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Influenza A viruses (IAV) can cause lung injury and acute respiratory distress syndrome (ARDS), which is characterized by accumulation of excessive fluid (edema) in the alveolar airspaces and leads to hypoxemia and death if not corrected. Clearance of excess edema fluid is driven mostly by the alveolar epithelial Na,K-ATPase and is crucial for survival of patients with ARDS. We therefore investigated whether IAV infection alters Na,K-ATPase expression and function in alveolar epithelial cells (AECs) and the ability of the lung to clear edema. IAV infection reduced Na,K-ATPase in the plasma membrane of human and murine AECs and in distal lung epithelium of infected mice. Moreover, induced Na,K-ATPase improved alveolar fluid clearance (AFC) in IAV-infected mice. We identified a paracrine cell communication network between infected and noninfected AECs and alveolar macrophages that leads to decreased alveolar epithelial Na,K-ATPase function and plasma membrane abundance and inhibition of AFC. We determined that the IAV-induced reduction of Na,K-ATPase is mediated by a host signaling pathway that involves epithelial type I IFN and an IFN-dependent elevation of macrophage TNF-related apoptosis-inducing ligand (TRAIL). Our data reveal that interruption of this cellular crosstalk improves edema resolution, which is of biologic and clinical importance to patients with IAV-induced lung injury., Introduction Influenza A viruses (IAV) infect cells in the alveolus and induce primary viral pneumonia, which can progress to acute respiratory distress syndrome (ARDS) with high mortality (1). IAV-induced lung [...]

The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic [Ca.sup.2+], initiated by the release of [Ca.sup.2+] from the sarcoplasmic reticulum and terminated by re-sequestration of [Ca.sup.2+] into the sarcoplasmic reticulum as the main mechanism of [Ca.sup.2+] removal. Re-sequestration requires active transport and is catalysed by the sarcoplasmic reticulum [Ca.sup.2+]-ATPase (SERCA), which has a key role in defining the contractile properties of skeletal and heart muscle tissue. The activity of SERCA is regulated by two small, homologous membrane proteins called phospholamban (PLB, also known as PLN) and sarcolipin (SLN) (1, 2). Detailed structural information explaining this regulatory mechanism has been lacking, and the structural features defining the pathway through which cytoplasmic [Ca.sup.2+] enters the intramembranous binding sites of SERCA have remained unknown. Here we report the crystal structure of rabbit SERCA1a (also known as ATP2A1) in complex with SLN at 3.1 Å resolution. The regulatory SLN traps the [Ca.sup.2+]-ATPase in a previously undescribed E1 state, with exposure of the [Ca.sup.2+] sites through an open cytoplasmic pathway stabilized by [Mg.sup.2+]. The structure suggests a mechanism for selective [Ca.sup.2+] loading and activation of SERCA, and provides new insight into how SLN and PLB inhibition arises from stabilization of this E1 intermediate state without bound [Ca.sup.2+]. These findings may prove useful in studying how autoinhibitory domains of other ion pumps modulate transport across biological membranes., The regulation of SERCA by SLN and PLB is a key determinant of the contractile properties of both skeletal and heart muscle tissue. In humans, SLN is primarily expressed in [...]

P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes, and are distinct from other ATPases in that the reaction cycle includes an autophosphorylation step. The best studied is [Ca.sup.2+]-ATPase from muscle sarcoplasmic reticulum (SERCA1a), a [Ca.sup.2+] pump that relaxes muscle cells after contraction, and crystal structures have been determined for most of the reaction intermediates (1,2). An important outstanding structure is that of the E1 intermediate, which has empty high-affinity [Ca.sup.2+]-binding sites ready to accept new cytosolic [Ca.sup.2+]. In the absence of [Ca.sup.2+] and at pH 7 or higher, the ATPase is predominantly in E1, not in E2 (low affinity for [Ca.sup.2+]) (3), and if millimolar [Mg.sup.2+] is present, one [Mg.sup.2+] is expected to occupy one of the [Ca.sup.2+]-binding sites with a millimolar dissociation constant (4,5). This [Mg.sup.2+] accelerates the reaction cycle (4), not permitting phosphorylation without [Ca.sup.2+] binding. Here we describe the crystal structure of native SERCA1a (from rabbit) in this E1 x [Mg.sup.2+] state at 3.0 [Anstrom] resolution in addition to crystal structures of SERCA1a in E2 free from exogenous inhibitors, and address the structural basis of the activation signal for phosphoryl transfer. Unexpectedly, sarcolipin (6), a small regulatory membrane protein of [Ca.sup.2+]-ATPase (7), is bound, stabilizing the E1 x [Mg.sup.2+] state. Sarcolipin is a close homologue of phospholamban, which is a critical mediator of β-adrenergic signal in [Ca.sup.2+] regulation in heart (for reviews, see, for example, refs 8-10), and seems to play an important role in muscle-based thermogenesis (11). We also determined the crystal structure of recombinant SERCA1a devoid of sarcolipin, and describe the structural basis of inhibition by sarcolipin/phospholamban. Thus, the crystal structures reported here fill a gap in the structural elucidation of the reaction cycle and provide a solid basis for understanding the physiological regulation of the calcium pump., New E2 (Supplementary Fig. 1) crystals of SERCAla free from exogenous inhibitors were grown in either 40 mM MgS[O.sub.4] (E2 x S[O.sub.4.sup.2-] crystals) or Mg[Cl.sub.2] (E2 crystals) at pH 6.5 [...]

Damage to mitochondria can lead to the depolarization of the inner mitochondrial membrane, thereby sensitizing impaired mitochondria for selective elimination by autophagy. However, fusion of uncoupled mitochondria with polarized mitochondria can compensate for damage, reverse membrane depolarization, and obviate mitophagy. Parkin, an E3 ubiquitin ligase that is mutated in monogenic forms of Parkinson's disease, was recently found to induce selective autophagy of damaged mitochondria. Here we show that ubiquitination of mitofusins Mfn1 and Mfn2, large GTPases that mediate mitochondrial fusion, is induced by Parkin upon membrane depolarization and leads to their degradation in a proteasome- and p97-dependent manner, p97, a AAA+ ATPase, accumulates on mitochondria upon uncoupling of Parkin-expressing cells, and both p97 and proteasome activity are required for Parkin-mediated mitophagy. After mitochondrial fission upon depolarization, Parkin prevents or delays refusion of mitochondria, likely by the elimination of mitofusins. Inhibition of Drp1mediated mitochondrial fission, the proteasome, or p97 prevents Parkin-induced mitophagy. doi/ 10.1083/jcb.201007013

Acid-base transport in the renal collecting tubule is mediated by two canonical cell types: the [beta]-intercalated cell secretes HC[O.sub.3] by an apical Cl:HC[O.sub.3] named pendrin and a basolateral vacuolar (V)-ATPase. Acid secretion is mediated by the [alpha]-intercalated cell, which has an apical V-ATPase and a basolateral Cl:HC[O.sub.3] exchanger (kAE1). We previously suggested that the [beta]-cell converts to the [alpha]-cell in response to acid feeding, a process that depended on the secretion and deposition of an extracellular matrix protein termed hensin (DMBT1). Here, we show that deletion of hensin from intercalated cells results in the absence of typical [alpha]-intercalated cells and the consequent development of complete distal renal tubular acidosis (dRTA). Essentially all of the intercalated cells in the cortex of the mutant mice are canonical [beta]-type cells, with apical pendrin and basolateral or diffuse/bipolar V-ATPase. In the medulla, however, a previously undescribed cell type has been uncovered, which resembles the cortical [beta]-intercalated cell in ultrastructure, but does not express pendrin. Polymerization and deposition of hensin (in response to acidosis) requires the activation of [beta]1 integrin, and deletion of this gene from the intercalated cell caused a phenotype that was identical to the deletion of hensin itself, supporting its critical role in hensin function. Because previous studies suggested that the conversion of [beta]- to [alpha]-intercalated cells is a manifestation of terminal differentiation, the present results demonstrate that this differentiation proceeds from HC[O.sub.3] secreting to acid secreting phenotypes, a process that requires deposition of hensin in the ECM. doi: 10.1073/pnas.1010364107

