Manganese selectivity of Pmr1, the yeast secretory pathway ion pump, is defined by residue Gln(783) in transmembrane segment 6 - Residue Asp(778) is essential for cation transport

We have solubilized and purified the histidine-tagged yeast secretory pathw ay/Golgi ion pump Pmr1 to near homogeneity in one step, using nickel affini ty chromatography, The purified pump demonstrates both Ca2+- and Mn2+-depen dent ATP hydrolysis and phosphoenzyme intermediate formation in forward (AT P) and reverse (P-i) directions. This preparation has allowed us to examine , in detail, the properties of mutations D778A and Q783A in transmembrane s egment M6 of Pmr1, In phenotypic screens of Ca2+ chelator and Mn2+ toxicity reported separately (Wei, Y,, Chen, J,, Rosas, G., Tompkins, D.A., Holt, P .A., and Rao, R. (2000) J. Biol. Chem. 275, XXXX-XXXX), D778A was a loss-of -function mutant apparently defective for transport of both Ca2+ and Mn2+, whereas mutant Q783A displayed a differential sensitivity consistent with t he selective loss of Mn2+ transport. We show that mutant D778A is devoid of cation-dependent ATP hydrolytic activity and phosphoenzyme formation from ATP, However, reverse phosphorylation from P-i is preserved but is insensit ive to inhibition by Ca2+ or Mn2+ ions, which is evidence for a specific in ability to bind cations in this mutant. We also show that Ca2+ can activate ATP hydrolysis in the purified Q783A mutant, with a half-maximal concentra tion of 0.06 mu M, essentially identical to that of wild type (0.07 mu M). Mn2+ activation of ATP hydrolysis was half-maximal at 0.02 mu M in wild typ e, establishing a normal selectivity profile of Mn2+ > Ca2+, Strikingly, Mn 2+-ATPase in the Q783A mutant was nearly abolished, even at concentrations of up to 10 mu M. These results were confirmed in assays of phosphoenzyme i ntermediates. Molecular modeling of the packing between helices M4 and M6 s uggests that residue Gln(783) in M6 may form a critical hydrophobic interac tion with Val(335) in M4, such that the Ala substitution modifies the packi ng or tilt of the helices and thus the ion pore. The data emphasize the cri tical role of transmembrane segment M6 in defiing the cation binding pocket of P-type ATPases.