Structure, function and regulation of the coated vesicle V-ATPase.

1992 ◽  
Vol 172 (1) ◽  
pp. 155-169
Author(s):  
M Forgac
Keyword(s):  
Molecular Mass ◽  
Critical Role ◽  
Coated Vesicle ◽  
Relative Molecular Mass ◽  
Macromolecular Complex ◽  
Subunit Structure ◽  
Chemical Labeling ◽  
Nucleotide Binding Sites ◽  
Vacuolar Acidification ◽  
A Subunit

The coated vesicle V-ATPase plays an important role in both receptor-mediated endocytosis and intracellular membrane traffic by providing the acidic environment required for ligand-receptor dissociation and receptor recycling. The coated vesicle V-ATPase is a macromolecular complex of relative molecular mass 750,000 composed of nine subunits arranged in two structural domains. The peripheral V1 domain, which has a relative molecular mass of 500,000, has the subunit structure 73(3)58(3)40(1)34(1)33(1) and possesses all the nucleotide binding sites of the V-ATPase. The integral Vo domain of relative molecular mass 250,000 has a subunit composition of 100(1)38(1)19(1)17(6) and possesses the pathway for proton conduction across the membrane. Reassembly studies have allowed us to probe the role of specific subunits in the V-ATPase complex while chemical labeling studies have allowed us to identify specific residues which play a critical role in catalysis. From both structural analysis and sequence homology, the vacuolar-type H(+)-ATPases resemble the F-type H(+)-ATPases. Unlike the F1 and Fo domains of the F-type ATPases, however, the V1 and Vo domains do not appear to function independently. The possible relevance of these observations to the regulation of vacuolar acidification is discussed.

scholarly journals The V-ATPase a3 Subunit: Structure, Function and Therapeutic Potential of an Essential Biomolecule in Osteoclastic Bone Resorption

2021 ◽  
Vol 22 (13) ◽  
pp. 6934
Author(s):  
Anh Chu ◽  
Ralph A. Zirngibl ◽  
Morris F. Manolson
Keyword(s):  
Bone Resorption ◽  
Proton Translocation ◽  
Critical Role ◽  
Lipid Binding ◽  
Bone Diseases ◽  
Osteoclastic Bone Resorption ◽  
Subunit Structure ◽  
A Subunit ◽  
A3 Subunit

This review focuses on one of the 16 proteins composing the V-ATPase complex responsible for resorbing bone: the a3 subunit. The rationale for focusing on this biomolecule is that mutations in this one protein account for over 50% of osteopetrosis cases, highlighting its critical role in bone physiology. Despite its essential role in bone remodeling and its involvement in bone diseases, little is known about the way in which this subunit is targeted and regulated within osteoclasts. To this end, this review is broadened to include the three other mammalian paralogues (a1, a2 and a4) and the two yeast orthologs (Vph1p and Stv1p). By examining the literature on all of the paralogues/orthologs of the V-ATPase a subunit, we hope to provide insight into the molecular mechanisms and future research directions specific to a3. This review starts with an overview on bone, highlighting the role of V-ATPases in osteoclastic bone resorption. We then cover V-ATPases in other location/functions, highlighting the roles which the four mammalian a subunit paralogues might play in differential targeting and/or regulation. We review the ways in which the energy of ATP hydrolysis is converted into proton translocation, and go in depth into the diverse role of the a subunit, not only in proton translocation but also in lipid binding, cell signaling and human diseases. Finally, the therapeutic implication of targeting a3 specifically for bone diseases and cancer is discussed, with concluding remarks on future directions.

Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H(+)-ATPases

2000 ◽  
Vol 203 (1) ◽  
pp. 71-80 ◽  
Author(s):  
M. Forgac
Keyword(s):  
Molecular Mass ◽  
Proton Transport ◽  
Atp Hydrolysis ◽  
Transport Processes ◽  
Site Directed Mutagenesis ◽  
Eukaryotic Cells ◽  
Coated Vesicle ◽  
Proton Pumps ◽  
Reversible Dissociation ◽  
Vacuolar Acidification

The vacuolar H(+)-ATPases (or V-ATPases) are a family of ATP-dependent proton pumps that carry out acidification of intracellular compartments in eukaryotic cells. This review is focused on our work on the V-ATPases of clathrin-coated vesicles and yeast vacuoles. The coated-vesicle V-ATPase undergoes trafficking to endosomes and synaptic vesicles, where it functions in receptor recycling and neurotransmitter uptake, respectively. The yeast V-ATPase functions to acidify the central vacuole and is necessary both for protein degradation and for coupled transport processes across the vacuolar membrane. The V-ATPases are multisubunit complexes composed of two functional domains. The V(1) domain is a 570 kDa peripheral complex composed of eight subunits of molecular mass 73–14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V(o) domain is a 260 kDa integral complex composed of five subunits of molecular mass 100-17 kDa (subunits a, d, c, c' and c”) that is responsible for proton translocation. To explore the function of individual subunits in the V-ATPase complex as well as to identify residues important in proton transport and ATP hydrolysis, we have employed a combination of chemical modification, site-directed mutagenesis and in vitro reassembly. A central question concerns the mechanism by which vacuolar acidification is controlled in eukaryotic cells. We have proposed that disulfide bond formation between conserved cysteine residues at the catalytic site of the V-ATPase plays an important role in regulating V-ATPase activity in vivo. Other regulatory mechanisms that are discussed include reversible dissociation and reassembly of the V-ATPase complex, changes in the tightness of coupling between proton transport and ATP hydrolysis, differential targeting of V-ATPases within the cell and control of the Cl(−) conductance that is necessary for vacuolar acidification.

Extraction of polyethylene glycols with dicarbolide solutions in nitrobenzene

1990 ◽  
Vol 55 (8) ◽  
pp. 1959-1967 ◽  
Author(s):  
Petr Vaňura ◽  
Pavel Selucký
Keyword(s):  
Polyethylene Glycol ◽  
Molecular Mass ◽  
Extraction Constant ◽  
Metal Extraction ◽  
Polyethylene Glycols ◽  
Relative Molecular Mass ◽  
Published Data ◽  
Average Molecular Mass ◽  
Peg 400 ◽  
Extraction Constants

The extraction of polyethylene glycol of average molecular mass 400 (PEG 400) with dicarbolide solution in nitrobenzene and of longer-chain polyethylene glycol, of average molecular mass 1 500 (PEG 1 500), with chlorinated dicarbolide solution in nitrobenzene was studied. During the extraction of PEG 400, the polyethylene glycol solvates the Horg+ ion in the organic phase giving rise to the HLorg+ species (L is polyethylene glycol). The obtained value of the extraction constant Kex(HLorg+) = 933 is consistent with published data of metal extraction. Extraction of PEG 1 500 was treated applying the simplified assumption that the thermodynamic behaviour of PEG 1 500 is the same as that of n molecules of polyethylene glycol with relative molecular mass 1 500/n, each solvating one cation. For this model, the value of n = 3.2 ± 1.1 and the values of the extraction constants of the HL1/n,org+ and HL2/n,org+ species were obtained by using the adapted program LETAGROP. This value of n is consistent with published extraction data in the presence of polyethylene glycol with a relative molecular mass from 200 to 1 000.

scholarly journals Hph1 and Hph2 Are Novel Components of the Sec63/Sec62 Posttranslational Translocation Complex That Aid in Vacuolar Proton ATPase Biogenesis

2010 ◽  
Vol 10 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Francisco J. Piña ◽  
Allyson F. O'Donnell ◽  
Silvere Pagant ◽  
Hai Lan Piao ◽  
John P. Miller ◽  
...  
Keyword(s):  
Environmental Stress ◽  
Complex Function ◽  
Content Type ◽  
Growth Defects ◽  
Proton Atpase ◽  
Vacuolar Acidification ◽  
Atpase Subunits ◽  
Specific Proteins ◽  
A Subunit ◽  
Split Ubiquitin

ABSTRACT Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1 Δ hph2 Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity. sec71 Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1 Δ hph2 Δ and hph1 Δ hph2 Δ sec71 Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1 Δ hph2 Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.