Some microbes, among them a few species of cyanobacteria, are able to excavate carbonate minerals, from limestone to biogenic carbonates, including coral reefs, in a bioerosive activity that directly links biological and geological parts of the global carbon cycle. The physiological mechanisms that enable such endolithic cyanobacteria to bore, however, remain unknown. In fact, their boring constitutes a geochemical paradox, in that photoautotrophic metabolism will tend to precipitate carbonates, not dissolve them. We developed a stable microbe/mineral boring system based on a cyanobacterial isolate, strain BC008, with which to study the process of microbial excavation directly in the laboratory. Measurements of boring into calcite under different light regimes, and an analysis of photopigment content and photosynthetic rates along boring filaments, helped us reject mechanisms based on the spatial or temporal separation of alkali versus Acid-generating metabolism (i.e., photosynthesis and respiration). Instead, extracellular [Ca.sup.2+] imaging of boring cultures in vivo showed that BC008 was able to take up [Ca.sup.2+] at the excavation front, decreasing the local extracellular ion activity product of calcium carbonate enough to promote spontaneous dissolution there. Intracellular [Ca.sup.2+] was then transported away along the multicellular cyanobacterial trichomes and excreted at the distal borehole opening into the external medium. Inhibition assays and gene expression analyses indicate that the uptake and transport was driven by P-type [Ca.sup.2+]-ATPases. We believe such a chemically simple and biologically sophisticated mechanism for boring to be unparalleled among bacteria. bioerosion | calcium metabolism | endoliths | microbialites doi: 10.1073/pnas.1011884108

Phospholemman (PLM) is a major target for phosphorylation mediated by both PKA (at Ser68) and PKC (at both Ser63 and Ser68) in the heart. In intact cardiac myocytes, PLM associates with and inhibits [Na.sup.+]-[K.sup.+]-ATPase (NKA), mainly by reducing its affinity for internal [Na.sup.+]. The inhibition is relieved upon PLM phosphorylation by PKA or PKC. The aim here was to distinguish the role of the Ser63 and Set68 PLM phosphorylation sites in mediating kinase-induced modulation of NKA function. We expressed wild-type (WT) PLM and S63A, S68A, and AA (Ser63 and Ser68 to alanine double mutant) PLM mutants in HeLa cells that stably express rat NKA-[[alpha].sub.1] and we measured the effect of PKA and PKC activation on NKA-mediated intracellular [Na.sup.+] concentration decline. PLM expression (WT or mutant) significantly decreased the apparent NKA affinity for internal [Na.sup.+] and had no significant effect on the maximum pump rate ([V.sub.max]). PKA activation with forskolin (20 [micro]M) restored NKA [Na.sup.+] affinity in cells expressing WT but not AA PLM and did not affect [V.sub.max] in either case. Similarly, PKC activation with 300 nM phorbol 12,13-dibutyrate increased NKA [Na.sup.+] affinity in cells expressing WT, S63A, and S68A PLM and had no effect in cells expressing AA PLM. Neither forskolin nor phorbol 12,13-dibutyrate affected NKA function in the absence of PLM. We conclude that PLM phosphorylation at either Set63 or Ser68 is both necessary and sufficient for completely relieving the PLM-induced NKA inhibition. FXYD; apparent Na affinity; PKA; PKC doi: 10.1152/ajpcell.00027.2010.

FtsK is a homohexameric, RecA-like dsDNA translocase that plays a key role in bacterial chromosome segregation. The FtsK regulatory [gamma]-subdomain determines directionality of translocation through its interaction with specific 8 base pair chromosomal sequences [(KOPS); FtsK Orienting / Polarizing Sequence(s)] that are cooriented with the direction of replication in the chromosome. We use millisecond-resolution ensemble translocation and ATPase assays to analyze the assembly, initiation, and translocation of FtsK. We show that KOPS are used to initiate new translocation events rather than reorient existing ones. By determining kinetic parameters, we show sigmoidal dependences of translocation and ATPase rates on ATP concentration that indicate sequential cooperative coupling of ATP hydrolysis to DNA motion. We also estimate the ATP coupling efficiency of translocation to be 1.632.11 bp of dsDNA translocated/ATP hydrolyzed. The data were used to derive a model for the assembly, initiation, and translocation of FtsK hexamers. ASCE ATPase | DNA translocation | FtsK translocase | stopped-flow | triplex displacement doi/ 10.1073/pnas.1007518107

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics. In this study, we investigated the role of polar residues within or near the predicted transmembrane [alpha]-helices 1 and 6 of BCRP in drug transport. We substituted [Asn.sup.387], [Gln.sup.398], [Asn.sup.629], and [Thr.sup.642] with Ala, [Thr.sup.402] with Ala and Arg, and [Tyr.sup.645] with Phe, and the mutants were stably expressed in human embryonic kidney-293 or Flp-In-293 cells. Immunoblotting and confocal microscopy analysis revealed that all of the mutants were well expressed and predominantly targeted to the plasma membrane. While T402A and T402R showed a significant global reduction in the efflux of mitoxantrone, Hoechst 33342, and BODIPY-prazosin, N629A exhibited significantly increased efflux activities for all of the substrates. N387A and Q398A displayed significantly impaired efflux for mitoxantrone and Hoechst 33342, but not for BODIPY-prazosin. In contrast, T642A and Y645F showed a moderate reduction in Hoechst 33342 efflux only. Drug resistance profiles of human embryonic kidney-293 cells expressing the mutants generally correlated with the efflux data. Furthermore, N629A was associated with a marked increase, and N387A and T402A with a significant reduction, in BCRP ATPase activity. Mutations of some of the polar residues may cause conformational changes, as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin. The inward-facing homology model of BCRP indicated that [Thr.sup.402] within transmembrane 1 may be important for helical interactions, and [Asn.sup.629] may be involved in BCRP-substrate interaction. In conclusion, we have demonstrated the functional importance of some of these polar residues in BCRP activity. adenosine 5'-triphosphate-binding cassette efflux transporter; multi-drug resistance; site-directed mutagenesis; structure-function of adenosine 5'-triphosphate-binding cassette transporter doi: 10.1152/ajpcell.00160.2010.

The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the [gamma]-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase, p400 is recruited to DSBs by a mechanism that is independent of ATM but requires mdcl. Further, the destabilization of nucleosomes by p400 is required for the RNF8-dependent ubiquitination of chromatin, and for the subsequent recruitment of brca1 and 53BP1 to DSBs. These results identify p400 asa novel DNA damage response protein and demonstrate that p400-mediated alterations in nucleosome and chromatin structure promote both chromatin ubiquitination and the accumulation of brca1 and 53BP1 at sites of DNA damage. doi/ 10.1083/jcb.201001160

Bacillus subtilis is able to utilize arabinopolysaccharides derived from plant biomass. Here, by combining genetic and physiological analyses we characterize the AraNPQ importer and identify primary and secondary transporters of B. subtilis involved in the uptake of arabinosaccharides. We show that the ABC-type importer AraNPQ is involved in the uptake of [alpha]-l,5-arabinooligosaccharides, at least up to four L-arabinosyl units. Although this system is the key transporter for [alpha]-l,5-arabinotriose and [alpha]-l,5-arabinotetraose, the results indicate that [alpha]-l,5-arabinobiose also is translocated by the secondary transporter AraE. This broad-specificity proton symporter is the major transporter for arabinose and also is accountable for the uptake of xylose and galactose. In addition, MsmX is shown to be the ATPase that energizes the incomplete AraNPQ importer. Furthermore, the results suggest the existence of at least one more unidentified MsmX-dependent ABC importer responsible for the uptake of nonlinear [alpha]-l,2- and [alpha]-l,3-arabinooligosaccharides. This study assigns MsmX as a multipurpose B. subtilis ATPase required to energize different saccharide transporters, the arabinooligosaccharide-specific AraNPQ-MsmX system, a putative MsmX-dependent ABC transporter specific for nonlinear arabinooligosaccharides, and the previously characterized maltodextrin-specific MdxEFG-MsmX system. doi: 10.1128/JB.00832-10

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity. doi: 10.1128/JB.00557-10

