100-kDa Subunit Research Articles

Different roles of two groEL homologues in methylotrophic utiliser of dichloromethane Methylorubrum extorquens DM4.

The genome of methylotrophic bacteria Methylorubrum extorquens DM4 contains two homologous groESL operons encoding the 60-kDa and 10-kDa subunits of GroE heat shock chaperones with highly similar amino acid sequences. To test a possible functional redundancy of corresponding GroEL proteins we attempted to disrupt the groEL1 and groEL2 genes. Despite the large number of recombinants analysed and the gentle culture conditions the groEL1-lacking mutant was not constructed suggesting that the loss of GroEL1 was lethal for cells. At the same time the ∆groEL2 strain was viable and varied from the wild-type by increased sensitivity to acid, salt and desiccation stresses as well as by the impaired growth with a toxic halogenated compound-dichloromethane (DCM). The evaluation of activity of putative PgroE1 and PgroE2 promoters using the reporter gene of green fluorescent protein (GFP) showed that the expression of groESL1 operon greatly prevails (about two orders of magnitude) over those of groESL2 under all tested conditions. However the above promoters demonstrated differential regulation in response to stresses. The expression from PgroE1 was heat-inducible, while the activity of PgroE2 was upregulated upon acid shock and cultivation with DCM. Based on these results we conclude that the highly conservative groESL1 operon (old locus tags METDI5839-5840) encodes the housekeeping chaperone essential for fundamental cellular processes. On the contrary the second pair of paralogues (METDI4129-4130) is dispensable, but corresponding GroE2 chaperone promotes the tolerance to acid and salt stresses, in particular, during the growth with DCM.

Development of endogenous promoters that drive high-level expression of introduced genes in the model diatom Phaeodactylum tricornutum

The marine diatom Phaeodactylum tricornutum is attractive for basic and applied diatom research. We isolated putative endogenous gene promoters derived from genes that are highly expressed in P. tricornutum: the fucoxanthin chlorophyll a/c-binding protein (FCP) C gene, the vacuolar ATP synthase 16-kDa proteolipid subunit (V-ATPase C) gene, the clumping factor A gene and the solute carrier family 34 member 2 gene. Five putative promoter regions were isolated, linked to an antibiotic resistance gene (Sh ble) and transformed into P. tricornutum. Using quantitative RT-PCR, the promoter activities in the transformants were analyzed and compared to that of the diatom endogenous gene promoter, the FCP A gene promoter which has been used for the transformation of P. tricornutum. Among the five isolated potential promoters, the activity of the V-ATPase C gene promoter was approximately 2.73 times higher than that of the FCP A gene promoter. The V-ATPase C gene promoter drove the expression of Sh ble mRNA transcripts under both light and dark conditions at the stationary phase. These results suggest that the V-ATPase C gene promoter is a novel tool for the genetic engineering of P. tricornutum.

