Cloning, expression and crystallization of VMA13p, an essential subunit of the vacuolar H<sup>+</sup>-ATPase of<i>Saccharomyces cerevisiae</i>

Sagermann, Martin; Matthews, Brian W.

doi:10.1107/s0907444900000950

Cited by 4 publications

(4 citation statements)

References 16 publications

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“…The purified protein was found to fully reconstitute V-ATPase activity when added back to vacuolar membranes isolated from vma13⌬ yeast mutants (unpublished work). Crystals were obtained as described (9) in space group P3 2 21 with one molecule per asymmetric unit.…”

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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.

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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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mentioning

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.

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126

127

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“…Only the 100 kDa VHA-a is encoded by two different isoforms (Vph1 and Stv1) [13]. In general the subunit-isogenes of the V-ATPase have a very high degree of similarity within each species [16,39]. In a converse manner, the sequences of the VHA-a isogenes are very heterogenic in S. cerevisiae and A. thaliana (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Occurrence of FRET between VHA-A/YFP and VHA-B/CFP supports the same structural arrangement in V-ATPase. Following crystalization of isolated yeast VHA-H [16], the structure was fitted in 3D reconstructions of plant V-ATPase based on electron microscopic analysis [43] and suggests localization of the C-terminus of VHA-H to the head structure in proximity to VHA-B. In vivo -FRET in protoplasts expressing VHA-B/CFP and VHA-H-YFP confirms the orientation of the C-termini of VHA-B and H in close vicinity.…”

Section: Discussionmentioning

confidence: 99%

“…Mutant yeast cells devoid of VHA-a or complemented with modified VHA-a variants exhibited the conditional phenotype of V-ATPase deficiency [15]. Another novel subunit in plants that was only addressed recently is VHA-H. VMA13p being the yeast orthologue of VHA-H was cloned and represents the only crystallized subunit of V-ATPase at present [16]. VHA-H is considered to activate and regulate V-ATPase by functionally coupling ATP hydrolysis to proton flow through the V o -domain [17].…”

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confidence: 99%

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Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex

et al. 2004

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Background: Vacuolar H + -ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed.

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2000

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In order to keep subscribers up‐to‐date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly‐published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (3 weeks journals ‐ search completed 21st June 2000)

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Cloning, expression and crystallization of VMA13p, an essential subunit of the vacuolar H⁺-ATPase ofSaccharomyces cerevisiae

Cited by 4 publications

References 16 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

Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex

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Cloning, expression and crystallization of VMA13p, an essential subunit of the vacuolar H+-ATPase ofSaccharomyces cerevisiae

Cited by 4 publications

References 16 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

Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex

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Cloning, expression and crystallization of VMA13p, an essential subunit of the vacuolar H⁺-ATPase ofSaccharomyces cerevisiae