Intestinal HC[O.sup.-.sub.3] secretion and NaCl absorption are essential for counteracting dehydration in marine teleost fsh. We investigated how these two processes are coordinated in toadfish. HC[O.sup.-.sub.3] stimulated a luminal positive short-circuit current ([I.sub.sc]) in intestine mounted in Ussing chamber, bathed with the same saline solution on the external and internal sides of the epithelium. The [I.sub.sc] increased proportionally to the HC[O.sup.-.sub.3] in the bath up to 80 mM NaHC[O.sub.3], and it did not occur when NaHC[O.sub.3] was replaced with [Na.sup.+]-gluconate or with NaHC[O.sub.3] in [Cl.sup.-]-free saline. HC[O.sup.-.sub.3] (20 mM) induced a ~2.5-fold stimulation of [I.sub.sc], and this [HC[O.sup.-.sub.3]] was used in all subsequent experiments. The HC[O.sup.-.sub.3]-stimulated [I.sub.sc] was prevented or abolished by apical application of 10 [micro]M bumetanide (a specific inhibitor of NKCC) and by 30 [micro]M 4-catechol estrogen ICE; an inhibitor of soluble adenylyl cyclase (sAC)]. The inhibitory effects of bumetanide and CE were not additive. The HC[O.sup.-.sub.3]-stimulated [I.sub.sc] was prevented by apical bafilomycin (1 [micro]M) and etoxolamide (1 mM), indicating involvement of V-[H.sup.+]-ATPase and carbonic anhydrases, respectively. Immunohistochemistry and Western blot analysis confirmed the presence of an NKCC2-like protein in the apical membrane and subapical area of epithelial intestinal cells, of [Na.sup.+]/[K.sup.+]-ATPase in basolateral membranes, and of an sAC-like protein in the cytoplasm. We propose that sAC regulates NKCC activity in response to luminal HC[O.sup.-.sub.3], and that V-[H.sup.+]-ATPase and intracellular carbonic anhydrase are essential for transducing luminal HC[O.sup.-.sub.3] into the cell by C[O.sub.2]/ HC[O.sup.-.sub.3] hydration/dehydration. This mechanism putatively coordinates HC[O.sup.-.sub.3] secretion with NaC1 and water absorption in toadfish intestine. NKCC; cAMP; carbonic anhydrase; bumetanide; proton pump doi: 10.1152/ajpregu.00761.2009.

Cardiolipin is an ever-present component of the energy-conserving inner membranes of bacteria and mitochondria. Its modulation of the structure and dynamism of the bilayer impacts on the activity of their resident proteins, as a number of studies have shown. Here we analyze the consequences cardiolipin has on the conformation, activity, and localization of the protein translocation machinery. Cardiolipin tightly associates with the SecYEG protein channel complex, whereupon it stabilizes the dimer, creates a high-affinity binding surface for the SecA ATPase, and stimulates ATP hydrolysis. In addition to the effects on the structure and function, the subcellular distribution of the complex is modified by the cardiolipin content of the membrane. Together, the results provide rare and comprehensive insights into the action of a phospholipid on an essential transport complex, which appears to be relevant to a broad range of energy-dependent reactions occurring at membranes. doi/ 10.1073/pnas.0914680107

To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal substrates that have engaged the spliceosome. Whereas DExD/H box ATPases have been implicated in rejecting suboptimal substrates, the mechanism for discarding suboptimal substrates has remained obscure. Corroborating evidence that suboptimal, mutated lariat intermediates can be exported to the cytoplasm for turnover, we have found that the ribosome can translate mutated lariat intermediates. By glycerol gradient analysis, we have found that the spliceosome can dissociate mutated lariat intermediates in vivo in a manner that requires the DEAH box ATPase Prp43p. Through an in vitro assay, we demonstrate that Prp43p promotes the discard of suboptimal and optimal 5' exon and lariat intermediates indiscriminately. Finally, we demonstrate a requirement for Prp43p in repressing splicing at a cryptic splice site. We propose a model for the fidelity of exon ligation in which the DEAH box ATPase Prp22p slows the flow of suboptimal intermediates through exon ligation and Prp43p generally promotes discard of intermediates, thereby establishing a pathway for turnover of stalled intermediates. Because Prp43p also promotes spliceosome disassembly after exon ligation, this work establishes a parallel between the discard of suboptimal intermediates and the dissociation of a genuine excised intron product. RNA helicase | RNA processing | small nuclear RNA | ribonucleoprotein particle | Saccharomyces cerevisiae doi/ 10.1073/pnas.0906022107

Proton-transporting cells are located in several tissues where they acidify the extracellular environment. These cells express the vacuolar [H.sup.+]-ATPase (V-ATPase) B1 subunit (ATP6V1B1) in their plasma membrane. We provide here a comprehensive catalog of the proteins that are expressed in these cells, after their isolation by enzymatic digestion and fluorescence-activated cell sorting (FACS) from transgenic B1-enhanced green fluorescent protein (EGFP) mice. In these mice, type A and B intercalated cells and connecting segment cells of the kidney, and narrow and clear cells of the epididymis, which all express ATP6V1B1, also express EGFP, while all other cell types are negative. The proteome of renal and epididymal EGFP-positive ([EGFP.sup.+]) cells was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and compared with their respective EGFP-negative ([EGFP.sup.-]) cell populations. A total of 2,297 and 1,564 proteins were detected in [EGFP.sup.+] cells from the kidney and epididymis, respectively. Out of these proteins, 202 and 178 were enriched by a factor greater than 1.5 in [EGFP.sup.+] cells compared with [EGFP.sup.-] cells, in the kidney and epididymis respectively, and included subunits of the V-ATPase (B1, a4, and A). In addition, several proteins involved in intracellular trafficking, signaling, and cytoskeletal dynamics were identified. A novel common protein that was enriched in renal and epididymal [EGFP.sup.+] cells is the progesterone receptor, which might be a potential candidate for the regulation of V-ATPase-dependent proton transport. These proteomic databases provide a framework for comprehensive future analysis of the common and distinct functions of V-ATPase-B1-expressing cells in the kidney and epididymis. intercalated cells; mass spectrometry; proteome; proton secretion; clear cells doi: 10.1152/ajpcell.00552.2009.

The vesicular adenosine triphosphatase (v-ATPase) is a proton pump that acidifies intracellular compartments. In addition, mutations in components of the membrane-bound v-ATPase V0 sector cause acidification-independent defects in yeast, worm, fly, zebrafish, and mouse. In this study, we present a dual function for the neuron-specific V0 subunit a 1 orthologue v100 in Drosophila melanogaster. A v100 mutant that selectively disrupts proton translocation rescues a previously characterized synaptic vesicle fusion defect and vesicle fusion with early endosomes. Correspondingly, V100 selectively interacts with syntaxins on the respective target membranes, and neither synaptic vesicles nor early endosomes require v100 for their acidification. In contrast, V100 is required for acidification once endosomes mature into degradative compartments. As a consequence of the complete loss of this neuronal degradation mechanism, photoreceptors undergo slow neurodegeneration, whereas selective rescue of the acidification-independent function accelerates cell death by increasing accumulations in degradation-incompetent compartments. We propose that V100 exerts a temporally integrated dual function that increases neuronal degradative capacity. doi/ 10.1083/jcb.201003062

Hsp104 is a ring-forming AAA+ machine that recognizes both aggregated proteins and prion-fibrils as substrates and, together with the Hsp70 system, remodels substrates in an ATP-dependent manner. Whereas the ability to disaggregate proteins is dependent on the Hsp104 M-domain, the location of the M-domain is controversial and its exact function remains unknown. Here we present cryoEM structures of two Hsp104 variants in both crosslinked and noncrosslinked form, in addition to the structure of a functional Hsp104 chimera harboring T4 lysozyme within the M-domain helix L2. Unexpectedly, we found that our Hspl04 chimera has gained function and can solubilize heat-aggregated [beta]-galactosidase ([beta]-gal) in the absence of the Hsp70 system. Our fitted structures confirm that the subunit arrangement of Hsp104 is similar to other AAA+ machines, and place the M-domains on the Hsp104 exterior, where they can potentially interact with large, aggregated proteins. ATPase | AAA+ | chaperone | disaggregasse | CIpB doi/ 10.1073/pnas.1003572107

Type IV secretion (T4S) systems are involved in several secretion processes, including secretion of virulence factors, such as toxins or transforming molecules, or bacterial conjugation whereby two mating bacteria exchange genetic material. T4S systems are generally composed of 12 protein components, three of which, termed VirB4, VirB11, and VirD4, are ATPases. VirB4 is the largest protein of the T4S system, is known to play a central role, and interacts with many other T4S system proteins. In this study, we have biochemically characterized the protein TraB, a VirB4 homologue from the pKM101 conjugation T4S system. We demonstrated that TraB is a modular protein, composed of two domains, both able to bind DNA in a non-sequence-specific manner. Surprisingly, both TraB N- and C-terminal domains can bind ATP, revealing a new degenerated nucleotide-binding site in the TraB N-terminal domain. TraB purified from the membrane forms stable dimers and is unable to hydrolyze ATP while, when purified from the soluble fraction, TraB can form hexamers capable of hydrolyzing ATP. Remarkably, both the N- and C-terminal domains display ATP-hydrolyzing activity. These properties define a new class of VirB4 proteins. doi: 10.1128/JB.01384-09

