sequenced. The gene encodes a hydrophilic protein of 382 amino acids with a calculated molecular weight of 43,989. Hydropathy plots revealed no apparent transmembrane segments and a rather high helix content was detected. A cDNA encoding most of the C subunit of the V-ATPase of human brain was also cloned and sequenced. The deduced amino acid sequence of this gene is almost identical to the bovine polypeptide with only one change of tyrosine 336 that was replaced by histidine in the human gene. Two polypeptide fragments derived from subunit E of V-ATPase from chromaffin granules were sequenced and found to be identical to the predicted amino acid sequence of this subunit from bovine kidney. These observations support the idea that the amino acid sequences of corresponding subunits from different V-ATPases are highly conserved. Unlike the A and B subunits of V-ATPases, that are homologous to the beta and alpha subunits of F-ATPases, subunits C and E showed no homology with analogous subunits of the F-ATPase family. It is proposed that the addition of the C and gamma subunits to the respective V- and F-ATPases during evolution defined them as two separate families of H(+)-ATPases.     

Functional characterization of the N-terminal domain of subunit H (Vma13p) of the yeast vacuolar ATPase

The yeast Saccharomyces cerevisiae vacuolar H(+)-ATPase (V-ATPase) is a multisubunit complex responsible for acidifying intracellular organelles and is highly regulated. One of the regulatory subunits, subunit H, is encoded by the VMA13 gene in yeast and is composed of two domains, the N-terminal domain (amino acids (aa) 1-352) and the C-terminal domain (aa 353-478). The N-terminal domain is required for the activation of the complex, whereas the C-terminal domain is required for coupling ATP hydrolysis to proton translocation (Liu, M., Tarsio, M., Charsky, C. M., and Kane, P. M. (2005) J. Biol. Chem. 280, 36978-36985). Experiments with epitope-tagged copies of Vma13p revealed that there is only one copy of Vma13p/subunit H per V-ATPase complex. Analysis of the N-terminal domain shows that the first 179 amino acids are not required for the activation and full function of the V-ATPase complex and that the minimal region of Vma13p/subunit H capable of activating the V-ATPase is aa 180-353 of the N-terminal domain. Subunit H is expressed as two splice variants in mammals, and deletion of 18 amino acids in yeast Vma13p corresponding to the mammalian subunit H beta isoform results in reduced V-ATPase activity and significantly lower coupling of ATPase hydrolysis to proton translocation. Intriguingly, the yeast Vma13p mimicking the mammalian subunit H beta isoform is functionally equivalent to Vma13p lacking the entire C-terminal domain. These results suggest that the mammalian V-ATPase complexes with subunit H splice variant SFD-alpha or SFD-beta are likely to have different activities and may perform distinct cellular functions.     

Effects of Subchronic Clozapine and Haloperidol on Striatal Glutamatergic Synapses

Abstract: Subchronic treatment with haloperidol increases the number of asymmetric glutamate synapses associated with a perforated postsynaptic density in the striatum. To characterize these synaptic changes further, the effects of subchronic (28 days) administration of an atypical antipsychotic, clozapine (30 mg/kg, s.c.), or a typical antipsychotic, haloperidol (0.5 mg/kg, s.c.), on the binding of [³H]MK-801 to the NMDA receptor-linked ion channel complex and on the in situ hybridization of riboprobes for NMDAR2A and 2B subunits and splice variants of the NMDAR1 subunit were examined in striatal preparations from rats. The density of striatal glutamate immunogold labeling associated with nerve terminals of all asymmetric synapses and the immunoreactivity of those asymmetric synapses associated with a perforated postsynaptic density were also examined by electron microscopy. Subchronic neuroleptic administration had no effect on [³H]MK-801 binding to striatal membrane preparations. Both drugs increased glutamate immunogold labeling in nerve terminals of all asymmetric synapses, but only haloperidol increased the density of glutamate immunoreactivity within nerve terminals of asymmetric synapses containing a perforated postsynaptic density. Whereas subchronic administration of clozapine, but not haloperidol, resulted in a significant increase in the hybridization of a riboprobe that labels all splice variants of the NMDAR1 subunit, both drugs significantly decreased the abundance of NMDAR1 subunit mRNA containing a 63-base insert. Neither drug altered mRNA for the 2A subunit, but clozapine significantly increased hybridization of a probe for the 2B subunit. The data suggest that some neuroleptic effects may be mediated by glutamatergic systems and that typical and atypical antipsychotics can have varying effects on the density of glutamate in presynaptic terminals and on the expression of specific NMDA receptor splice variant mRNAs. Alternatively, NMDAR1 subunit splice variants may differentially respond to interactions with glutamate.     

