Genetic Probing of the First and Second Transmembrane Helices of the Plasma Membrane H+-ATPase from Saccharomyces cerevisiae

Seto-Young, Donna; Hall, Michael J.; Na, Songqing; Haber, James E.; Perlin, David S.

doi:10.1074/jbc.271.1.581

Cited by 23 publications

(20 citation statements)

References 51 publications

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“…While similar mutations have been described previously for the yeast ATPase (29,30,32,(35)(36)(37), they have been scattered throughout the 100-kDa polypeptide, with no discernible structural or functional pattern (Fig. 5).…”

Section: Discussionmentioning

confidence: 94%

Stalk Segment 4 of the Yeast Plasma Membrane H+-ATPase

Ambesi

Miranda

Allen

et al. 2000

Journal of Biological Chemistry

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In the P 2 -type ATPases, there is growing evidence that four ␣-helical stalk segments connect the cytoplasmic part of the molecule, responsible for ATP binding and hydrolysis, to the membrane-embedded part that mediates cation transport. The present study has focused on stalk segment 4, which displays a significant degree of sequence conservation among P 2 -ATPases. When sitedirected mutants in this region of the yeast plasma membrane H ؉ -ATPase were constructed and expressed in secretory vesicles, more than half of the amino acid substitutions led to a severalfold decrease in the rate of ATP hydrolysis, although they had little or no effect on the coupling between hydrolysis and transport. Strikingly, mutant ATPases bearing single substitutions of 13 consecutive residues from Ile-359 through Gly-371 were highly resistant to inorganic orthovanadate, with IC 50 values at least 10-fold above those seen in the wild-type enzyme. Most of the same mutants also displayed a significant reduction in the K m for MgATP and an increase in the pH optimum for ATP hydrolysis. Taken together, these changes in kinetic behavior point to a shift in equilibrium from the E 2 conformation of the ATPase toward the E 1 conformation. The residues from Ile-359 through Gly-371 would occupy three full turns of an ␣-helix, suggesting that this portion of stalk segment 4 may provide a conformationally active link between catalytic sites in the cytoplasm and cation-binding sites in the membrane. P-type ATPases, which are found throughout prokaryotic and eukaryotic cells, use the energy from ATP hydrolysis to pump inorganic cations across cell membranes. In the most abundant subfamily, designated P 2 , the 100-kDa ATPase polypeptide is embedded in the lipid bilayer by four hydrophobic segments at the N-terminal end of the molecule and six at the C-terminal end (1). The central hydrophilic region protrudes into the cytoplasm and contains catalytic sites for ATP binding and formation of the characteristic ␤-aspartyl phosphate intermediate.It was proposed more than a decade ago that conformational changes in the catalytic portion of the P-type ATPases are transmitted into the membrane via a stalk-like region made up of the cytoplasmic extensions from some, but not necessarily all, of the transmembrane segments (2). Recently, the stalk has been directly visualized in cryoelectron microscopic studies of the sarcoplasmic reticulum Ca 2ϩ -ATPase (3) and the plasma membrane H ϩ

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Section: Discussionmentioning

confidence: 94%

Stalk Segment 4 of the Yeast Plasma Membrane H+-ATPase

Ambesi

Miranda

Allen

et al. 2000

Journal of Biological Chemistry

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“…Proton Pumping by Reconstituted Vesicles-Plasma membranes were isolated as described previously (23), and the H ϩ -ATPase was reconstituted into asolectin vesicles by a modification of the procedure of SetoYoung et al (24). Membranes (175 g) were resuspended (on ice) in 400 l of extraction buffer (10 mM Hepes-Tris, pH 7.0, 0.1 M KCl, 45% glycerol, 0.2 mM EDTA, 1 mM dithiothreitol) containing 10 mg/ml asolectin and 1 mg/ml phosphatidylserine.…”

Section: Methodsmentioning

confidence: 99%

“…The pelleted vesicles were resuspended in 200 l of cold dilution buffer. Proton transport measurements were made as described previously (24).…”