The productivity of higher plants as a major source of food and energy is linked to their ability to buffer changes in the concentrations of essential and toxic ions. Transport across the tonoplast is energized by two proton pumps, the vacuolar [H.sup.+]-ATPase (V-ATPase) and the vacuolar [H.sup.+]-pyrophosphatase (V-PPase), however, their functional relation and relative contributions to ion storage and detoxification are unclear. We have identified an Arabidopsis mutant in which energization of vacuolar transport solely relies on the activity of the V-PPase. The vha-a2 vha-a3 double mutant, which lacks the two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a, is viable but shows day-length-dependent growth retardation. Nitrate content is reduced whereas nitrate assimilation is increased in the vha-a2 vha-a3 mutant, indicating that vacuolar nitrate storage represents a major growth-limiting factor. Zinc is an essential micronutrient that is toxic at excess concentrations and is detoxified via a vacuolar [Zn.sup.2+]/[H.sup.+]-antiport system. Accordingly, the double mutant shows reduced zinc tolerance. In the same way the vacuolar [Na.sup.+]/[H.sup.+]-antiport system is assumed to be an important component of the system that removes sodium from the cytosol. Unexpectedly, salt tolerance and accumulation are not affected in the vhaa2 vha-a3 double mutant. In contrast, reduction of V-ATPase activity in the trans-Golgi network/early endosome (TGN/EE) leads to increased salt sensitivity. Taken together, our results show that during gametophyte and embryo development V-PPase activity at the tonoplast is sufficient whereas tonoplast V-ATPase activity is limiting for nutrient storage but not for sodium tolerance during vegetative and reproductive growth. proton-pump | pH-homeostasis | vacuole | nitrate | salt tolerance www.pnas.org/cgi/doi/10.1073/pnas.0913035107

The [Na.sup.+]/[K.sup.+]-ATPase mediates electrogenic transport by exporting three [Na.sup.+] ions in exchange for two [K.sup.+] ions across the cell membrane per adenosine triphosphate molecule. The location of two [Rb.sup.+] ions in the crystal structures of the [Na.sup.+]/[K.sup.+]-ATPase has defined two 'common' cation binding sites, I and II, which accommodate [Na.sup.+] or [K.sup.+] ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this 'unique' [Na.sup.+] binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human [Na.sup.+]/[K.sup.+]-ATPase [[alpha].sub.2] subunit decreases the affinity for extracellular and intracellular [Na.sup.+], in agreement with previous biochemical studies. Apparently, the [DELTA]YY deletion changes [Na.sup.+] affinity at site III but leaves the common sites unaffected, whereas the more extensive [DELTA]KETYY deletion affects the unique site and the common sites as well. In the absence of extracellular [K.sup.+], the [DELTA]YY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were [Na.sup.+] dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under [Na.sup.+]/[Na.sup.+] exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular [Na.sup.+] release/binding mechanism. In analogy to the mechanism proposed for the [H.sup.+] leak currents of the wild-type [Na.sup.+]/[K.sup.+]-ATPase, we suggest that the [DELTA]YY deletion lowers the energy barrier for the intracellular [Na.sup.+] occlusion reaction, thus destabilizing the [Na.sup.+]-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the [Na.sup.+]/[K.sup.+]-ATPase [alpha] subunit represents a structural and functional relay between [Na.sup.+] binding site III and the intracellular cation occlusion gate. doi/ 10.1085/jgp.200910301

Many bacterial proteins, including most secretory proteins, are translocated across the plasma membrane by the interplay of the cytoplasmic SecA ATPase and a protein-conducting channel formed by the SecY complex. SecA catalyzes the sequential movement of polypeptide segments through the SecY channel. How SecA interacts with a broad range of polypeptide segments is unclear, but structural data raise the possibility that translocation substrates bind into a 'clamp' of SecA. Here, we have used disulfide bridge cross-linking to test this hypothesis. To analyze polypeptide interactions of SecA during translocation, two cysteines were introduced into a translocation intermediate: one that cross-links to the SecY channel and the other one for cross-linking to a cysteine placed at various positions in SecA. Our results show that a translocating polypeptide is indeed captured inside SecA's clamp and moves in an extended conformation through the clamp into the SecY channel. These results define the polypeptide path during SecA-mediated protein translocation and suggest a mechanism by which ATP hydrolysis by SecA is used to move a polypeptide chain through the SecY channel. disulfide bridge cross-linking | protein translocation | SecY | secretion | SecA damp doi/10.1073/pnas.0910550106

Sarco(endo)plasmic reticulum [Ca.sup.2+] ATPase (SERCA) [Ca.sup.2+] transporters pump cytosolic [Ca.sup.2+] into the endoplasmic reticulum, maintaining a [Ca.sup.2+] gradient that controls vital cell functions ranging from proliferation to death. To meet the physiological demand of the cell, SERCA activity is regulated by adjusting the affinity for [Ca.sup.2+] ions. Of all SERCA isoforms, the housekeeping SERCA2b isoform displays the highest [Ca.sup.2+] affinity because of a unique C-terminal extension (2b-tail). Here, an extensive structure-function analysis of SERCA2b mutants and SERCAla2b chimera revealed how the 2b-tail controls [Ca.sup.2+] affinity. Its transmembrane (TM) segment (TM11) and luminal extension functionally cooperate and interact with TM7/TM10 and luminal loops of SERCA2b, respectively. This stabilizes the [Ca.sup.2+]-bound E1 conformation and alters [Ca.sup.2+]-transport kinetics, which provides the rationale for the higher apparent [Ca.sup.2+] affinity. Based on our NMR structure of TM11 and guided by mutagenesis results, a structural model was developed for SERCA2b that supports the proposed 2b-tail mechanism and is reminiscent of the interaction between the [alpha]-and [beta]-subunits of [Na.sup.+],[K.sup.+]-ATPase. The 2b-tail interaction site may represent a novel target to increase the [Ca.sup.2+] affinity of malfunctioning SERCA2a in the failing heart to improve contractility. endoplasmic reticulurn | [Ca.sup.2+]-ATPase | ion transporter | phospholarnban I P-type ATPase www.pnas.org/cgi/doi/10.1073/pnas.0906797106

This work shows that vesicles derived from the blue marlin heater organ retain a sarcoplasmic reticulum (SR) [Ca.sup.2+]-ATPase that can interconvert different forms of energy. During the hydrolysis of ATP part of the energy is always converted into heat, and the other part can be converted into work ([Ca.sup.2+] transport) or heat, depending on the temperature and the presence of KCl in the reaction medium. At 15[degrees]C, where KCl stimulates the activity approximately threefold, measurements of the amount of heat released per mole of ATP hydrolyzed ([DELTA][H.sup.cal]) show similar values (approximately -11 kcal/mol) in the presence or absence of a [Ca.sup.2+] gradient. At 25[degrees]C, KCl activates the enzyme to the same extent as at 15[degrees]C, but inhibits the production of extra heat by SR [Ca.sup.2+]-ATPase when a [Ca.sup.2+] gradient is built up across the membrane. The [DELTA][H.sup.cal] values found in the presence of a [Ca.sup.2+]-gradient were -26.2 [+ or -] 2.9 kcal/mol (n = 7) in control experiments and -16.1 [+ or -] 1.5 (n = 14) in the presence of 100 mM KCl. At 35[degrees]C, KCl has a smaller effect (~1.5-fold) on activating the enzyme. Similar to SR [Ca.sup.2+]-ATPase from mammals, at this temperature the enzyme produces almost twice the amount of heat per mole of ATP hydrolyzed in the presence of a [Ca.sup.2+] gradient and KCl has no effect at all on this increment. These data suggest that the marlin SR [Ca.sup.2+]-ATPase may play an important role in heater organ thermogenesis and that KCl has the potential for regulating the heat production catalyzed by the enzyme. billfish; regional endothermy; SERCA1; Makaira nigricans doi: 10.1152/ajpregu.90993.2008.

