Building the Stator of the Yeast Vacuolar-ATPase

To determine which subunit C domain binds EG with high affinity, we have generated C head and C foot and characterized their interaction with subunit EG heterodimer. Our findings indicate that the high affinity site for EGC interaction is C head . In addition, we provide evidence that the EGC head interaction greatly stabilizes EG heterodimer.2 is a ubiquitous multisubunit enzyme that couples the free energy of ATP hydrolysis to active proton transport. In all eukaryotic cells, V-ATPase function sets up an electrochemical potential and acidifies intracellular compartments (1-5). These functions make the V-ATPase a crucial enzyme involved in intracellular traffic, vesicular transport, endo/exocytosis, and pH homeostasis. In the specialized cells of higher eukaryotes, plasma membrane-associated V-ATPases serve to acidify the extracellular space. The malfunction of the V-ATPase in these cells has been linked to a range of diseases including osteoporosis, diabetes, renal tubular acidosis, and cancer (6 -9). As shown in EM images and reconstructions, the V-ATPase is bilobular in overall structure, typical of the rotary ATPases (10 -13). These two lobes are composed of distinct functional domains, a soluble catalytic sector, V 1 , and a membrane integral proton pore, V o . The V 1 sector contains subunits A 3 B 3 (C)DE 3 FG 3 H, and the V o is made of ac 3-4 cЈcЉde. Most V-ATPase subunits have functional and structural counterparts in the A-and F-type motors; however, only the nucleotide-binding and proteolipid subunits share significant sequence identity. Much like in the F-ATP synthase, the AB subunits come together in an alternating arrangement to form a catalytic hexamer with nucleotide-binding sites located at the AB interfaces (14, 15). ATP hydrolysis-driven rotation of a central rotor domain, composed of subunits DFd and a ring of the proteolipid subunits, c 3-4 cЈcЉ, is coupled to proton translocation along the interface of the proteolipid ring and the C-terminal domain of the a subunit. During catalysis, three peripheral stalks (each formed by a subunit EG heterodimer) in conjunction with subunits C and H and the N-terminal domain of subunit a (a NT ), resist the torque of rotation, thus keeping the A 3 B 3 hexamer static and allowing for rotation to be productive.Unlike F-ATPase, the activity of eukaryotic V-ATPase is regulated by a unique mechanism of reversible dissociation, a process first described for the enzymes from yeast and insect (16, 17) but more recently also found in cells of higher animals (18 -20). In yeast under conditions of nutrient deprivation, the V 1 sector dissociates from the V o sector, and both are functionally silenced (21,22). Although dissociated, a single V 1 subunit, C, is released into the cytosol and is reincorporated into the enzyme upon nutrient readdition (16). Because the C subunit has been proposed to be a molecular switch for controlled enzyme dissociation, the interactions between the C subunit and its intraenzyme binding partners are of particular interest.The crystal ...

Section: Resultsmentioning

confidence: 99%

Domain Characterization and Interaction of the Yeast Vacuolar ATPase Subunit C with the Peripheral Stator Stalk Subunits E and G

Oot

Wilkens

2010

“…Conclusion-Given the available evidence from electron microscopy (8 -10, 19), chemical cross-linking (12,13), and protein biochemistry (11,14), it is likely that the E and G subunits, as a heterodimer, bind at the periphery of the three B subunits to form the peripheral stalks or stators of the V-ATPase enzyme. Furthermore, it has been shown that the E and G subunits connect the V 1 -ATPase domain to the membrane-bound domain of the a subunit as well as the V-ATPasespecific stalk subunits C and H (12, 13, 40 -43).…”

Section: Analysis Of the E And G Subunit Copy Number Usingmentioning

confidence: 99%

“…There is evidence that these elongated proteins densities are formed by the E and G subunits. First, it has been shown that these two subunits are able to form an elongated, heterodimeric complex with equimolar stoichiometry (11), and second, chemical cross-linking from cysteines on the surface of the B subunits indicates close proximity to both E and G subunits from residues distributed between the bottom of the V 1 and the very top (12,13). Recently, Kane and co-workers (14) have shown that there are at least two E and two G subunits per V 1 -ATPase complex.…”

mentioning

confidence: 99%

Stoichiometry of the Peripheral Stalk Subunits E and G of Yeast V1-ATPase Determined by Mass Spectrometry

