Evidence That the NH2 Terminus of Vph1p, an Integral Subunit of the V0 Sector of the Yeast V-ATPase, Interacts Directly with the Vma1p and Vma13p Subunits of the V1Sector

Landolt-Marticorena, Carolina; Williams, Kimberly; Correa, Judy; Chen, Weimin; Manolson, Morris F.

doi:10.1074/jbc.m000207200

Cited by 96 publications

(84 citation statements)

References 67 publications

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“…It remains to be determined whether this is the result of steric interference at the interface between subunits A and H or a transmitted structural change. Landolt-Marticorena et al (33) also demonstrated that subunit H forms a direct interaction with subunit a of the V 0 domain in vitro and in vivo.…”

Section: Structural Comparisons With Other Proteinsmentioning

confidence: 99%

Crystal structure of the regulatory subunit H of the V-type ATPase of Saccharomyces cerevisiae

Sagermann

Stevens²,

Matthews³

2001

Proc. Natl. Acad. Sci. U.S.A.

126

127

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In contrast to the F-type ATPases, which use a proton gradient to generate ATP, the V-type enzymes use ATP to actively transport protons into organelles and extracellular compartments. We describe here the structure of the H-subunit (also called Vma13p) of the yeast enzyme. This is the first structure of any component of a V-type ATPase. The H-subunit is not required for assembly but plays an essential regulatory role. Despite the lack of any apparent sequence homology the structure contains five motifs similar to the so-called HEAT or armadillo repeats seen in the importins. A groove, which is occupied in the importins by the peptide that targets proteins for import into the nucleus, is occupied here by the 10 amino-terminal residues of subunit H itself. The structural similarity suggests how subunit H may interact with the ATPase itself or with other proteins. A cleft between the amino-and carboxyl-terminal domains also suggests another possible site of interaction with other factors.T he vacuolar proton-translocating ATPases play an important role in the acidification of extracelluar compartments and organelles and are found in most eukaryotic cells. In contrast to the F-type ATPases, which generate ATP from a proton gradient across a membrane, the V-type enzymes use ATP to acidify compartments for receptor-mediated endocytosis, intracellular trafficking, and protein degradation (1, 2). The enzyme is composed of two functionally distinct complexes V 1 and V 0 (Fig. 1). V 0 is integral to the membrane and is thought to consist of at least five different subunits with a total molecular mass of about 260 kDa. This complex is involved in translocation of protons across the membrane. The V 1 complex is more hydrophilic and is composed of at least eight different subunits totaling a molecular mass of about 570 kDa. Because of the relatively high sequence identity between the catalytic subunits ␣ and ␤ of the F-type ATPases and subunits B and A of the V-type ATPases, the V 1 complex is thought to use ATP to drive the proton translocation across the membrane. The rotary catalytic mechanism of ATP hydrolysis and proton transportation (3, 4) is thought to be very similar among the two classes of enzymes, but the different subunit stoichiometry and architectural appearance in electron micrographs suggests that the V-type ATPases are more complex (5, 6).Because the V-type enzymes are involved in key biochemical processes, such as the regulation of pH, and also because they consume rather than generate energy, their function is likely to be under tight control. Previous studies have revealed that most components of the V-type ATPase are essential for assembly of the enzyme complex (1). Characterization of subunit H, also known as Vma13p, however, indicates that this polypeptide is essential for the activity of the enzyme but not for targeting or assembly (7). The regulatory function of subunit H recently was demonstrated by Parra et al. (8). Binding of the subunit to isolated, cytosolic V 1 particles inhibits CaATP hydro...

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Section: Structural Comparisons With Other Proteinsmentioning

confidence: 99%

Crystal structure of the regulatory subunit H of the V-type ATPase of Saccharomyces cerevisiae

Sagermann

Stevens²,

Matthews³

2001

Proc. Natl. Acad. Sci. U.S.A.

126

127

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“…Crosslinking, two-hybrid screening, coimmunoprecipitation, and electron microscopy showed that the a-subunit isoforms directly interact with A-, E-, H-, C-, and G-subunits of the V 1 sector [9][10][11][12][13] as well as with c-and d-subunits of the V o sector. 2,[14][15][16] Recently, it has also been shown that the V-ATPase a-subunit directly assembles with proteins that are not part of the V-ATPase complex including: (i) aldolase B, 17,18 (ii) phosphofrucktokinase-1, 19,20 (iii) calmodulin, 21 and (iv) t-SNARES syntaxin and SNAP-25.…”