Type-IV P-type ATPases (P4-ATPases) are putative phospholipid translocases, or flippases, that translocate specific phospholipid substrates from the exofacial to the cytosolic leaflet of membranes to generate phospholipid asymmetry. In addition, the activity of Drs2p, a P4-ATPase from Saccharomyces cerevisiae, is required for vesicle-mediated protein transport from the Golgi and endosomes, suggesting a role for phospholipid translocation in vesicle budding. Drs2p is necessary for translocation of a fluorescent phosphatidylserine analogue across purified Golgi membranes. However, a flippase activity has not been reconstituted with purified Drs2p or any other P4-ATPase, so whether these ATPases directly pump phospholipid across the membrane bilayer is unknown. Here, we show that Drs2p can catalyze phospholipid translocation directly through purification and reconstitution of this P4-ATPase into proteoliposomes. The noncatalytic subunit, Cdc50p, also was reconstituted in the proteoliposome, although at a substoichiometric concentration relative to Drs2p. In proteoliposomes containing Drs2p, a phosphatidylserine analogue was actively flipped across the liposome bilayer to the outer leaflet in the presence of [Mg.sup.2+]-ATP, whereas no activity toward the phosphatidylcholine or sphingomyelin analogues was observed. This flippase activity was mediated by Drs2p, because protein-free liposomes or proteoliposomes reconstituted with a catalytically inactive form of Drs2p showed no translocation activity. These data demonstrate for the first time the reconstitution of a flippase activity with a purified P4-ATPase. flippase | membrane asymmetry | P4-ATPase | proteoliposome | Cdc50p

Oxidative stress has been implicated in diverse disease states and aging. To date, induction of cellular responses to combat oxidative stress has been characterized largely at the transcriptional level, with emphasis on Nrf2-mediated activation of antioxidant response elements. In this study, we demonstrate that OLA1, a novel Obg-like ATPase, functions as a negative regulator of the cellular antioxidant response independent of transcriptional processes. Knockdown of OLA1 in human cells elicited an increased resistance to oxidizing agents including tert-butyl hydroperoxide (tBH) and diamide without affecting cell proliferation, baseline apoptosis, or sensitivity to other cytotoxic agents that target the mitochondria, cytoskeleton, or DNA. Conversely, overexpression of OLA1 increased cellular sensitivity to tBH and diamide. When challenged with oxidants, OLA1-knockdown cells had decreased production of intracellular reactive oxygen species and exhibited less depletion of reduced glutathione. Surprisingly, knockdown of OLA1 caused only minimal genomic response; no changes were found in the mRNA levels of genes encoding antioxidant enzymes, enzymes that produce antioxidants (including glutathione), or other genes known to respond to Nrf2. Moreover, when de novo protein synthesis was blocked by cycloheximide in OLA1-knockdown cells, they continued to demonstrate increased resistance to both tBH and diamide. These data demonstrate that OLA1 suppresses the antioxidant response through nontranscriptional mechanisms. The beneficial effects observed upon OLA1-knockdown suggest that this regulatory ATPase is a potential novel target for antioxidative therapy. drug target | oxidative stress | posttranslational regulation

Cardiac [Na.sup.+]-[K.sup.+]-ATPase (NKA) regulates intracellular [Na.sup.+], which in turn affects intracellular [Ca.sup.2+] and contractility via the [Na.sup.+]/[Ca.sup.2+] exchanger. Extracellular [K.sup.+] concentration ([[K.sup.+]]) is a central regulator of NKA activity. Phospholemman (PLM) has recently been recognized as a critical regulator of NKA in the heart. PLM reduces the intracellular [Na.sup.+] affinity of NKA, an effect relieved by PLM phosphorylation. Here we tested whether the NKA [[alpha].sub.1]-vs. [[alpha].sub.2]-isoforms have different external [K.sup.+] sensitivity and whether PLM and PKA activation affects the NKA affinity for [K.sup.+] in mouse cardiac myocytes. We measured the external [[K.sup.+]] dependence of the pump current generated by the ouabain resistant NKA isoform in myocytes from wild-type (WT) mice (i.e., current due to NKA-[[alpha].sub.1]) and mice in which the NKA isoforms have swapped ouabain affinities ([[alpha].sub.1] is ouabain sensitive and [[alpha].sub.2] is ouabain resistant) to assess current due to NKA-[[alpha].sub.2]. We found that NKA-[[alpha].sub.1] has a higher affinity for external [K.sup.+] than NKA-[[alpha].sub.2] [half-maximal pump activation ([K.sub.0.5]) = 1.5 [+ or -] 0.1 vs. 2.9 [+ or -] 0.3 mM]. The apparent external [K.sup.+] affinity of NKA was significantly lower in myocytes from WT vs. PLM-knockout mice ([K.sub.0.5] = 2.0 [+ or -] 0.2 vs. 1.05 [+ or -] 0.08 mM). However, PKA activation by isoproterenol (1 [micro]M) did not alter the [K.sub.0.5] of NKA for external [K.sup.+] in WT myocytes. We conclude that 1) NKA-[[alpha].sub.1] has higher affinity for [K.sup.+] than NKA-[[alpha].sub.2] in cardiac myocytes, 2) PLM decreases the apparent external [K.sup.+] affinity of NKA, and 3) phosphorylation of PLM at the cytosolic domain does not alter apparent extracellular [K.sup.+] affinity of NKA. voltage-clamp; phosphorylation

Transcriptionally silent genes are maintained in inaccessible chromatin. Accessibility of these genes requires their modification by chromatin remodeling complexes (CRCs), which are recruited to promoters by sequence-specific DNA-binding proteins. Early B-cell factor (EBF), which is crucial for B-cell lineage specification, reprograms mb-1 (Ig-[alpha]) promoters by increasing chromatin accessibility and initiating the loss of DNA methylation. In turn, this facilitates promoter activation by Pax5. Here, we investigated the roles of ATP-dependent CRCs in these mechanisms. Fusion of EBF and Pax5 with the ligand-binding domain of ER[alpha] allowed for 4-hydroxytamoxifen-dependent, synergistic activation of mb-1 transcription in plasmacytoma cells. Knock-down of the SWI/SNF ATPases Brg1 and Brm inhibited transcriptional activation by EBF:ER and Pax5-ER. In contrast, knock-down of the Mi-2/NuRD complex subunit Mi-2[beta] greatly enhanced chromatin accessibility and mb-1 transcription in response to the activators. The reduction of Mi-2[beta] also propagated DNA demethylation in response to EBF:ER and Pax5:ER, resulting in fully unmethylated mb-1 promoters. In EBF- or EBF/Pax5-deficient fetal liver cells, both EBF and Pax5 were required for efficient demethylation of mb-1 promoters. Together, our data suggest that Mi-2/NuRD is important for the maintenance of hypermethylated chromatin in B cells. We conclude that SWI/SNF and Mi-2/NuRD function in opposition to enable or limit the reprogramming of genes by EBF and Pax5 during B-cell development. DNA methylation | mb-1 promoter | Cd19 promoter | chromatin accessibility

The [H.sup.+]-[K.sup.+]-ATPase [[alpha].sub.2] (HK[alpha]2) gene of the renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5'-flanking region confers basal transcriptional activity in murine inner medullary collecting duct (mIMCD3) cells and that NF-[KAPPA]B and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the -144/-135 Sp element influences basal HKe[alpha]2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for -154/-127 revealed Sp 1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (CHIP) assays demonstrated that Spl, but not Sp3, binds to this promoter region of the HK[alpha]2 gene in mIMCD3 cells in vivo. HK[alpha]2 minimal promoter-luciferase constructs with point mutations in the -144/-135 Sp element exhibited much lower activity than the wild-type promoter in transient transfection assays. Overexpression of Spl, but not Sp3, trans-activated an HK[alpha]2 proximal promoterluciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, small interfering RNA knockdown of Sp 1 inhibited endogenous HK[alpha]2 mRNA expression, and binding of Spl to chromatin associated with the proximal HK[alpha]2 promoter without altering the binding or regulatory influence of NF-[KAPPA]B p65 or CREB-1 on the proximal HKc[alpha]2 promoter. We conclude that Spl plays an important and positive role in controlling basal HK[alpha]2 gene expression in mIMCD3 cells in vivo and in vitro. chromatin; transcription; collecting duct; transcription factor; gene expression