Phosphorylation of a constitutive serine inhibits BK channel variants containing the alternate exon “SRKR”

BK Ca²⁺-activated K⁺ currents exhibit diverse properties across tissues. The functional variation in voltage- and Ca²⁺-dependent gating underlying this diversity arises from multiple mechanisms, including alternate splicing of Kcnma1, the gene encoding the pore-forming (α) subunit of the BK channel, phosphorylation of α subunits, and inclusion of β subunits in channel complexes. To address the interplay of these mechanisms in the regulation of BK currents, two native splice variants, BK₀ and BK_SRKR, were cloned from a tissue that exhibits dynamic daily expression of BK channel, the central circadian pacemaker in the suprachiasmatic nucleus (SCN) of mouse hypothalamus. The BK₀ and BK_SRKR variants differed by the inclusion of a four–amino acid alternate exon at splice site 1 (SRKR), which showed increased expression during the day. The functional properties of the variants were investigated in HEK293 cells using standard voltage-clamp protocols. Compared with BK₀, BK_SRKR currents had a significantly right-shifted conductance–voltage (G-V) relationship across a range of Ca²⁺ concentrations, slower activation, and faster deactivation. These effects were dependent on the phosphorylation state of S642, a serine residue within the constitutive exon immediately preceding the SRKR insert. Coexpression of the neuronal β4 subunit slowed gating kinetics and shifted the G-V relationship in a Ca²⁺-dependent manner, enhancing the functional differences between the variants. Next, using native action potential (AP) command waveforms recorded from SCN to elicit BK currents, we found that these splice variant differences persist under dynamic activation conditions in physiological ionic concentrations. AP-induced currents from BK_SRKR channels were significantly reduced compared with BK₀, an effect that was maintained with coexpression of the β4 subunit but abolished by the mutation of S642. These results demonstrate a novel mechanism for reducing BK current activation under reconstituted physiological conditions, and further suggest that S642 is selectively phosphorylated in the presence of SRKR.     

Defined sites of interaction between subunits E (Vma4p), C (Vma5p), and G (Vma10p) within the stator structure of the vacuolar H+-ATPase

Vacuolar H(+)-ATPases (V-ATPases) are multi-subunit membrane proteins that couple ATP hydrolysis to the extrusion of protons from the cytoplasm. Although they share a common macromolecular architecture and rotational mechanism with the F(1)F(0)-ATPases, the organization of many of the specialized V-ATPase subunits within this rotary molecular motor remains uncertain. In this study, we have identified sequence segments involved in linking putative stator subunits in the Saccharomyces V-ATPase. Precipitation assays revealed that subunits Vma5p (subunit C) and Vma10p (subunit G), expressed as glutathione-S-transferase fusion proteins in E. coli, are both able to interact strongly with Vma4p (subunit E) expressed in a cell-free system. GST-Vma10p also associated with Vma2p and Vma1p, the core subunits of the ATP-hydrolyzing domain, and was able to self-associate to form a dimer. Mutations within the first 19-residue region of Vma4p, which disrupted interaction with Vma5p in vitro, also prevented the Vma4p polypeptide from restoring V-ATPase function in a complementation assay in vivo. These mutations did not prevent assembly of Vma5p (subunit C) and Vma2p (subunit B) into an inactive complex at the vacuolar membrane, indicating that Vma5p must make multiple interactions involving other V-ATPase subunits. A second, highly conserved region of Vma4p between residues 19 and 38 is involved in binding Vma10p. This region is highly enriched in charged residues, suggesting a role for electrostatic effects in Vma4p-Vma10p interaction. These protein interaction studies show that the N-terminal region of Vma4p is a key factor not only in the stator structure of the V-ATPase rotary molecular motor, but also in mediating interactions with putative regulatory subunits.     