Metal Nanoparticles Dispersion By Chaperonin Complexes

Introduction GroEL is the best characterized chaperonin; it is found in the cytoplasm of Escherichia coli and is essential for cell viability and growth. GroEL is a large cylindrical protein complex comprising two heptameric rings of identical 57-kDa subunits, and these rings are stacked back to back. GroES is a single heptameric ring of identical 10-kDa subunits. GroEL encapsulates the substrates to the cavities, GroEL binds a wide variety of substrate proteins in non-native states, and forms a binary complex, which then binds seven ATP molecules with positive cooperativity and GroES to the same (cis) GroEL ring, to form the cis-ternary complex. The binding of GroES induces the encapsulation of the substrate protein into an enlarged cavity (horizonal width of 5-6nm and vertical width of 6-7nm) inside the cis-ring, which is capped by GroES. In the cis-cavity, non-native protein initiates folding without the risk of aggregation. Following hydrolysis of ATP in the cisring, the products of this cycle, ADP and folded substrate protein, are released with GroES, as another similar folding-active assembly is formed in the opposite trans ring. On the other hand, the hydrolysis-defective mutant GroEL(D52/398A) can bind two GroES in the presence of ATP, producing a symmetric 1:2 GroEL-GroES complex (the “football”-shaped complex) with half-time of ~150 h (~6 days). The property of the GroEL(D52/398A) looks beneficial for dispersing aggregative nanoparticles and hydrophobic compounds. FePt, Pt, CdSe/ZnS and Au were used to encapsulate in GroEL(D52/398A) as metal nanoparicles because these nanoparticles are used to engineering manufactures such as semiconductors, devices and batteries. Experiments Pt (average φ2 nm), FePt (φ4 nm), CdSe/ZnS (φ7nm and 6 nm) and Au (φ10 nm, 4 nm and 2 nm) were used to encapsulate in GroEL(D52/398A) as metal nanoparicles. In the case of nanoparticle encapsulation in one side of GroEL(D52/398A) cavities, 0.1-1.0 µM GroEL(D52/398A), 0.2-2.0 µM GroES (2-fold mol of GroEL), and 1 mM ATP were added to the sonicated nanoparticle solution. For two different nanoparticle encapsulation in both sides of GroEL(D52/398A) cavities, GroEL(D52/398A) which is bound to first nanoparticle was added to the same molar of GroES and 5 mM ADP. The bullet-shaped GroEL(D52/398A) encapsulating first nanoparticle was mixed with the second nanoparticle solution for 1 minute, then, GroES and 1 mM ATP were added to form the football-shaped complex. GroEL(D52/398A) complexes were stained with 1% phosphotungstic acid (pH 4.0) for the observation with the transmission electron microscopy JEM 2000EX or JEM 2100 (JEOL Co., Ltd.). For protein crystalizaion, the sample solution of metal nanoparticle encapsulating GroEL(D52/398A) complexes was added to 1% ammonium molybdate/0.2 % PEG1500 with equal amount volume. Then, the mixture was dropped on TEM grid or flesh cleaved mica and air dried. Fixed protein was observed using with TEM or scanning probe microscopy SPM 9600 (Shimazu Corp.). Analysis was performed using with gel filtration HPLC system equipped with a G3000SWXL (TOSOH) and elution buffer which was consisted of 20 mM HEPES/KOH (pH 7.0), 100 mM KCl, 5 mM MgCl2 and 50 mM Na2SO4 at a flow rate of 0.5 ml/min. The eluate was monitored using a UV detector set at the wavelength 280 nm. Fluorescence intensity was detected at an excitation wavelength of 370 nm and emission at 490 nm for CdSe/ZnS λ490 or 575 nm emission wavelength for CdSe/ZnS λ575 was used. Results and discussion From TEM images, the highest FePt nanoparticles encapsulation percentage of GroEL(D52/398A) was about 95% in this experimental condition. Pt, CdSe/ZnS and Au nanoparticles were encapsulated in GroEL(D52/398A) complexes, too. GroEL(D52/398A) wasn’t seemed to select metal nanoparticle. Two CdSe/ZnS nanoparticles encapsulation of GroEL(D52/398A) of analysis indicated FL emission peaks of both wave length at 490 nm and 575 nm around 12 min of retention time which GroEL(D52/398A) elution time with UV detector. This result showed a possibly that GroEL(D52/398A) could encapsulate two particles in each their cavities. In a presence of protein crystallization reagent, metal nanoparticles encapsulating GroEL(D52/398A) complexes showed tendency to fill up with themselves on TEM grid or mica as an experimental flat plane. These experiments are continued to make monolayer aligns of metal nanoparticle encapsulating GroEL(D52/398A) complexes on flat plated of manufactures. These results from a sequential experiments of GroEL(D52/398A) suggest a potential that GroEL(D52/398A) will support to construct nano manufactures.

The involvement of clathrin-mediated endocytosis and two Sid-1-like transmembrane proteins in double-stranded RNA uptake in the Colorado potato beetle midgut.