Goldfish (Carassius auratus) acclimated to 7[degrees]C and exposed to hypoxia (~10 mmHg) for 7 days exhibited a pronounced remodeling of the gill consisting of the removal of an interlamellar cell mass (ILCM). Subsequent experiments were designed to assess the impact of gill remodeling and the associated increase in functional lamellar surface area on the distribution of branchial ionocytes and [Cl.sup.-] flux across the gill. Despite the increased functional lamellar surface area during hypoxia, there was no corresponding increase in [Cl.sup.-] loss or efflux of the extracellular marker polyethylene glycol (PEG 4000). However, when hypoxic fish were returned to normoxic water for 12 h, rates of [Cl.sup.-] and PEG efflux were markedly stimulated in keeping with an increased surface area for solute movement. Similarly, the rate of branchial [Cl.sup.-1] uptake was reduced (105 [+ or -] 22 vs. 45 [+ or -] 8 [micro]mol x [kg.sup.-1] x [h.sup.-1]) in normoxic and hypoxic fish, respectively, but then stimulated (345 [micro]mol x [kg.sup.-1] x [h.sup.-1] ) upon reestablishment of normoxic conditions. Hypoxia (7 days) was accompanied by a significant decrease in the total cross-sectional area of branchial ionocytes owing to a decrease in their numbers and individual sizes. Thus, despite experiencing an increase in functional lamellar surface area, hypoxic goldfish limit branchial [Cl.sup.-] loss likely by a hypoxia-mediated decrease in paracellular permeability. In normoxic fish, the ionocytes were largely confined to the outer edges of the ILCM. During hypoxia, preexisting ionocytes migrated with the shrinking ILCM, while a smaller proportion of newly differentiated cells appeared below the surface of the ILCM. The capacity to maintain a population of ionocytes in contact with the water is an appropriate strategy to retain ionoregulatory capabilities regardless of whether the lamellae are uncovered or covered. hypoxia; ion regulation; C1 balance; mitochondrion-rich cell; ionocyte; [Na.sup.+ /[K.sup.+]-ATPase; interlamellar cell mass

The transporter associated with antigen processing (TAP) represents a focal point in the immune recognition of virally or malignantly transformed cells by translocating proteasomal degradation products into the endoplasmic reticulum--lumen for loading of MHC class I molecules. Based on a number of experimental data and the homology to the bacterial ABC exporter Sav1866, we constructed a 3D structural model of the core TAP complex and used it to examine the interface between the transmembrane and nucleotide-binding domains (NBD) by cysteine-scanning and cross-linking approaches. Herein, we demonstrate the functional importance of the newly identified X-loop in the NBD in coupling substrate binding to downstream events in the transport cycle. We further verified domain swapping in a heterodimeric ABC half-transporter complex by cysteine cross-linking. Strikingly, either substrate binding or translocation can be blocked by cross-linking the X-loop to coupling helix 2 or 1, respectively. These results resolve the structural arrangement of the transmission interface and point to different functions of the cytosolic loops and coupling helices in substrate binding, signaling, and transport. membrane protein structure | traffic ATPase | substrate recognition | conformational change | cysteine cross-linking

We hypothesized that testosterone at physiological levels enhances cardiac contractile responses to stimulation of both [[alpha].sub.1]- and [[beta].sub.1]-adrenoceptors by increasing [Ca.sup.2+] release from the sarcoplasmic reticulum (SR) and speedier removal of [Ca.sup.2+] from cytosol via [Ca.sup.2+]-regulatory proteins. We first determined the left ventricular developed pressure, velocity of contraction and relaxation, and heart rate in perfused hearts isolated from control rats, orchiectomized rats, and orchiectomized rats without and with testosterone replacement (200 [micro]g/100 g body wt) in the presence of norepinephrine ([10.sup.-7] M), the [[alpha].sub.1]-adrenoceptor agonist phenylephrine ([10.sup.-6] M), or the nonselective [beta]-adrenoceptor agonist isoprenaline ([10.sup.-7] M) in the presence of 5 x [10.sup.-7] M ICI-118,551, a [[beta].sub.2]-adrenoceptor antagonist. Next, we determined the amplitudes of intracellular [Ca.sup.2+] concentration transients induced by electrical stimulation or caffeine, which represent, respectively, [Ca.sup.2+] release via the ryanodine receptor (RyR) or releasable [Ca.sup.2+] in the SR, in ventricular myocytes isolated from the three groups of rats. We also measured [sup.45][Ca.sup.2+] release via the RyR. We then determined the time to 50% decay of both transients, which represents, respectively, [Ca.sup.2+] reuptake by sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA) and removal via the sarcolemmal [Na.sup.+]/[Ca.sup.2+] exchanger (NCX). We correlated [Ca.sup.2+] removal from the cytosol with activities of SERCA and its regulator phospholamban as well as NCX. The results showed that testosterone at physiological levels enhanced positive inotropic and lusitropic responses to stimulation of [[alpha].sub.1]- and [[beta].sub.1]-adrenoceptors via the androgen receptor. The increased contractility and speedier relaxation were associated with increased [Ca.sup.2+] release via the RyR and faster [Ca.sup.2+] removal out of the cytosol via SERCA and NCX. orchiectomy; androgen receptor; left ventricular developed pressure; velocity of contraction and relaxation; electrical-induced intracellular [Ca.sup.2+] concentration transients; caffeine-induced intracellular [Ca.sup.2+] concentration transients; ryanodine receptor; sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase; [Na.sup.+]/[Ca.sup.2+] exchanger

It is important to maintain internal pH homeostasis in biological systems. In our previous studies, [H.sup.+]-ATPase-rich (HR) cells were found to be responsible for proton secretion in the skin of zebrafish embryos during development. In this study, zebrafish embryos were exposed to acidic and basic waters to investigate the regulation of HR cell acid secretion during pH disturbances. Our results showed that the function of HR cells on the skin of zebrafish embryos can be upregulated in pH 4 water not only by increasing the cell number but also by enlarging the acid-secreting function of single cells. We also identified an 'alveolar-type' apical opening under scanning electron microscopy observations of the apical membrane of HR cells, and the density and size of the alveolar type of apical openings were also increased in pH 4 water, p63 and PCNA immunostaining results also showed that additional HR cells in pH 4 water may be differentiated not only from ionocyte precursor cells but also newly proliferating epithelial stem cells. ionocytes; acid-base regulation; ion regulation; proliferation; differentiation

Acidic luminal pH and low [HC[O.sup.-.sub.3]] maintain sperm quiescent during maturation in the epididymis. The vacuolar [H.sup.+]-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HC[O.sup.-.sub.3], induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and nonhuman primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-l-[beta]-D-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by [N.sup.6]-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [[sup.32]P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK. vas deferens; male reproductive tract; metabolism; soluble adenylyl cyclase; acid secretion

Transition metals, although being essential cofactors in many physiological processes, are toxic at elevated concentrations. Among the membrane-embedded transport proteins that maintain appropriate intracellular levels of transition metals are ATP-driven pumps belonging to the P-type ATPase superfamily. These metal transporters may be differentiated according to their substrate specificities, where the majority of pumps can extrude either silver and copper or zinc, cadmium, and lead. In the present report, we have established the substrate specificities of nine previously uncharacterized prokaryotic transition-metal P-type ATPases. We find that all of the newly identified exporters indeed fall into one of the two above-mentioned categories. In addition to these exporters, one importer, Pseudomonas aeruginosa Q91147, was also identified. This protein, designated HmtA (heavy metal transporter A), exhibited a different substrate recognition profile from the exporters. In vivo metal susceptibility assays, intracellular metal measurements, and transport experiments all suggest that HmtA mediates the uptake of copper and zinc but not of silver, mercury, or cadmium. The substrate selectivity of this importer ensures the high-affinity uptake of essential metals, while avoiding intracellular contamination by their toxic counterparts. membrane proteins | transition-metal homeostasis | heavy metal transporters | [P.sub.1B] ATPases

Many gram-negative plant and animal pathogenic bacteria employ a type III secretion (T3S) system to inject effector proteins into the cytosol of eukaryotic host cells. The membrane-spanning T3S apparatus is associated with an ATPase that presumably provides the energy for the secretion process. Here, we describe the role of the predicted ATPase HrcN from the plant pathogenic bacterium Xanthomonas campestris pathovar vesicatoria. We show that HrcN hydrolyzes ATP in vitro and is essential for T3S and bacterial pathogenicity. Stability of HrcN in X. campestris pv. vesicatoria depends on the conserved HrcL protein, which interacts with HrcN in vitro and in vivo. Both HrcN and HrcL bind to the inner membrane protein HrcU and specifically localize to the bacterial membranes under T3S-permissive conditions. Protein-protein interaction studies revealed that HrcN also interacts with the T3S substrate specificity switch protein HpaC and the global T3S chaperone HpaB, which promotes secretion of multiple effector proteins. Using an in vitro chaperone release assay, we demonstrate that HrcN dissociates a complex between HpaB and the effector protein XopF1 in an ATP-dependent manner, suggesting that HrcN is involved in the release of HpaB-bound effectors. Effector release depends on a conserved glycine residue in the HrcN phosphate-binding loop, which is crucial for enzymatic activity and protein function during T3S. There is no experimental evidence that T3S can occur in the absence of the ATPase, in contrast to recent findings reported for animal pathogenic bacteria.

