Structure-Function Relationships in Membrane Segment 5 of the Yeast Pma1 H+-ATPase

Dutra, Marcio B; Ambesi, Anthony; Slayman, Carolyn W.

doi:10.1074/jbc.273.28.17411

Cited by 27 publications

(25 citation statements)

References 40 publications

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“…Trypsinolysis-Limited trypsinolysis was performed on isolated secretory vesicles as described previously (30). Vesicles were suspended at 0.5 mg/ml in 20 mM Tris-HCl, pH 7.0, and 5 mM MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%

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: Methodsmentioning

confidence: 99%

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 present study, we focused on the M6 segment of gastric H ϩ ,K ϩ -ATPase ␣-subunit and studied the role of the side chain of each amino acid residue from Cys 815 to Leu 833 in the M6 segment in the partial reactions of H ϩ ,K ϩ -ATPase to determine the affinity for cations. For this purpose, we performed alanine-scanning mutagenesis, which had been used previously to study the structure-function relationships of specific domain(s) of membrane proteins, including ion pumps and ion channels, especially for the binding site of drugs, ligands, and proteins (17)(18)(19)(20)(21) cDNAs of ␣-and ␤-Subunits of H ϩ ,K ϩ -ATPase-cDNAs of the ␣-and ␤-subunits of H ϩ ,K ϩ -ATPase were prepared from rabbit gastric mucosa as described elsewhere (6). The ␣-and ␤-subunit cDNAs were digested with EcoRI and XhoI.…”

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Alanine-scanning Mutagenesis of the Sixth Transmembrane Segment of Gastric H+,K+-ATPase α-Subunit

Asano¹,

Io²,

Kimura³

et al. 2001

Journal of Biological Chemistry

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The sixth transmembrane (M6) segment of the catalytic subunit plays an important role in the ion recognition and transport in the type II P-type ATPase families. In this study, we singly mutated all amino acid residues in the M6 segment of gastric H ؉ ,K ؉ -ATPase ␣-subunit with alanine, expressed the mutants in HEK-293 cells, and studied the effects of the mutation on the functions of H ؉ ,K ؉ -ATPase; overall K ؉ -stimulated ATPase, phosphorylation, and dephosphorylation. Four mutants, L819A, D826A, I827A, and L833A, completely lost the K ؉ -ATPase activity. Mutant L819A was phosphorylated but hardly dephosphorylated in the presence of K ؉ , whereas mutants D826A, I827A, and L833A were not phosphorylated from ATP. We found that almost all of these amino acid residues, which are important for the function, are located on the same side of the ␣-helix of the M6 segment. In addition, we found that amino acids involved in the phosphorylation are located exclusively in the cytoplasmic half of the M6 segment and those involved in the K ؉ -dependent dephosphorylation are in the luminal half. Several mutants such as I821A, L823A, T825A, and P829A partly retained the K ؉ -ATPase activity accompanying the decrease in the rate of phosphorylation.

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“…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%