Reconstitution in Vitro of the V1 Complex from the Yeast Vacuolar Proton-translocating ATPase

130

177

The vacuolar (H ؉ )-ATPases (or V-ATPases) are structurally related to the F 1 F 0 ATP synthases of mitochondria, chloroplasts and bacteria, being composed of a peripheral (V 1 ) and an integral (V 0 ) domain. To further investigate the arrangement of subunits in the V-ATPase complex, covalent cross-linking has been carried out on the V-ATPase from clathrin-coated vesicles using three different cross-linking reagents. Crosslinked products were identified by molecular weight and by Western blot analysis using polyclonal antibodies raised against individual V-ATPase subunits. In the intact V 1 V 0 complex, evidence for cross-linking of subunits C and E, D and F, as well as E and G by disuccinimidyl glutarate was obtained, while in the free V 1 domain, cross-linking of subunits H and E was also observed. Subunits C and E as well as D and E could be cross-linked by 1-ethyl-3-(dimethylaminopropyl)carbodiimide, while subunits a and E could be cross-linked by 4-(N-maleimido)benzophenone. It was further demonstrated that it is possible to treat the V-ATPase with potassium iodide and MgATP in such a way that while subunits A, B, and H are nearly quantitatively removed, significant amounts of subunits C, D, E, and F remain attached to the membrane, suggesting that one or more of these latter subunits are in contact with the V 0 domain. In addition, treatment of the V-ATPase with cystine, which modifies Cys-254 of the catalytic A subunit, results in dissociation of subunit H, suggesting communication between the catalytic nucleotide binding site and subunit H. Finally, the stoichiometry of subunits F, G, and H were determined by quantitative amino acid analysis. Based on these and previous observations, a new structural model of the V-ATPase from clathrincoated vesicles is proposed.The vacuolar (H ϩ )-ATPases (or V-ATPases) 1 are a family of ATP-dependent proton pumps that carry out proton transport across both intracellular membranes and, in some cases, the plasma membrane (1-6). Acidification of intracellular compartments is important for such processes as protein degradation, intracellular protein targeting, and receptor-mediated endocytosis (1-6), while V-ATPases in the plasma membrane function in renal acidification, bone resorption, and tumor metastasis (7-9).The V-ATPase is a heteroligomeric complex of molecular mass approximately 800 kDa composed of at least 13 different subunits arranged into two separate domains (1-6). The peripheral V 1 domain has a molecular mass of about 570 kDa and contains eight different subunits of molecular mass 70 (A), 60 (B), 57 (H), 40 (C), 34 (D), 33 (E), 14 (F), and 16 (G) kDa. The integral V 0 domain has a molecular mass of 260 kDa and contains five different subunits of molecular mass 100 (a), 38 (d), 19 (cЉ), and 17 (c, cЈ) kDa. Functional studies indicate that the V 1 domain is responsible for ATP hydrolysis while the V 0 domain carries out proton transport (1-6). We have previously demonstrated that each V-ATPase complex contains three copies each of the A and B subunits, six co...

Section: Discussionsupporting

confidence: 82%

Subunit Interactions in the Clathrin-coated Vesicle Vacuolar (H+)-ATPase Complex

Vasilyeva

Forgac

1999

130

177

“…Early data obtained from yeast two-and three-hybrid experiments and native subcomplex isolation in subunit deficient mutant strains, as well as co-immunoprecipitation, have helped identify important interactions between the different subunits of yeast V-ATPase (16,38,48,49). In addition, neighboring subunits have been identified by chemical cross-linking experiments (17,19) in native complexes.…”

Section: Discussionmentioning

confidence: 99%

“…Several reports have already indicated that these subunits might interact (16,17,38). However, very few data exist on the isolated subunits.…”

Section: Expression and Purification Of The Recombinant Subunits-mentioning

confidence: 99%

Building the Stator of the Yeast Vacuolar-ATPase

Féthière

Venzke

Diepholz

et al. 2004

The vacuolar (H ؉ )-ATPase (or V-ATPase) is a membrane protein complex that is structurally related to F 1 and F 0 ATP synthases. The V-ATPase is composed of an integral domain (V 0 ) and a peripheral domain (V 1 ) connected by a central stalk and up to three peripheral stalks. The number of peripheral stalks and the proteins that comprise them remain controversial. We have expressed subunits E and G in Escherichia coli as maltose binding protein fusion proteins and detected a specific interaction between these two subunits. This interaction was specific for subunits E and G and was confirmed by co-expression of the subunits from a bicistronic vector. The EG complex was characterized using size exclusion chromatography, cross-linking with short length chemical cross-linkers, circular dichroism spectroscopy, and electron microscopy. The results indicate a tight interaction between subunits E and G and revealed interacting helices in the EG complex with a length of about 220 Å. We propose that the V-ATPase EG complex forms one of the peripheral stators similar to the one formed by the two copies of subunit b in F-ATPase.Vacuolar ATPases (V-ATPases) 1 are ATP-dependent proton pumps that play an important role in the pH homeostasis of various intracellular compartments to allow several cellular processes such as endocytosis and intracellular transport to take place (1-3). They are also present at the plasma membrane of various specialized cells where they function to regulate intracellular pH (4 -7). Thereby, they participate in a variety of physiological and pathophysiological states such as acid-base balance in the kidney, bone resorption by osteoclasts, and metastatic invasion by tumor cells to name a few (reviewed in Refs. 8 and 9).V-ATPases are multisubunit protein complexes composed of 13 polypeptide chains. Electron microscopy images as well as analysis of subunit stoichiometry have revealed some analogy in the overall bilobed structure of V-ATPase and F-ATPase (10, 11). It is now well established that the VATPase segregates into two main functional domains: V 0 , which forms the core of the proton translocating machinery associated with the membrane (subunits accЈcЉd), is functionally homologous to the F 0 domain of the F-ATPases; and V 1 , which contains the catalytic sites (subunits A-H), is functionally homologous to the F 1 domain of F-ATPases. ATP hydrolysis takes place in the A 3 B 3 hexamer of V 1 , and the energy provided by this reaction is transmitted to V 0 via the connecting regions to drive proton translocation. Similarly to the F-type ATPase, a rotational mechanism linking these two functionalities has been proposed (12, 13).Despite these similarities, V-ATPases differ significantly from F-ATPases in some respect. In particular, electron microscopy has revealed significant protrusions in the connecting elements linking V 0 to V 1 (14, 15), indicating a more complex structure for this region. In addition, extensive experimental characterization of these connecting elements using different techni...