RNA interference (RNAi) is a powerful tool in entomology and shows promise as a crop protection strategy, but variability in its efficiency across different insect species limits its applicability. For oral uptake of the double-stranded RNA (dsRNA), the RNAi trigger, two different mechanisms are known: systemic RNA interference deficient-1 (Sid-1) transmembrane channel-mediated uptake and clathrin-mediated endocytosis. So far, a wide range of experiments has been conducted, confirming the involvement of one of the pathways in dsRNA uptake, but never both pathways in the same species. We investigated the role of both pathways in dsRNA uptake in the Colorado potato beetle, Leptinotarsa decemlineata, known to have an efficient RNAi response. Through RNAi-of-RNAi experiments, we demonstrated the contribution of two different sid-1-like (sil) genes, silA and silC, and clathrin heavy chain and the 16kDa subunit of the vacuolar H(+) ATPase (vha16), elements of the endocytic pathway, to the RNAi response. Furthermore, the sid-1-like genes were examined through phylogenetic and hydrophobicity analysis. This article reports for the first time on the involvement of two pathways in dsRNA uptake in an insect species and stresses the importance of evaluating both pathways through a well-devised reporter system in any future experiments on cellular dsRNA uptake.

Molybdenum-containing membrane-bound formate dehydrogenase isolated from Citrobacter sp. S-77 having high stability against oxygen, pH, and temperature

Membrane-bound formate dehydrogenase (FDH) was purified to homogeneity from a facultative anaerobic bacterium Citrobacter sp. S-77. The FDH from Citrobacter sp. S-77 (FDHS77) was a monomer with molecular mass of approximately 150kDa. On SDS-PAGE, the purified FDHS77 showed as three different protein bands with molecular mass of approximately 95, 87, and 32kDa, respectively. Based on the N-terminal amino acid sequence analysis, the sequence alignments observed for the 87kDa protein band were identical to that of the large subunit of 95kDa, indicating that the purified FDHS77 consisted of two subunits; a 95kDa large subunit and a 32kDa small subunit. The purified FDHS77 in this purification did not contain a heme b subunit, but the FDHS77 showed significant activity for formate oxidation, determined by the Vmax of 30.4U/mg using benzyl viologen as an electron acceptor. The EPR and ICP-MS spectra indicate that the FDHS77 is a molybdenum-containing enzyme, displaying a remarkable O2-stability along with thermostability and pH resistance. This is the first report of the purification and characterization of a FDH from Citrobacter species.

Top‐down protein identification of proteasome proteins with nanoLC‐FT‐ICR‐MS employing data‐independent fragmentation methods

A comparison of different data-independent fragmentation methods combined with LC coupled to high-resolution FT-ICR-MS/MS is presented for top-down MS of protein mixtures. Proteins composing the 20S and 19S proteasome complexes and their PTMs were identified using a 15 T FT-ICR mass spectrometer. The data-independent fragmentation modes with LC timescales allowed for higher duty-cycle measurements that better suit online LC-FT-ICR-MS. Protein top-down dissociation was effected by funnel-skimmer collisionally activated dissociation (FS-CAD) and CASI (continuous accumulation of selected ions)-CAD. The N-termini for 9 of the 14 20S proteasome proteins were found to be modified, and the α3 protein was found to be phosphorylated; these results are consistent with previous reports. Mass-measurement accuracy with the LC-FT-ICR system for the 20- to 30-kDa 20S proteasome proteins was 1 ppm. The intact mass of the 100-kDa Rpn1 subunit from the 19S proteasome complex regulatory particle was measured with a deviation of 17 ppm. The CASI-CAD technique is a complementary tool for intact-protein fragmentation and is an effective addition to the growing inventory of dissociation methods that are compatible with online protein separation coupled to FT-ICR-MS.

Bacterial expression and functional reconstitution of human heparanase

Human heparanase is a heparan sulfate degrading enzyme located in the extracellular matrix playing a decisive role in angiogenesis and tumor metastasis. Translated as a 65kDa inactive prae-form, the protein is processed into an 8kDa and a 50kDa subunit which form a non-covalently associated active heterodimer. We have expressed the two subunits separately in Escherichia coli which yielded active human heparanase upon reconstitution. The two purified subunits folded independently and secondary structure analysis by far-UV CD spectroscopy gave 33.1/11.1% α/β content for the 50kDa subunit and 6.9/49% α/β content for the 8kDa subunit. This heparanase expression system is easy and can be used for efficient screening for enzyme inhibitors.

