Functional role of aspartyl and glutamyl residues in the membrane segments of the yeast PMA1 ATPase: Interaction with DCCD

Padmanabha, K.; Pardo, Juan Pablo; Петров, В. А.; Gupta, Soma Sen; Slayman, Carolyn W.

doi:10.1007/bf02818996

Cited by 5 publications

(4 citation statements)

References 14 publications

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“…In a subsequent study, the site of inhibition was provisionally identified as Glu-129 in M1, based on labeling with [ 14 C]DCCD (51). To follow up these results, we have recently examined the sensitivity of the yeast PMA1 H ϩ -ATPase to DCCD, taking advantage of the above-described mutants in which Asp and Glu residues throughout the transmembrane segments were substituted one at a time by neutral amino acids (52). The pattern of DCCD sensitivity proved to be quite complex in the mutants, with fractional reductions in rate constants in E129Q and E129L, D143N, and E703Q and E703L.…”

Section: Mechanism Of Proton Transport: Role Of Charged Residues In Mmentioning

confidence: 99%

Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase

Петров¹,

Padmanabha

Nakamoto³

et al. 2000

Journal of Biological Chemistry

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As defined by hydropathy analysis, the membranespanning segments of the yeast plasma membrane H E703Q, E703L, E703D) showed significantly reduced pumping over a wide range of hydrolysis values and thus appeared to be partially uncoupled. At Glu-803, by contrast, one mutant (E803N) was almost completely uncoupled, while another (E803Q) pumped protons at an enhanced rate relative to the rate of ATP hydrolysis. Both Glu-703 and Glu-803 occupy positions at which amino acid substitutions have been shown to affect transport by mammalian P-ATPases. Taken together, the results provide growing evidence that residues in membrane segments 5 and 8 of the P-ATPases contribute to the cation transport pathway and that the fundamental mechanism of transport has been conserved throughout the group.

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Section: Mechanism Of Proton Transport: Role Of Charged Residues In Mmentioning

confidence: 99%

Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase

Петров¹,

Padmanabha

Nakamoto³

et al. 2000

Journal of Biological Chemistry

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“…Based on recent evidence from site-directed mutagenesis, M5 and M6 are also structurally and functionally important in the Pma1 H ϩ -ATPase of S. cerevisiae. Several residues in both membrane segments are required for normal biogenesis of the ATPase, and others play a role in the conformational changes that accompany the reaction cycle (17,18). Asp-730, located near the middle of M6, appears to be especially critical, since replacement by Asn or Val leads to a complete failure of newly synthesized ATPase to reach the secretory vesicles responsible for delivering it to the plasma membrane (18).…”

mentioning

confidence: 99%

“…Several residues in both membrane segments are required for normal biogenesis of the ATPase, and others play a role in the conformational changes that accompany the reaction cycle (17,18). Asp-730, located near the middle of M6, appears to be especially critical, since replacement by Asn or Val leads to a complete failure of newly synthesized ATPase to reach the secretory vesicles responsible for delivering it to the plasma membrane (18). To investigate this finding in greater detail, we have examined the effect of the D730N and D730V mutations on the folding and subcellular localization of the ATPase, and have gone on to search for compensatory mutations in M5.…”

mentioning

confidence: 99%

Evidence for a Salt Bridge between Transmembrane Segments 5 and 6 of the Yeast Plasma-membrane H+-ATPase

Gupta

DeWitt

Allen

et al. 1998

Journal of Biological Chemistry

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The plasma-membrane H+-ATPase of Saccharomyces cerevisiae, which belongs to the P2 subgroup of cation-transporting ATPases, is encoded by the PMA1 gene and functions physiologically to pump protons out of the cell. This study has focused on hydrophobic transmembrane segments M5 and M6 of the H+-ATPase. In particular, a conserved aspartate residue near the middle of M6 has been found to play a critical role in the structure and biogenesis of the ATPase. Site-directed mutants in which Asp-730 was replaced by an uncharged residue (Asn or Val) were abnormally sensitive to trypsin, consistent with the idea that the proteins were poorly folded, and immunofluorescence confocal microscopy showed them to be arrested in the endoplasmic reticulum. Similar defects are known to occur when either Arg-695 or His-701 in M5 is replaced by a neutral residue (Dutra, M. B., Ambesi, A., and Slayman, C. W. (1998) J. Biol. Chem. 273, 17411-17417). To search for possible charge-charge interactions between Asp-730 and Arg-695 or His-701, double mutants were constructed in which positively and negatively charged residues were swapped or eliminated. Strikingly, two of the double mutants (R695D/D730R and R695A/D730A) regained the capacity for normal biogenesis and displayed near-normal rates of ATP hydrolysis and ATP-dependent H+ pumping. These results demonstrate that neither Arg-695 nor Asp-730 is required for enzymatic activity or proton transport, but suggest that there is a salt bridge between the two residues, linking M5 and M6 of the 100-kDa polypeptide.