“…The E subunit is not the main catalytic subunit of V-ATPase, but it was reported that the E subunit was essential for ATP hydrolysis (4) and might neighbor the A 3 B 3 complex of the main catalytic domain (12). The exact position of the E subunit in the tertiary structure of V-ATPase and the role of the subunit in enzymatic function remain to be clarified.…”

Section: Table I Comparison Of the Compositions Of D And E Subunits Imentioning

confidence: 99%

“…The rotation of the ␥ subunit is thought to be physicochemically coupled with H ϩ translocation in the F 0 domain (membrane domain). In V-ATPase, the homologue of the ␥ subunit has not yet been identified, although some candidates have been recommended, such as the D subunit (11) or a pair of D and E subunits (12). On the other hand, Xie reported that at least the A, B, C, E, and G subunits, but not the D or F subunits, were essential for ATP hydrolysis (4).…”

mentioning

confidence: 99%

Tissue Specificity of E Subunit Isoforms of Plant Vacuolar H+-ATPase and Existence of Isotype Enzymes

Kawamura¹,

Arakawa²,

Maeshima³

et al. 2000

Immunoblot analyses and partial amino acid sequencings revealed that both the 40-(E1) and 37-kDa (E2) subunits of V-ATPase in the pea epicotyl were E subunit isoforms. Similarly, both the 35-(D1) and 29-kDa (D2) subunits were D subunit isoforms, although the similarity of the amino acid sequences is still unknown. In immunoblot analyses, two or three E subunit isoforms with molecular masses ranging from 29 to 40 kDa were detected in other plants. Two isotypes of V-ATPase from the pea epicotyl were separated by ion exchange chromatography and had subunit compositions differing only in the ratio of E1 and E2. There was a difference in the V max and K m of ATP hydrolysis between the two isotypes. E1 was scarcely detected in crude membrane fractions from the leaf and cotyledon, while E2 was detected in fractions from all of the tissues examined. The compositions of D subunit isoforms in the leaf and epicotyl were different, and the vacuolar membrane in the leaf did not contain D2. The efficiency of H ؉ pumping activity in the vacuolar membrane of the leaf was higher than that of the epicotyl. The results suggest that the presence of the isoforms of D and E subunits is characteristic to plants and that the isoforms are closely related to the enzymatic properties.Vacuoles of plant cells play a fundamental role in the regulation of cell turgor and in the transport and storage of ions and metabolites (1). Vacuolar H ϩ -ATPase (V-ATPase), 1 together with H ϩ -pyrophosphatase, generates an electrochemical gradient across the membrane and provides a driving force for secondary active transporters. V-ATPase is composed of two general domains, a peripheral catalytic domain (V 1 ) and a transmembranous proton channel (V 0 ) (2, 3). The V 1 domain of animal and yeast enzymes consists of eight subunits: three copies of A and B subunits and one copy of several other accessory subunits of C to H (4, 5). The V 0 domain consists of five subunits: six copies of the c subunit and one copy of other accessory subunits of a, cЈ, cЉ, and d (5). In the coated vesicle V-ATPase, the stoichiometry was determined to be A 3 B 3 C 1 D 1 E 1 a 1 c 6 (6). The tertiary structure of V-ATPase is similar to that of F-ATPase (7). High resolution analysis of the structure of the mitochondrial F 1 domain (peripheral domain) revealed that the central cavity of the F 1 domain is formed by alternating the ␣ and ␤ subunits (␣ 3 ␤ 3 ), which correspond to the B and A subunits in V-ATPase, respectively, and is occupied with ␥ subunit (8). The ␥ subunit has recently been demonstrated to rotate against the ␣ 3 ␤ 3 complex during ATP hydrolysis (9, 10). The rotation of the ␥ subunit is thought to be physicochemically coupled with H ϩ translocation in the F 0 domain (membrane domain). In V-ATPase, the homologue of the ␥ subunit has not yet been identified, although some candidates have been recommended, such as the D subunit (11) or a pair of D and E subunits (12). On the other hand, Xie reported that at least the A, B, C, E, and G subunits, but not the D or F su...