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2*

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P2 and other acidic phospholipids in a Ca(2+)-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P2, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P2 clustering are also capable of forming interconnections between PI(4,5)P2-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P2 and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P2-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.

Inhibition of vacuolar H+ ATPase enhances sensitivity to tamoxifen via up-regulation of CHOP in breast cancer cells

Resistance of estrogen receptor-positive breast cancer cells to tamoxifen represents a major barrier to the successful treatment of breast cancer. In the present study, we found that vacuolar H+ ATPase (vATPase) inhibitors, bafilomycin A1 and concanamycin A, sensitize tamoxifen-induced cell death. siRNA targeting ATP6V0C, a 16-kDa hydrophobic proteolipid subunit of vATPase that plays a central role in H+ transport, markedly increased cell death induced by tamoxifen. Interestingly, bafilomycin A1 induced up-regulation of DR4/DR5 and CHOP. Knock-down of CHOP by siRNA suppressed the cell death induced by bafilomycin A1 and tamoxifen, suggesting that bafilomycin A1-mediated CHOP activation sensitizes to tamoxifen. In addition, we found that bafilomycin A1 enhances TRAIL-induced cell death in breast cancer cells. Furthermore, we showed that combination of vATPase inhibitors with tamoxifen also effectively induced cell death in HER2- and ERα-overexpressing breast cancer cells. Overall, our results demonstrate that inhibition of vATPase can potentiate the apoptotic effects of tamoxifen through up-regulation of CHOP.

Altered V-ATPase expression in renal intercalated cells isolated from B1 subunit-deficient mice by fluorescence-activated cell sorting

Unlike human patients with mutations in the 56-kDa B1 subunit isoform of the vacuolar proton-pumping ATPase (V-ATPase), B1-deficient mice (Atp6v1b1(-/-)) do not develop metabolic acidosis under baseline conditions. This is due to the insertion of V-ATPases containing the alternative B2 subunit isoform into the apical membrane of renal medullary collecting duct intercalated cells (ICs). We previously reported that quantitative Western blots (WBs) from whole kidneys showed similar B2 protein levels in Atp6v1b1(-/-) and wild-type mice (Păunescu TG, Russo LM, Da Silva N, Kovacikova J, Mohebbi N, Van Hoek AN, McKee M, Wagner CA, Breton S, Brown D. Am J Physiol Renal Physiol 293: F1915-F1926, 2007). However, WBs from renal medulla (including outer and inner medulla) membrane and cytosol fractions reveal a decrease in the levels of the ubiquitous V-ATPase E1 subunit. To compare V-ATPase expression specifically in ICs from wild-type and Atp6v1b1(-/-) mice, we crossed mice in which EGFP expression is driven by the B1 subunit promoter (EGFP-B1(+/+) mice) with Atp6v1b1(-/-) mice to generate novel EGFP-B1(-/-) mice. We isolated pure IC populations by fluorescence-assisted cell sorting from EGFP-B1(+/+) and EGFP-B1(-/-) mice to compare their V-ATPase subunit protein levels. We report that V-ATPase A, E1, and H subunits are all significantly downregulated in EGFP-B1(-/-) mice, while the B2 protein level is considerably increased in these animals. We conclude that under baseline conditions B2 upregulation compensates for the lack of B1 and is sufficient to maintain basal acid-base homeostasis, even when other V-ATPase subunits are downregulated.