Section: Introductionmentioning

confidence: 99%

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

et al. 2010

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V-ATPase is a multisubunit membrane complex that functions as nanomotor coupling ATP hydrolysis with proton translocation across biological membranes. Recently, we uncovered details of the mechanism of interaction between the N-terminal tail of the V-ATPase a2-subunit isoform (a2N ) and ARNO, a GTP/GDP exchange factor for Arf-family small GTPases. Here, we describe the development of two methods for preparation of the a2N 1-402 recombinant protein in milligram quantities sufficient for further biochemical, biophysical, and structural studies. We found two alternative amphiphilic chemicals that were required for protein stability and solubility during purification: (i) non-detergent sulfobetaine NDSB-256 and (ii) zwitterionic detergent FOS-CHOLINEV R 12 (FC-12). Moreover, the other factors including mild alkaline pH, the presence of reducing agents and the absence of salt were beneficial for stabilization and solubilization of the protein. A preparation of a2N 1-402 in NDSB-256 was successfully used in pull-down and BIAcore TM protein-protein interaction experiments with ARNO, whereas the purity and quality of the second preparation in FC-12 was validated by size-exclusion chromatography and CD spectroscopy. Surprisingly, the detergent requirement for stabilization and solubilization of a2N 1-402 and its cosedimentation with liposomes were different from peripheral domains of other transmembrane proteins. Thus, our data suggest that in contrast to current models, so called ''cytosolic'' tail of the a2-subunit might actually be embedded into and/or closely associated with membrane phospholipids even in the absence of any obvious predicted transmembrane segments. We propose that a2N 1-402 should be categorized as an integral monotopic domain of the a2-subunit isoform of the V-ATPase.

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“…The amino-terminal domain of the a subunit has been shown previously to interact with both subunit A and subunit H of the V 1 domain (64) and has been suggested to form part of the peripheral stator connecting V 1 and V 0 (64,65). Based upon these results, it might have been predicted that the aminoterminal domain would play a more important role in controlling interactions between the V 1 and V 0 domains, including assembly, coupling, and regulation of dissociation.…”

Section: In Vivo Dissociation Of the V-atpase In Response To Glucose mentioning

confidence: 99%

The Amino-terminal Domain of the Vacuolar Proton-translocating ATPase a Subunit Controls Targeting and in Vivo Dissociation, and the Carboxyl-terminal Domain Affects Coupling of Proton Transport and ATP Hydrolysis

Kawasaki-Nishi¹,

Bowers²,

Nishi³

et al. 2001

Journal of Biological Chemistry

185

182

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The 100-kDa "a" subunit of the vacuolar proton-translocating ATPase (V-ATPase) is encoded by two genes in yeast, VPH1 and STV1. The Vph1p-containing complex localizes to the vacuole, whereas the Stv1p-containing complex resides in some other intracellular compartment, suggesting that the a subunit contains information necessary for the correct targeting of the V-ATPase. We show that Stv1p localizes to a late Golgi compartment at steady state and cycles continuously via a prevacuolar endosome back to the Golgi. V-ATPase complexes containing Vph1p and Stv1p also differ in their assembly properties, coupling of proton transport to ATP hydrolysis, and dissociation in response to glucose depletion. To identify the regions of the a subunit that specify these different properties, chimeras were constructed containing the cytosolic amino-terminal domain of one isoform and the integral membrane, carboxyl-terminal domain from the other isoform. Like the Stv1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Stv1p localized to the Golgi and the complex did not dissociate in response to glucose depletion. Like the Vph1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Vph1p localized to the vacuole and the complex exhibited normal dissociation upon glucose withdrawal. Interestingly, the V-ATPase complex containing the chimera with the carboxyl-terminal domain of Vph1p exhibited a higher coupling of proton transport to ATP hydrolysis than the chimera containing the carboxylterminal domain of Stv1p. Our results suggest that whereas targeting and in vivo dissociation are controlled by sequences located in the amino-terminal domains of the subunit a isoforms, coupling efficiency is controlled by the carboxyl-terminal region.The V-ATPases 1 are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells (1-8). Acidification of these compartments is crucial for such processes as receptor-mediated endocytosis, intracellular trafficking, the processing and degradation of macromolecules, and the coupled transport of small molecules. In addition, V-ATPases in the plasma membrane of specialized cells function in such processes as pH homeostasis (9), bone resorption (10), renal acidification (11), potassium transport (12), and tumor metastasis (13). In yeast, the VATPase functions to create the driving force for uptake of small molecules and ions into the vacuole (14) and is important for post-Golgi protein trafficking (15-17).The V-ATPase complex is composed of the following two domains: a soluble V 1 domain responsible for ATP hydrolysis and an integral V 0 domain responsible for proton translocation (1-8). The V 1 domain is a 500-kDa complex composed of eight different subunits (subunits A-H) of molecular masses 70 to 14 kDa, whereas the V 0 domain is a 250-kDa complex containing five different subunits (subunits a, d, c, cЈ, and cЉ) of molecular masses 100 to 16 kDa (1-8). The ...

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Evidence That the NH2 Terminus of Vph1p, an Integral Subunit of the V0 Sector of the Yeast V-ATPase, Interacts Directly with the Vma1p and Vma13p Subunits of the V1Sector

Cited by 96 publications

References 67 publications

Crystal structure of the regulatory subunit H of the V-type ATPase of Saccharomyces cerevisiae

Crystal structure of the regulatory subunit H of the V-type ATPase of Saccharomyces cerevisiae

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

The Amino-terminal Domain of the Vacuolar Proton-translocating ATPase a Subunit Controls Targeting and in Vivo Dissociation, and the Carboxyl-terminal Domain Affects Coupling of Proton Transport and ATP Hydrolysis

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