Towards a molecular description of cell transformation by avian sarcoma virus

1980 ◽  
Vol 210 (1180) ◽  
pp. 387-396 ◽  
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Molecular Mass ◽  
Cellular Protein ◽  
Transformed Cells ◽  
Relative Molecular Mass ◽  
Protein Kinase Activity ◽  
Avian Sarcoma Virus ◽  
Sarcoma Virus ◽  
Avian Sarcoma

The avian sarcoma virus transforming gene product has been identified and partially purified from extracts of transformed cells. It is a phosphoprotein with a relative molecular mass of 60 000 (pp60 src ) with two major sites of phosphorylation. pp60 src appears to be a cyclic-AMP-independent protein kinase as judged by protein phosphorylation with partly purified fractions. The specificity of the phosphorylation observed was judged by inhibition with anti-pp60 src IgG but not by normal IgG and by the fact that the protein kinase activity isolated from ts transformation-mutant infected cells was more thermolabile than that from wild-type transformed cells, thus showing more directly the origin of the enzymic activity. A cellular protein substrate of pp60 src has been identified as a 34000 molecular mass protein. These data together suggest that protein phosphorylation by pp60 src may be a function of the molecule that plays a major role in transformation.

Enzymatic deglycosylation of porcine thyroid peroxidase: effects on catalytic activity and immunoreactivity

1991 ◽  
Vol 124 (1) ◽  
pp. 107-114 ◽  
Author(s):  
Egberto G. Moura ◽  
Carmen C. Pazos-Moura ◽  
Naokata Yokoyama ◽  
Martha L. Dorris ◽  
Alvin Taurog
Keyword(s):  
Catalytic Activity ◽  
Molecular Mass ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Minor Role ◽  
Relative Molecular Mass ◽  
Thyroid Peroxidase ◽  
Autoimmune Thyroid ◽  
Tryptic Fragment ◽  
A Minor

Abstract Thyroid peroxidase is a heme-containing, membrane-bound, glycoprotein enzyme that catalyzes iodination and coupling in the thyroid gland. It is also the antigen for microsomal autoantibodies that are commonly found in the serum of patients with autoimmune thyroid disease. We examined the effect of deglycosylation on the catalytic functions and the immunoreactivity of this enzyme. A highly purified, solubilized, large tryptic fragment of porcine thyroid peroxidase, retaining all of the N-linked glycosylation sites of the native enzyme and displaying full catalytic activity was used. It was deglycosylated by treatment with N-glycanase under nondenaturing conditions. The loss in relative molecular mass after treatment, determined by gel electrophoresis, was about 75% of the estimated molecular weight of the glycan portion of porcine thyroid peroxidase. Lectin blots performed with horseradish peroxidase-conjugated concanavalin A showed a similar loss in relative molecular mass but some residual carbohydrate. The intensity of the carbohydrate stain was consistent with the loss of about 75% of the glycans. Despite this loss, three different assays for catalytic activity of porcine thyroid peroxidase were not significantly decreased. Immunoreactivity measured by immunoblotting and by enzyme-linked immunosorbent assay was also unimpaired. These findings suggest that N-glycanase-sensitive glycans in porcine thyroid peroxidase do not act as antigenic determinants and play a minor role, if any, in catalytic activity and, presumably therefore, in the maintenance of protein conformation.

Receptors for epidermal growth factor and insulin-like growth factor-I on preimplantation trophoderm of the pig

Development ◽  
1990 ◽  
Vol 110 (1) ◽  
pp. 221-227
Author(s):  
A.N. Corps ◽  
D.R. Brigstock ◽  
C.J. Littlewood ◽  
K.D. Brown
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Molecular Mass ◽  
Cross Linking ◽  
Relative Molecular Mass ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Igf I ◽  
Epidermal Growth ◽  
Growth Factor I