Section: Methodsmentioning

confidence: 99%

Genetic Probing of the Stalk Segments Associated with M2 and M3 of the Plasma Membrane H+-ATPase fromSaccharomyces cerevisiae

Soteropoulos¹,

Perlin²

1998

Journal of Biological Chemistry

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The stalk region of the H؉ -ATPase from Saccharomyces cerevisiae has been proposed to play a role in coupling ATP hydrolysis to proton transport. Genetic probing was used to examine the role of stalk segments S2 and S3, associated with M2 and M3, respectively. Saturation mutagenesis was used to explore the role of side group character at position Ile 183 in S2, at which an alanine substitution was shown previously to partially uncouple the enzyme (Wang, G., Tamas, M. J., Hall, M. J., Pascual-Ahuir, A., and Perlin, D. S. (1996) J. Biol. Chem. 271, 25438 -25445). Diverse side group substitutions were tolerated at this position, although three substitutions, I183N, I183R, and I183Y required second site mutations at the C terminus of the enzyme for stabilization. Substitution of glycine and proline at Ile 183 resulted in lethal phenotypes, suggesting that the backbone may be more important than side group at this position. Proline/glycine mutagenesis was used to study additional sites in S2 and S3. The substitution of proline at Gly 186 resulted in a lethal phenotype, whereas substitutions in S3 of proline or serine at Gly 270 and proline or glycine at Thr 287 resulted in viable mutants. Mutations G270P and T287P resulted in mutant enzymes that produced pronounced growth defects and ATP hydrolysis rates that were 35% and 60% lower than wild type enzyme, respectively. The mutant enzymes transported protons at rates consistent with their ATPase activity, suggesting that the growth defects observed were due to a reduced rate of ATP hydrolysis and not to uncoupling of proton transport. The prominent growth phenotypes produced by mutations G270P and T287P permitted the isolation of suppressor mutations, which restored wild type growth. Most of the suppressors either replaced the primary site mutation with alanine or restored the wild type residue by ectopic recombination with PMA2, both of which restore ␣-helical tendency. This study suggests that maintaining ␣-helical character is essential to S2 and may play an important role in S3.The plasma membrane H ϩ -ATPase from Saccharomyces cerevisiae is an electrogenic proton pump that is essential to cell survival. It plays a critical role in fungal cell physiology by helping to regulate intracellular pH and by generating the large electrochemical proton gradient necessary for nutrient uptake (1). The enzyme is a member of the P-type family of ion translocating ATPases that includes diverse members from plants, animals, and bacteria (2, 3). P-type ATPases generically consist of a membrane transport domain with 10 transmembrane segments, a large cytoplasmic ATP hydrolysis domain, and a narrow stalk domain that links the two larger domains. These enzymes couple ATP hydrolysis in the cytoplasmic domain to ion transport in the membrane-embedded domain forming an acylphosphate intermediate during catalysis (4, 5). The yeast H ϩ -ATPases couples energy with one proton transported per ATP hydrolyzed (6). There is ample evidence that dynamic changes in protein structure occur during ...

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“…Thus, the tagged molecules can be used with assurance in studies of folding, membrane insertion, oligomeric state, and subcellular localization (e.g., 5,6,9). Beyond their practical utility, the ndings add useful knowledge about the role of the N terminus of the ATPase.…”

Section: Glucose Regulation Of the Ha-and Myc-tagged Atpasesmentioning

confidence: 99%

“…3, 4). Recently, mutations have been identi ed that disrupt the folding of the ATPase and cause the polypeptide to be retained in endoplasmic reticulumlike intracellular membranes, rather than traveling to the cell surface (5)(6)(7)(8)(9). Signi cantly, the same mutations also block the biogenesis of coexpressed wild-type ATPase, and they behave genetically as dominant lethals.…”

Section: Introductionmentioning

confidence: 99%