ATP7B is a copper-transporting P-type ATPase present predominantly in liver. In basal copper, hepatic ATP7B is in a post-trans-Golgi network (TGN) compartment where it loads cytoplasmic Cu(I) onto newly synthesized ceruloplasmin. When copper levels rise, the protein redistributes via unique vesicles to the apical periphery where it exports intracellular Cu(I) into bile. We want to understand the mechanisms regulating the copper-sensitive trafficking of ATP7B. Earlier, our laboratory reported the presence of apical targeting/TGN retention information within residues 1-63 of human ATP7B; deletion of these residues resulted in a mutant protein that was not efficiently retained in the post-TGN in low copper and constitutively trafficked to the basolateral membrane of polarized, hepatic WIF-B cells with and without copper (13). In this study, we used mutagenesis and adenovirus infection of WIF-B cells followed by confocal immunofluorescence microscopy analysis to identify the precise retention/targeting sequences in the context of full-length ATP7B. We also analyzed the expression of selected mutants in livers of copper-deficient and -loaded mice. Our combined results clearly demonstrate that nine amino acids, [F.sub.37]AFDNVGY[E.sub.45], comprise an essential apical targeting determinant for ATP7B in elevated copper and participate in the TGN retention of the protein under low-copper conditions. The signal is novel, does not require phosphorylation, and is highly conserved in -24 species of ATP7B. Furthermore, N41S, which is part of the signal we identified, is the first and only Wilson disease-causing missense mutation in residues 1--63 of ATP7B. Expression of N41S-ATP7B in WIF-B cells severely disabled the targeting and retention of the protein. We present a working model of how this physiologically relevant signal might work. Wilson protein; WIF-B cells; mutagenesis; in vivo; trans-Golgi network

SecA is the ATPase that provides energy for translocation of precursor polypeptides through the SecYEG translocon in Escherichia coli during protein export. We showed previously that when SecA receives the precursor from SecB, the ternary complex is fully active only when two protomers of SecA are bound. Here we used variants of SecA and of SecB that populate complexes containing two protomers of SecA to different degrees to examine both the hydrolysis of ATP and the translocation of polypeptides. We conclude that the low activity of the complexes with only one protomer is the result of a low efficiency of coupling between ATP hydrolysis and translocation.

The 2 nanomotors of rotary ATP synthase, ionmotive [F.sub.o] and chemically active [F.sub.1], are mechanically coupled by a central rotor and an eccentric bearing. Both motors rotate, with 3 steps in [F.sub.1] and 10-15 in [F.sub.o]. Simulation by statistical mechanics has revealed that an elastic power transmission is required for a high rate of coupled turnover. Here, we investigate the distribution in the [F.sub.o][F.sub.1] structure of compliant and stiff domains. The compliance of certain domains was restricted by engineered disulfide bridges between rotor and stator, and the torsional stiffness ([kappa]) of unrestricted domains was determined by analyzing their thermal rotary fluctuations. A fluorescent magnetic bead was attached to single molecules of [F.sub.1] and a fluorescent actin filament to [F.sub.o][F.sub.1], respectively. They served to probe first the functional rotation and, after formation of the given disulfide bridge, the stochastic rotational motion. Most parts of the enzyme, in particular the central shaft in [F.sub.1], and the long eccentric bearing were rather stiff (torsional stiffness [kappa] > 750 pNnm). One domain of the rotor, namely where the globular portions of subunits [gamma] and [epsilon] of [F.sub.1] contact the c-ring of [F.sub.o], was more compliant ([kappa] [congruent to] 68 pNnm). This elastic buffer smoothes the cooperation of the 2 stepping motors. It is located were needed, between the 2 sites where the power strokes in [F.sub.o] and [F.sub.1] are generated and consumed. elasticity | F-ATPase | nanomotor

Helicobacter pylori infection of the gastric body induces transient hypochlorhydria and contributes to mucosal progression toward gastric carcinoma. Acid secretion is mediated by parietal cell H,K-ATPase, in which the catalytic a-subunit (HK[alpha]) promoter activity in transfected gastric epithelial [gastric adenocarcinoma (AGS)] cells is repressed by H. pylori through NF-[kappa]B p50 homodimer binding to the promoter. IL-1[beta], an acid secretory inhibitor whose mucosal level is increased by H. pylori, up-regulates HK[alpha] promoter activity in AGS cells. Because IL-1[beta] also activates NF-[kappa]B signaling, we investigated disparate HK[alpha] regulation by H. pylori and IL-1[beta], testing the hypothesis that IL-1[beta]-induced HK[alpha] promoter activation is mediated by the transcription factor Spl. DNase I footprinting revealed Spl binding to the HK[alpha] promoter at -56 to -39 bp. IL-1[beta] stimulated the activity of three HK[alpha] promoter constructs containing NF-[kappa]B and Spl sites transfected into AGS cells and also stimulated a construct containing only an Spl site. This stimulation was abrogated by mutating the HK[alpha] promoter Spl binding site. Gelshifi assays showed that IL-1[beta] increased Spl but not p50 binding to cognate HK[alpha] probes and that Spl also interacts with an HK[alpha] NF-[kappa]B site when bound to its cognate HK[alpha] cis-response element. H. pylori did not augment Spl binding to an HK[alpha] Spl probe, and small interfering RNA-mediated knockdown of Spl expression abrogated IL-1[beta]-induced HK[alpha] promoter stimulation. We conclude that IL-1[beta] upregulates HK[alpha] gene transcription by inducing Spl binding to HK[alpha] Spl and NF-[kappa]B sites and that the H. pylori perturbation of HK[alpha] gene expression is independent of Spl-mediated basal HK[alpha] transcription. adenosine 5'-triphosphatase; Helicobacter pylori; interleukin-1[beta]

Among other functions, ATP-dependent proteases degrade misfolded proteins and remove several key regulatory proteins necessary to activate stress responses. In Bacillus subtilis, ClpX, ClpE, and ClpC form homohexameric ATPases that couple to the ClpP peptidase. To understand where these peptidases and ATPases localize in living cells, each protein was fused to a fluorescent moiety. We found that ClpX-GFP (green fluorescent protein) and ClpP-GFP localized as focal assemblies in areas that were not occupied by the nucleoid. We found that the percentage of cells with ClpP-GFP loci increased following heat shock independently of protein synthesis. We determined that ClpE-YFP (yellow fluorescent protein) and ClpC-YFP formed loci coincident with nucleoid edges, usually near cell poles. Furthermore, we found that ClpQ-YFP (HslV) localized as small foci, usually positioned near the cell membrane. We found that ClpQ-YFP loci were dependent on the presence of the cognate hexameric ATPase ClpY (HslU). Moreover, we found that LonA-GFP is coincident with the nucleoid during normal growth and that LonA-GFP also localized to the forespore during development. We also investigated LonB-GFP and found that this protein localized to the forespore membrane early in development, followed by localization throughout the forespore later in development. Our comprehensive study has shown that in B. subtilis several ATP-fueled proteases occupy distinct subcellular locations. With these data, we suggest that substrate specificity could be determined, in part, by the spatial and temporal organization of proteases in vivo.

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. VirB4 was suggested to be an ATPase involved in energizing pilus assembly and substrate transport. However, conflicting experimental evidence concerning VirB4 ATP hydrolase activity was reported. Here, we demonstrate that TrwK is able to hydrolyze ATP in vitro in the absence of its potential macromolecular substrates and other T4SS components. The kinetic parameters of its ATPase activity have been characterized. The TrwK oligomerization state was investigated by analytical ultracentrifugation and electron microscopy, and its effects on ATPase activity were analyzed. The results suggest that the hexameric form of TrwK is the catalytically active state, much like the structurally related protein TrwB, the conjugative coupling protein.

Ribosome biogenesis takes place successively in the nucleolar, nucleoplasmic, and cytoplasmic compartments. Numerous nonribosomal factors transiently associate with the nascent ribosomes, but the mechanisms driving ribosome formation are mostly unknown. Here, we show that an energy-consuming enzyme, the AAA-type (ATPases associated with various cellular activities) ATPase Rix7, restructures a novel pre-60S particle at the transition from the nucleolus to nucleoplasm. Rix7 interacts genetically with Nsa1 and is targeted to the Nsa1-defined preribosomal particle. In vivo, Nsa1 cannot dissociate from pre-60S particles in rix7 mutants, causing nucleolar Nsa1 to escape to the cytoplasm, where it remains associated with aberrant 60S subunits. Altogether, our data suggest that Rix7 is required for the release of Nsa1 from a discrete preribosomal particle, thereby triggering the progression of 60S ribosome biogenesis.