Processing of cellulose synthase (AcsAB) from Gluconacetobacter hansenii 23769

The cellulose synthase protein (AcsAB) is encoded by a single gene in Gluconacetobacter hansenii ATCC 23769. We have examined the processing pattern of this enzyme and the localization of the cleavage products by heterologously expressing the truncated portions of the AcsAB protein and using specific antibodies generated against these regions. We found that the AcsAB protein is processed into three polypeptide subunits of molecular masses 46kDa, 34kDa and 95kDa. The 46kDa polypeptide (AcsAcat) harbors the conserved glycosyltransferase domain and hence contains the catalytic subunit of the enzyme. This polypeptide is localized in the cytoplasmic membrane. The 34kDa polypeptide (AcsAreg) is the regulatory subunit with the cyclic diGMP-binding PilZ domain. This polypeptide is largely cytoplasmic. The 95kDa subunit (AcsB) is of unknown function and contains a predicted signal peptide at its N-terminus. This subunit is localized in the outer membrane. In addition to this, we have also localized the AcsC protein in the outer membrane, confirming its predicted localization based on the OM-signal sequence at its N-terminus.

Osteopetrosis Mutation R444L Causes Endoplasmic Reticulum Retention and Misprocessing of Vacuolar H+-ATPase a3 Subunit

Osteopetrosis is a genetic bone disease characterized by increased bone density and fragility. The R444L missense mutation in the human V-ATPase a3 subunit (TCIRG1) is one of several known mutations in a3 and other proteins that can cause this disease. The autosomal recessive R444L mutation results in a particularly malignant form of infantile osteopetrosis that is lethal in infancy, or early childhood. We have studied this mutation using the pMSCV retroviral vector system to integrate the cDNA construct for green fluorescent protein (GFP)-fused a3(R445L) mutant protein into the RAW 264.7 mouse osteoclast differentiation model. In comparison with wild-type a3, the mutant glycoprotein localized to the ER instead of lysosomes and its oligosaccharide moiety was misprocessed, suggesting inability of the core-glycosylated glycoprotein to traffic to the Golgi. Reduced steady-state expression of the mutant protein, in comparison with wild type, suggested that the former was being degraded, likely through the endoplasmic reticulum-associated degradation pathway. In differentiated osteoclasts, a3(R445L) was found to degrade at an increased rate over the course of osteoclastogenesis. Limited proteolysis studies suggested that the R445L mutation alters mouse a3 protein conformation. Together, these data suggest that Arg-445 plays a role in protein folding, or stability, and that infantile malignant osteopetrosis caused by the R444L mutation in the human V-ATPase a3 subunit is another member of the growing class of protein folding diseases. This may have implications for early-intervention treatment, using protein rescue strategies.

Chaperonin Cofactors, Cpn10 and Cpn20, of Green Algae and Plants Function as Hetero-oligomeric Ring Complexes

The chloroplast chaperonin system of plants and green algae is a curiosity as both the chaperonin cage and its lid are encoded by multiple genes, in contrast to the single genes encoding the two components of the bacterial and mitochondrial systems. In the green alga Chlamydomonas reinhardtii (Cr), three genes encode chaperonin cofactors, with cpn10 encoding a single ∼10-kDa domain and cpn20 and cpn23 encoding tandem cpn10 domains. Here, we characterized the functional interaction of these proteins with the Escherichia coli chaperonin, GroEL, which normally cooperates with GroES, a heptamer of ∼10-kDa subunits. The C. reinhardtii cofactor proteins alone were all unable to assist GroEL-mediated refolding of bacterial ribulose-bisphosphate carboxylase/oxygenase but gained this ability when CrCpn20 and/or CrCpn23 was combined with CrCpn10. Native mass spectrometry indicated the formation of hetero-oligomeric species, consisting of seven ∼10-kDa domains. The cofactor "heptamers" interacted with GroEL and encapsulated substrate protein in a nucleotide-dependent manner. Different hetero-oligomer arrangements, generated by constructing cofactor concatamers, indicated a preferential heptamer configuration for the functional CrCpn10-CrCpn23 complex. Formation of heptamer Cpn10/Cpn20 hetero-oligomers was also observed with the Arabidopsis thaliana (At) cofactors, which functioned with the chloroplast chaperonin, AtCpn60α(7)β(7). It appears that hetero-oligomer formation occurs more generally for chloroplast chaperonin cofactors, perhaps adapting the chaperonin system for the folding of specific client proteins.