Kitagawa

Mazon

Heck

et al. 2008

The stoichiometry of yeast V 1 -ATPase peripheral stalk subunits E and G was determined by two independent approaches using mass spectrometry (MS). First, the subunit ratio was inferred from measuring the molecular mass of the intact V 1 -ATPase complex and each of the individual protein components, using native electrospray ionization-MS. The major observed intact complex had a mass of 593,600 Da, with minor components displaying masses of 553,550 and 428,300 Da, respectively. Second, defined amounts of V 1 -ATPase purified from yeast grown on 14 N-containing medium were titrated with defined amounts of 15 N-labeled E and G subunits as internal standards. Following protease digestion of subunit bands, 14 Nand 15 N-containing peptide pairs were used for quantification of subunit stoichiometry using matrix-assisted laser desorption/ ionization-time of flight MS. Results from both approaches are in excellent agreement and reveal that the subunit composition of yeast V 1 -ATPase is A 3 B 3 DE 3 FG 3 H.Vacuolar ATPases (V-ATPases, 3 V 1 V 0 -ATPases) are ATP hydrolysis-driven proton pumps found in the endomembrane system of eukaryotic organisms, where they function to acidify the interior of subcellular organelles such as lysosomes, early and late endosomes, clathrin-coated vesicles, the Golgi, the plant tonoplast, and the yeast vacuole (1-4). In higher organisms, the V-ATPase complex can also be found in the plasma membrane of polarized cells involved in acid secretion such as the ruffled membrane of bone osteoclasts or the apical membrane of renal intercalated cells. The vacuolar ATPase is a large, multisubunit complex, which can be divided into a water-soluble ATPase domain and a membrane-bound proton pore. The two domains are termed V 1 and V 0 , respectively, in analogy to the F 1 and F 0 of the related F 1 F 0 -ATP synthase. In yeast, the V 1 -ATPase domain contains subunits AB(C)DEFGH, whereas the membrane-bound V 0 is made of subunits accЈcЉde. Much like the F-ATP synthase, the V-ATPase is a rotary molecular motor enzyme (5, 6); ATP hydrolysis taking place on the A subunits of the A 3 B 3 catalytic domain is coupled to proton translocation across the membrane domain via rotation of a central stalk made of subunits D, F, and d and a proteolipid ring (subunits c, cЈ, and cЉ). The remaining subunits C, E, G, and H are involved in forming a peripheral stator domain that provides a structural link between the catalytic domain (A 3 B 3 ) and the membrane-bound a subunit. In the related F-ATP synthase, it is now well established that there is a single peripheral stalk, which, in the case of the bacterial enzyme, is formed by two copies of the membrane-anchored b subunits and the ␦ subunit (7). The situation in the vacuolar ATPase, however, is more complicated in that there appear to be multiple peripheral stalks that connect the catalytic domain to the membranebound a subunit, possibly via the V-ATPase-specific H and C subunits. Using electron microscopy and single particle image analysis, we have previously shown ...

“…Instability of subunit E-S78A could have occurred if the mutation disrupted interactions between subunits E and G. Early association between subunits E and G is strong and essential for biosynthetic assembly of V 1 V o and V 1 complexes in yeast (58), and in the absence of subunit G (vma10⌬ cells), subunit E is degraded (71). Dimer formation between subunits E and G has been shown (33), and it may be equivalent to the b-b dimer of the F 1 F 0 from Escherichia coli (72), where each individual subunit is essential for assembly of F 1 (73) and has a specialized function within the peripheral stalk (74). Functional parallels may exist between Ala-79 of the b dimer of E. coli and the residues Tyr-46 and Ser-78 of the yeast V-ATPase subunits G and E, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…By making contact with all components of the peripheral stalk(s), subunit E (and possibly G) may represent the foundation for the peripheral stalk(s) (17,28,29). Subunits E and G assemble into E-G dimers (33), and two E-G dimers, associated each with a B subunit, could represent the two peripheral masses detected by two-dimensional electron microscopy of the yeast V 1 complex (34).…”

Section: Vacuolar Hmentioning

confidence: 99%

Mutational Analysis of the Stator Subunit E of the Yeast V-ATPase

Owegi

Carenbauer

Wick

et al. 2005

Subunit E is a component of the peripheral stalk(s) that couples membrane and peripheral subunits of the V-ATPase complex. In order to elucidate the function of subunit E, site-directed mutations were performed at the amino terminus and carboxyl terminus. Except for S78A and D233A/T202A, which exhibited V 1 V o assembly defects, the function of subunit E was resistant to mutations. Most mutations complemented the growth phenotype of vma4⌬ mutants, including T6A and D233A, which only had 25% of the wild-type ATPase activity. Residues Ser-78 and Thr-202 were essential for V 1 V o assembly and function. The mutation S78A destabilized subunit E and prevented assembly of V 1 subunits at the membranes. Mutant T202A membranes exhibited 2-fold increased V max and about 2-fold less of V 1 V o assembly; the mutation increased the specific activity of Vacuolar Hϩ -ATPase (V-ATPase) 1 proton pumps are present on vacuoles, lysosomes, endosomes, secretory vesicles, and Golgi of all eukaryotic cells where they maintain the acidic pH required for the multiple cellular processes achieved in these organelles (1-3). In kidneys, osteoclasts, neutrophils, and other specialized cells (3), V-ATPases are located also on the plasma membrane, where ATP-driven proton translocation to the extracellular space supports urine acidification, bone resorption, and cytosolic pH regulation among other processes.Amino acid sequence conservation and heterologous genetic complementation of V-ATPase subunits from mammals and plants in yeast (5-11) have shown that V-ATPases are structurally and functionally highly conserved pumps. The yeast V-ATPase complex consists of 14 different subunits organized into two domains, V 1 and V o (1-3). ATP hydrolysis is catalyzed in V 1 , which is peripherally bound to the cytosolic side of the membrane and consists of subunits A-H. Integral to the membrane is V o , which forms the proton transporting domain and consists of subunits a, c, cЈ, cЉ, d, and e (2, 3, 12). Connecting V 1 to V o are one central stalk made of subunits D and F and one to three peripheral stalks consisting of subunits C, E, G, H, and the amino terminus domain of the V o subunit a (13-17).V-ATPases operate by a rotary mechanism of proton transport (18, 19) similar to that of the F-ATPases (20, 21), and both molecular motors share functional homolog subunits involved in rotation and catalysis (22). Similar to subunits ␤ and ␣ of the mitochondrial F-ATPase enzyme, subunits A and B form a hexamer where ATP binds and is hydrolyzed by the V 1 domain. Subunit D is functionally equivalent to ␥ of F 1 and constitutes the rotating central stalk tightly associated with the proteolipid rotor in V o . Comparable with subunits a and c from F 0 , the ring of proteolipid subunits (c, cЈ, and cЉ) and the V o subunit a form the path for proton transport across the membrane. Despite an overall structural similarity, there are important differences that distinguish V-ATPases from F-ATPases. One difference is the possibility of two or three peripheral stalks per V...