Alanine-scanning Mutagenesis along Membrane Segment 4 of the Yeast Plasma Membrane H+-ATPase EFFECTS ON STRUCTURE AND FUNCTION

Abstract

Membrane segment 4 of P-type cation pumps has been suggested to play a critical role in the coupling of ATP hydrolysis to ion translocation. In this study, structurefunction relationships in M4 of the yeast (Saccharomyces cerevisiae) plasma membrane H ؉ -ATPase have been explored by alanine-scanning mutagenesis. Mutant enzymes were expressed behind an inducible heat-shock promoter in yeast secretory vesicles, as described previously (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940 -7949). One substitution (I329A) led to arrest of the enzyme at an early stage of biogenesis, and three others (G333A, L338A, G349A) reduced ATP hydrolysis to near-background levels. The remaining 26 mutants were expressed well enough in secretory vesicles (44 -121% of wild type) and had sufficient ATPase activity (16 -123% of wild type) to be characterized in detail. When acridine orange fluorescence quenching was used to measure rates of ATP-dependent proton pumping over a range of ATP concentrations, only minor changes were seen. In kinetic studies, however, seven of the mutant enzymes (I331A, I332A, V334A, V336A, V341A, V342A, and M346A) were resistant to vanadate inhibition, and three of them (I332A, V336A, and V341A) also had a decreased K m and increased pH optimum for ATP hydrolysis. Limited trypsinolysis was used to probe the structure of two different Val-336 substitutions, V336A, described above, and V336R, which displayed little or no ATPase activity. Both were cleaved at a relatively normal rate to give a pattern of fragments essentially identical to that seen with the wild-type enzyme. However, while vanadate, ADP, and ATP were able to protect the wild-type and V336A enzymes against trypsinolysis, the V336R ATPase was protected only by ADP and ATP. Taken together, the data suggest that key residues in the M4 segment may help to communicate the E 1 -E 2 conformational change to ion-binding sites in the membrane.The plasma membrane H ϩ

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

“…3. As reported earlier (25), the relationship between the initial rate of acridine orange fluorescence quenching and the rate of hydrolysis was approximately linear in the wild-type control. Significantly, the data points for E703A and E703D fell along the same straight line, consistent with normal coupling between H ϩ pumping and ATP hydrolysis.…”

“…Rather, the increased K i , decreased K m , and altered pH optimum can more reasonably be accounted for by a slowing of the E 1 P-to-E 2 P conformational change; as a result, the ATPase accumulates in E 1 , which has a relatively high affinity for ATP and protons and a relatively low affinity for orthovanadate. This idea is supported by the fact that mutations at three positions in M4 lead to a similar set of kinetic changes (25). ATP-dependent Proton Transport-Given the fact that membrane segment 5 is believed to play a direct role in cation translocation in the Ca 2ϩ -and Na ϩ ,K ϩ -ATPases, it was of particular interest to explore the proton-pumping ability of the yeast M5 mutants.…”

“…One unit is defined as 1 mol of P i /min. c The initial rate of fluorescence quenching (proton transport) was determined as previously described (25). One unit is defined as 1% of total fluorescence quenching/min.…”

Epitope‐Tagged Constructs of the Yeast Plasma‐Membrane H+‐ATPase

“…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.…”

“…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.…”