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“…In our earlier study, conservative substitution D730E was reasonably expressed in secretory vesicles (48% of the wild type level). This mutant retained low ATP hydrolyzing activity (15%) but undetectable H + transport [23, 51]. Although other mutations at Asp-730 in the S. cerevisiae H + -ATPase led to unfolding of the enzyme and retention in the endoplasmic reticulum, the essential role of Asp-730 in H + -binding and transport is obvious and comes from several lines of evidence including: (i) expressed in yeast, the A. thaliana H + -ATPase mutants D684N, D684A, and D684R reached the plasma membrane and retained the ability to hydrolyze ATP, however these mutant ATPases were unable to pump protons suggesting a defect in the transition from E1P to E2P [52–53]; (ii) Asp-730 in M6 is strictly conserved in all plant, yeast and protozoa H + -ATPases; in the mammalian Ca 2+ -ATPases (Asp800 in SERCA1), Na + , K + -ATPases (Asp808, in the pig enzyme) and the H + , K + -ATPase (Asp824 in the human enzyme); (iii) similarly, in the yeast Pmr1 Ca 2+ , Mn 2+ -ATPase corresponding residue (Asp-778) is responsible for cation transport [54]; and finally, (iv) based on the high resolution crystal structure for the SERCA1 ATPase in the E1 conformation, Asp-800 clearly participates in binding of Ca 2+ [5].…”

Section: Discussionmentioning

confidence: 99%

Structure–function relationships in membrane segment 6 of the yeast plasma membrane Pma1 H+-ATPase

Miranda

Pardo

Петров

2011

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The crystal structures of the Ca2+- and H+-ATPases shed light into the membrane embedded domains involved in binding and ion-translocation. Consistent with site-directed mutagenesis, these structures provided additional evidence that membrane-spanning segments M4, M5, M6 and M8 are the core through which cations are pumped. In the present study, we have used alanine/serine scanning mutagenesis to study the structure-function relationships within M6 (Leu-721-Pro-742) of the yeast plasma membrane ATPase. Of the 22 mutants expressed and analyzed in secretory vesicles, alanine substitutions at two well conserved residues (Asp-730 and Asp-739) led to a complete block in biogenesis; in the mammalian P-ATPases, residues corresponding to Asp-730 are part of the cation-binding domain. Two other mutants (V723A and I736A) displayed a dramatic 20 fold increase in the IC50 for inorganic orthovanadate compared to the wild-type control, accompanied by a significant reduction in the Km for Mg-ATP, and an alkaline shift in the pH optimum for ATP hydrolysis. This behavior is apparently due to a shift in equilibrium from the E2 conformation of the ATPase towards the E1 conformation. By contrast, the most striking mutants lying toward the extracellular side in a helical structure (L721A, I722A, F724A, I725A, I727A and F728A), were expressed in secretory vesicles but had a severe reduction of ATPase activity. Moreover, all of these mutants but one (F728A) were unable to support yeast growth when the wild-type chromosomal PMA1 gene was replaced by the mutant allele. Surprisingly, in contrast to M8 where mutations S800A and E803Q (Guerra, G. et al., Biochim. Biophys. Acta 1768: 2383–2392, 2007) led to a dramatic increase in the apparent stoichiometry of H+ transport, three substitutions (A726S, A732S and T733A) in M6 showed a reduction in the apparent coupling ratio. Taken together, these results suggest that M6 residues play an important role in protein stability and function, and probably are responsible for cation binding and stoichiometry of ion transport as suggested by homology modeling.

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Functional role of aspartyl and glutamyl residues in the membrane segments of the yeast PMA1 ATPase: Interaction with DCCD

Cited by 5 publications

References 14 publications

Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase

Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase

Evidence for a Salt Bridge between Transmembrane Segments 5 and 6 of the Yeast Plasma-membrane H+-ATPase

Structure–function relationships in membrane segment 6 of the yeast plasma membrane Pma1 H+-ATPase

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