125I-labelled epidermal growth factor (125I-EGF) and 125I-labelled insulin-like growth factor-I (125I-IGF-I) bound to trophoderm cells from pig blastocysts obtained on days 15–19 of pregnancy. Specific binding was detected on freshly isolated cell suspensions and on cells cultured for several days. The binding of 125I-EGF was inhibited by increasing concentrations of EGF, but not by various other growth factors and hormones. Chemical cross-linking of 125I-EGF to its receptors using disuccinimidyl suberate (DSS) revealed a radiolabelled band of relative molecular mass 160,000, similar to that identified as the EGF receptor in other cell types. The binding of 125I-IGF-I was inhibited by both IGF-I and insulin, indicating that the receptors were either type I IGF receptors or insulin receptors. Cross-linking of 125I-IGF-I to serum-free supernatants from trophoderm cultures showed that the cells secreted an IGF-binding protein, giving a complex of relative molecular mass about 45,000. The presence of receptors for EGF and IGF/insulin suggests that these factors could be involved in regulating the growth and development of the early blastocyst.

scholarly journals Physical and kinetic characterization of recombinant human cholesteryl ester transfer protein

1996 ◽  
Vol 320 (1) ◽  
pp. 39-47 ◽  
Author(s):  
Daniel T CONNOLLY ◽  
Jonathan McINTYRE ◽  
Deborah HEUVELMAN ◽  
Edward E REMSEN ◽  
Russell E McKINNIE ◽  
...  
Keyword(s):  
Cholesteryl Ester ◽  
Molecular Mass ◽  
Cholesteryl Ester Transfer Protein ◽  
Mammalian Cells ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Relative Molecular Mass ◽  
Transfer Protein ◽  
Ldl Particles ◽  
Carrier Mechanism

Cholesteryl ester transfer protein (CETP) mediates the exchange of triglycerides (TGs), cholesteryl esters (CEs) and phospholipids (PLs) between lipoproteins in the plasma. In order to better understand the lipid transfer process, we have used recombinant human CETP expressed in cultured mammalian cells, purified to homogeneity by immunoaffinity chromatography. Purified recombinant CETP had a weight-average relative molecular mass (Mw) of 69561, determined by sedimentation equilibrium, and a specific absorption coefficient of 0.83 litre·g-1· cm-1. The corresponding hydrodynamic diameter (Dh) of the protein, determined by dynamic light scattering, was 14 nm, which is nearly twice the expected value for a spheroidal protein of this molecular mass. These data suggest that CETP has a non-spheroidal shape in solution. The secondary structure of CETP was estimated by CD to contain 32% α-helix, 35% β-sheet, 17% turn and 16% random coil. Like the natural protein from plasma, the recombinant protein consisted of several glycoforms that could be only partially deglycosylated using N-glycosidase F. Organic extraction of CETP followed by TLC showed that CE, unesterified cholesterol (UC), PL, TG and fatty acids (FA) were associated with the pure protein. Quantitative analyses verified that each mol of CETP contained 1.0 mol of cholesterol, 0.5 mol of TG and 1.3 mol of PL. CETP mediated the transfer of CE, TG, PL, and UC between lipoproteins, or between protein-free liposomes. In dual-label transfer experiments, the transfer rates for CE or TG from HDL to LDL were found to be proportional to the initial concentrations of the respective ligands in the donor HDL particles. Kinetic analysis of CE transfer was consistent with a carrier mechanism, having a Km of 700 nM for LDL particles and of 2000 nM for HDL particles, and a kcat of 2 s-1. The Km values were thus in the low range of the normal physiological concentration for each substrate. The carrier mechanism was verified independently for CE, TG, PL and UC in ‘half-reaction’ experiments.

scholarly journals Isolation and characterization of PEP5, a gene essential for vacuolar biogenesis in Saccharomyces cerevisiae.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 739-752 ◽  
Author(s):  
C A Woolford ◽  
C K Dixon ◽  
M F Manolson ◽  
R Wright ◽  
E W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mass ◽  
Open Reading Frame ◽  
Relative Molecular Mass ◽  
Cell Fractionation ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Genomic Dna Library

Abstract pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation. Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment. DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D. Deletion/disruption of the PEP5 gene did not kill the cells. The resulting strains grow very slowly at 37 degrees. The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase. Apparently normal precursors forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen. Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells. Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein.

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