Vacuolar H+-ATPase binding to microfilaments: regulation in response to phosphatidylinositol 3-kinase activity and detailed characterization of the actin-binding site in subunit B

Vacuolar H(+)-ATPase (V-ATPase) binds microfilaments, and that interaction may be mediated by an actin binding domain in subunit B of the enzyme. To test for possible physiologic functions of the actin binding activity of V-ATPase, early responses of resorbing osteoclasts to inhibition of phosphatidylinositol 3-kinase activity by wortmannin and LY294002 were examined. Rapid co-localization between V-ATPase and F-actin was demonstrated by immunocytochemistry, and corresponding association between V-ATPase and F-actin in immunoprecipitations and pelleting assays was detected. This response was reversed as osteoclasts recovered resorptive activity after inhibitors were removed. By expressing and characterizing fusion proteins containing segments of the actin-binding amino-terminal regions of the B subunits of V-ATPase, we mapped the actin-binding site to a 44-amino acid domain. An 11-amino acid segment with a sequence similar to the actin-binding site of human profilin I was detected within this region. 13-Mers containing these profilin-like segments bound actin in fluorescent anisotropy studies and competed with profilin for binding to actin. Using site-directed mutagenesis, the 11-amino acid profilin-like actin-binding motifs (amino acids 49-59 of B1 and 55-65 of B2) were replaced with an 11-amino acid spacer with a sequence based on the homologous sequence from subunit B of Pyrococcus horikoshii, an organism that lacks an actin cytoskeleton. These substitutions eliminated the actin-binding activity of the B subunit fusion proteins. In summary, binding between V-ATPase and F-actin in osteoclasts occurs in response to blocking phosphatidylinositol 3-kinase activity. This response was fully reversible. The actin binding activities of the B subunits of V-ATPase required 11-amino acid actin-binding motifs that are similar in sequence to the actin-binding site of mammalian profilin I.     

Inactive forms of the catalytic subunit of protein kinase A are expressed in the brain of higher primates

It is well documented that the beta-gene of the catalytic (C) subunit of protein kinase A encodes a number of splice variants. These splice variants are equipped with a variable N-terminal end encoded by alternative use of several exons located 5' to exon 2 in the human, bovine and mouse Cbeta gene. In the present study, we demonstrate the expression of six novel human Cbeta mRNAs that lack 99 bp due to loss of exon 4. The novel splice variants, designated CbetaDelta4, were identified in low amounts at the mRNA level in NTera2-N cells. We developed a method to detect CbetaDelta4 mRNAs in various cells and demonstrated that these variants were expressed in human and Rhesus monkey brain. Transient expression and characterization of the CbetaDelta4 variants demonstrated that they are catalytically inactive both in vitro against typical protein kinase A substrates such as kemptide and histone, and in vivo against the cAMP-responsive element binding protein. Furthermore, co-expression of CbetaDelta4 with the regulatory subunit (R) followed by kinase activity assay with increasing concentrations of cAMP and immunoprecipitation with extensive washes with cAMP (1 mm) and immunoblotting demonstrated that the CbetaDelta4 variants associate with both RI and RII in a cAMP-independent fashion. Expression of inactive C subunits which associate irreversibly with R may imply that CbetaDelta4 can modulate local cAMP effects in the brain by permanent association with R subunits even at saturating concentrations of cAMP.     

Analysis of Maxi-K alpha subunit splice variants in human myometrium

Background  

Large-conductance, calcium-activated potassium (Maxi-K) channels are implicated in the modulation of human uterine contractions and myometrial Ca^{2 +} homeostasis. However, the regulatory mechanism(s) governing the expression of Maxi-K channels with decreased calcium sensitivity at parturition are unclear. The objectives of this study were to investigate mRNA expression of the Maxi-K alpha subunit, and that of its splice variants, in human non-pregnant and pregnant myometrium, prior to and after labour onset, to determine whether altered expression of these splice variants is associated with decreased calcium sensitivity observed at labour onset.     

Structure and Function of the Vacuolar H+-ATPase: Moving from Low-Resolution Models to High-Resolution Structures

In the absence of a high-resolution structure for the vacuolar H⁺-ATPase, a number of approaches can yield valuable information about structure/function relationships in the enzyme. Electron microscopy can provide not only a representation of the overall architecture of the complex, but also a low-resolution map onto which structures solved for individually expressed subunits can be fitted. Here we review the possibilities for electron microscopy of the Saccharomyces V-ATPase and examine the suitability of V-ATPase subunits for expression in high yield prokaryotic systems, a key step towards high-resolution structural studies. We also review the role of experimentally-derived structural models in understanding structure/function relationships in the V-ATPase, with particular reference to the complex of proton-translocating 16 kDa proteolipids in the membrane domain of the V-ATPase. This model in turn makes testable predictions about the sites of binding of bafilomycins and the functional interactions between the proteolipid and the single-copy membrane subunit Vph1p, with implications for the constitution of the proton translocation pathway.