Regulatory mechanisms of acetylcholine synthesis and release by T cells

AimsMuscarinic and nicotinic acetylcholine (ACh) receptors are expressed in immune cells. ACh synthesized by choline acetyltransferase (ChAT) and released in T cells binds to these receptors. Furthermore, we have recently demonstrated the involvement of mediatophore, a homooligomer of a 16-kDa proteolipid subunit of vacuolar H+-ATPase, in ACh release from T cells. In this study, we investigated the effects of phorbol 12-myristate 13-acetate (PMA), dibutyryl cAMP (dbcAMP) and FK506, an immunosuppressant calcineurin inhibitor, on lymphocytic cholinergic activity in T cells. Main methodsWe determined the content and release of ACh in human leukemic T cell line MOLT-3 cells using a sensitive and specific radioimmunoassay for ACh. In addition, expression of ChAT mRNA and ChAT activity were investigated using reverse-transcription-polymerase chain reaction and Fonnum method, respectively. Key findingsPhytohemagglutinin (PHA), a T-cell activator, up-regulated ChAT mRNA expression, synthesis and release of ACh. PMA, a protein kinase C (PKC) activator, and dbcAMP, a protein kinase A (PKA) activator, also increased ChAT activity and ACh synthesis by up-regulating ChAT gene expression. FK506 inhibited PHA-induced up-regulation of ChAT mRNA expression, suggesting the involvement of calcineurin-mediated pathways in ChAT gene transcription. SignificanceActivation of PKC and PKA up-regulates ACh synthesis in T cells, and immunological activation triggers ChAT gene transcription through calcineurin-mediated pathways.

Crystal Structure of Archaeal Chromatin Protein Alba2-Double-stranded DNA Complex from Aeropyrum pernix K1

All thermophilic and hyperthermophilic archaea encode homologs of dimeric Alba (Sac10b) proteins that bind cooperatively at high density to DNA. Here, we report the 2.0 Å resolution crystal structure of an Alba2 (Ape10b2)-dsDNA complex from Aeropyrum pernix K1. A rectangular tube-like structure encompassing duplex DNA reveals the positively charged residues in the monomer-monomer interface of each dimer packing on either side of the bound dsDNA in successive minor grooves. The extended hairpin loop connecting strands β3 and β4 undergoes significant conformational changes upon DNA binding to accommodate the other Alba2 dimer during oligomerization. Mutational analysis of key interacting residues confirmed the specificity of Alba2-dsDNA interactions.

Mediatophore regulates acetylcholine release from T cells

Immunological stimulation of T cells by phytohemagglutinin (PHA) enhances the synthesis and release of acetylcholine (ACh), suggesting a role for the lymphocytic cholinergic system in the regulation of immune function. In the present study, we used two human leukemic T cell lines as models to investigate whether mediatophore, a homooligomer of a 16-kDa subunit homologous to the proteolipid subunit c of vacuolar H+-ATPase (V-ATPase), is involved in mediating ACh release from T cells. Immunohistochemical analysis revealed the presence of mediatophore in the cytoplasm and on the plasma membrane of both T cell lines. Mediatophore gene expression was up-regulated by immunological T cell activation by PHA. Transfection of anti-mediatophore small interference RNA down-regulated mediatophore gene expression and significantly reduced ACh release. These results suggest that T cells express mediatophore, which then plays a key role in mediating ACh release, and that mediatophore expression is regulated by immunological stimulation.