A majority of ATP in the brain is formed in the mitochondria through oxidative phosphorylation of ADP with the [F.sub.1][F.sub.0]-ATP (ATPase) enzyme. This ATP production rate plays central roles in brain bioenergetics, function and neurodegeneration. In vivo [sup.31]P magnetic resonance spectroscopy combined with magnetization transfer (MT) is the sole approach able to noninvasively determine this ATP metabolic rate via measuring the forward ATPase reaction flux ([F.sub.f,ATPase]). However, previous studies indicate lack of quantitative agreement between [F.sub.f,ATPase] and oxidative metabolic rate in heart and liver. In contrast, recent work has shown that [F.sub.f,ATPase] might reflect oxidative phosphorylation rate in resting human brains. We have conducted an animal study, using rats under varied brain activity levels from light anesthesia to isoelectric state, to examine whether the in vivo [sup.31]P MT approach is suitable for measuring the oxidative phosphorylation rate and its change associated with varied brain activity. Our results conclude that the measured [F.sub.f,ATPase] reflects the oxidative phosphorylation rate in resting rat brains, that this flux is tightly correlated to the change of energy demand under varied brain activity levels, and that a significant amount of ATP energy is required for 'housekeeping' under the isoelectric state. These findings reveal distinguishable characteristics of ATP metabolism between the brain and heart, and they highlight the importance of in vivo [sup.31]P MT approach to potentially provide a unique and powerful neuroimaging modality for noninvasively studying the cerebral ATP metabolic network and its central role in bioenergetics associated with brain function, activation, and diseases. brain metabolism | In vivo [sup.31P]MRS | ATPase | ATP synthesis | magnetization transfer

As in other P-type ATPases, metal binding to transmembrane metal-binding sites (TM-MBS) in [Cu.sup.+]-ATPases is required for enzyme phosphorylation and subsequent transport. However, [Cu.sup.+] does not access [Cu.sup.+]-ATPases in a free (hydrated) form but is bound to a chaperone protein. [Cu.sup.+] transfer from [Cu.sup.+] chaperones to regulatory cytoplasmic metal-binding domains (MBDs) present in these ATPases has been described, but there is no evidence of a proposed subsequent [Cu.sup.+] movement from the MBDs to the TM-MBS. Alternatively, we postulate the parsimonious [Cu.sup.+] transfer by the chaperone directly to TM-MBS. Testing both models, the delivery of [Cu.sup.+] by Archaeoglobus fulgidus [Cu.sup.+] chaperone CopZ to the corresponding [Cu.sup.+]-ATPase, CopA, was studied. As expected, CopZ interacted with and delivered the metal to CopA MBDs. [Cu.sup.+]-loaded MBDs, acting as metal donors, were unable to activate CopA or a truncated CopA lacking MBDs. Conversely, [Cu.sup.+]-loaded CopZ activated the CopA ATPase and CopA constructs in which MBDs were rendered unable to bind [Cu.sup.+]. Furthermore, under nonturnover conditions, CopZ transferred [Cu.sup.+] to the TM-MBS of a CopA lacking MBDs. These data are consistent with a model where MBDs serve a regulatory function without participating in metal transport and the chaperone delivers [Cu.sup.+] directly to transmembrane transport sites of [Cu.sup.+]-ATPases. CopA | CopZ | Cu homeostasis | Cu-ATPase | metal binding

Cation-transporting P-type ATPases show a high degree of structural and functional homology. Nevertheless, for many members of this large family, the molecular mechanism of transport is unclear; namely, whether transport is electrogenic or not and if countertransport is involved remains to be established. In a few well-studied cases such as the [Na.sup.+]-[K.sup.+]-ATPase, plasma membrane [Ca.sup.2+] ATPase (PMCA) and sarcoplasmic reticulum [Ca.sup.2]. ATPase (SERCA) countertransport has been clearly demonstrated. New data based on the crystal structure of SERCA now strongly indicate that countertransport could be mandatory for all P-type ATPases. This concept should be verified for other known and for all newly characterized P-type ATPases.

The yeast ABC transporter Pdr5 plays a major role in drug resistance against a large number of structurally unrelated compounds. Although Pdr5 has been extensively studied, many important aspects regarding its molecular mechanisms remain unresolved. For example, a striking degeneration of conserved amino acid residues exists in the nucleotide binding domains (NBDs), but their functional relevance is unknown. Here, we performed in vivo and in vitro experiments to address the functional asymmetry of NBDs. It became evident by ATPase activity and drug transport studies that catalysis at only one of the two NBD composite sites is crucial for protein function. Furthermore, mutations of the proposed 'catalytic carboxylate' (E1036) and the 'catalytic dyad histidine' (H1068) were characterized. Although a mutation of the glutamate abolished ATPase activity and substrate transport, mutation of H1068 had no influence on ATP consumption. However, the H1068A mutation abolished rhodamine transport in vivo and in vitro, while leaving the transport of other substrates unaffected. By contrast to mammalian P-glycoprotein (P-gp), the ATPase activity of yeast Pdr5 is not stimulated by the addition of substrates, indicating that Pdr5 is an uncoupled ABC transporter that constantly hydrolyses ATP to ensure active substrate transport. Taken together, our data provide important insights into the molecular mechanism of Pdr5 and suggest that not solely the transmembrane domains dictate substrate selection. ATPase activity | multidrug resistance | substrate recognition

The [H.sup.+]/ATP ratio is an important parameter for the energy balance of all cells and for the coupling mechanism between proton transport and ATP synthesis. A straightforward interpretation of rotational catalysis predicts that the [H.sup.+]/ATP coincides with the ratio of the c-subunits to [beta]-subunits, implying that, for the chloroplast and Escherichia coli ATPsynthases, numbers of 4.7 and 3.3 are expected. Here, the energetics described by the chemiosmotic theory was used to determine the [H.sup.+]/ATP ratio for the two enzymes. The isolated complexes were reconstituted into liposomes, and parallel measurements were performed under identical conditions. The internal phase of the liposomes was equilibrated with the acidic medium during reconstitution, allowing to measure the internal pH with a glass electrode. An acid-base transition was carried out and the initial rates of ATP synthesis or ATP hydrolysis were measured with luciferin/luciferase as a function of [DELTA]pH at constant Q = [ATP]/([ADP][[P.sub.i]]). From the shift of the equilibrium [DELTA]pH as a function of Q the standard Gibbs free energy for phosphorylation, [DELTA][G.sup.0.sub.p]; and the [H.sup.+]/ATP ratio were determined. It resulted [DELTA] [G.sup.0.sub.p]' = 38 [+ or -] 3 kJ*[mol.sup.-1] and [H.sup.+]/ATP = 4.0 [+ or -] 0.2 for the chloroplast and [H.sup.+]/ATP = 4.0 [+ or -] 0.3 for the E. coil enzyme, indicating that the thermodynamic [H.sup.+]/ATP ratio is the same for both enzymes and that it is different from the subunit stoichiometric ratio. ATPase | FOF1 | proteoliposomes | chemipsmotic theory

Polyunsaturated fatty acids (PUFAs) can have strong effects on hibernation and daily torpor in mammals. High dietary PUFA contents were found to increase proneness for torpor, decrease body temperatures, prolong torpor bout duration, and attenuate hibernation mass loss. The mechanism by which PUFAs enhance torpor and hibernation is unknown, however. On the basis of a review of the literature, and on reexamining our own data on alpine marmots, we propose that effects on hibernation are not due to PUFAs in general, but to shifts in the ratio of n-6 PUFAs to n-3 PUFAs in membrane phospholipids. Specifically, high ratios of n-6 to n-3 PUFAs increase the activity of the [Ca.sup.2+]-[Mg.sup.2+] pump in the sarcoplasmic reticulum of the heart (SERCA) and counteract [Q.sub.10] effects on SERCA activity at low tissue temperatures. Therefore, high n-6 to n-3 PUFA ratios in cardiac myocyte membranes appear to protect the hibernating heart from arrhythmia, which in hypothermic nonhibernators is caused by massive increases in cytosolic [Ca.sup.2+]. The resulting reduced risk of cardiac arrest during hypothermia may explain why increased dietary uptake of n-6 PUFAs, but not of n-3 PUFAs, can strongly enhance the propensity for hibernation, and allows heterotherms to reach lower body temperatures, with associated increased energy savings. Therefore, at least for herbivorous hibernators, such as marmots, linoleic acid (C18:2 n-6)--the dietary source of all n-6 PUFAs--appears to represent a crucial and limited resource in natural environments. hypothermia; sarcoplasmic reticulum of the heart; heart; [Ca.sup.2+] ATPase