Definition of Membrane Topology and Identification of Residues Important for Transport in Subunit a of the Vacuolar ATPase

Subunit a of the vacuolar H(+)-ATPases plays an important role in proton transport. This membrane-integral 100-kDa subunit is thought to form or contribute to proton-conducting hemichannels that allow protons to gain access to and leave buried carboxyl groups on the proteolipid subunits (c, c', and c″) during proton translocation. We previously demonstrated that subunit a contains a large N-terminal cytoplasmic domain followed by a C-terminal domain containing eight transmembrane (TM) helices. TM7 contains a buried arginine residue (Arg-735) that is essential for proton transport and is located on a helical face that interacts with the proteolipid ring. To further define the topology of the C-terminal domain, the accessibility of 30 unique cysteine residues to the membrane-permeant reagent N-ethylmaleimide and the membrane-impermeant reagent polyethyleneglycol maleimide was determined. The results further define the borders of transmembrane segments in subunit a. To identify additional buried polar and charged residues important in proton transport, 25 sites were individually mutated to hydrophobic amino acids, and the effect on proton transport was determined. These and previous results identify a set of residues important for proton transport located on the cytoplasmic half of TM7 and TM8 and the lumenal half of TM3, TM4, and TM7. Based upon these data, we propose a tentative model in which the cytoplasmic hemichannel is located at the interface of TM7 and TM8 of subunit a and the proteolipid ring, whereas the lumenal hemichannel is located within subunit a at the interface of TM3, TM4, and TM7.

Complete 1H, 13C and 15N NMR assignments for donor-strand complemented AafA, the major pilin of aggregative adherence fimbriae (AAF/II) from enteroaggregative E. coli

Aggregative adherence fimbriae (AAF) are the primary adhesive factors of enteroaggregative Escherichia coli (EAEC) and are required for intestinal colonization. They mediate binding to extracellular matrix proteins of the enteric mucosa and display proinflammatory effects on epithelial cells in vitro. Among the simplest of bacterial fimbriae, these passive hairlike appendages are composed primarily of a single 16-kDa structural and adhesive subunit, AafA. Oligomerization occurs by incorporating the N-terminal strand of each AafA subunit into an otherwise incomplete β-sheet of an adjacent AafA subunit. We have engineered a highly soluble AafA monomer by positioning the N-terminal "donor strand" at the C-terminus, following a turn and short linker that were introduced to allow access of the donor strand to the recipient cleft of the same subunit. The resulting "donor-strand complemented" AafA subunit, or AafA-dsc folds autonomously, is monodisperse in solution, and yields high quality NMR spectral data. Here, we report the (1)H, (13)C, and (15)N chemical shift assignments for AafA-dsc.

A Rationale for Osteoclast Selectivity of Inhibiting the Lysosomal V-ATPase a3 Isoform

Osteoclastic bone resorption can be completely abolished by inhibiting the vacuolar H(+)-ATPase (V-ATPase), a proton pump composed of at least 12 different subunits. However, V-ATPases are ubiquitous and it is unclear whether the osteoclast V-ATPase has a unique composition that would allow its selective inhibition. Aiming to answer this question, we compared human osteoclasts and monocytic THP.1 cells with respect to the localization of the a3 isoform of the 116-kDa subunit, which is indispensable for bone resorption, and sensitivity to SB242784, a V-ATPase inhibitor that prevents experimentally induced osteoporosis. By immunofluorescence, a3 was essentially nondetectable in THP.1 cells, while in osteoclasts a3 was highly upregulated and localized to lysosomes in nonresorbing osteoclasts. We isolated the lysosomal compartment from both sources as latex bead-containing phagolysosomes and compared them. Osteoclast phagolysosomes and THP.1 phagolysosomes both contained a3 and a1; however, the a3/a1 ratio was 3.8- to 11.2-fold higher in osteoclast phagolysosomes. Importantly, the V-ATPase-dependent acidification of phagolysosomes from both sources was essentially equally sensitive to SB242784. Thus, we observed no indication of a qualitative uniqueness of the osteoclast V-ATPase; rather, the high a3-level in osteoclasts may represent an upregulation of the common lysosomal V-ATPase. Our results, together with the reported phenotype of a3 deficiency and the reported efficacy of SB242784 in vivo, suggest that V-ATPase structure-independent mechanisms render bone resorption more sensitive than lysosomal function to V-ATPase inhibition. One such mechanism may be compensation of a3 by a1, which may be sufficient for retaining lysosomal function but not bone resorption.

V-ATPase Membrane Sector Associates with Synaptobrevin to Modulate Neurotransmitter Release

Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.