Manganese selectivity of Pmr1, the yeast secretory pathway ion pump, is defined by residue Gln783 in transmembrane segment 6: Residue Asp778 is essential for cation transport

Debjani Mandal, Thomas B Woolf, Rajini Rao

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Abstract

We have solubilized and purified the histidine-tagged yeast secretory pathway/Golgi ion pump Pmr1 to near homogeneity in one step, using nickel affinity chromatography. The purified pump demonstrates both Ca2+-and Mn2+-dependent ATP hydrolysis and phosphoenzyme intermediate formation in forward (ATP) and reverse (P(i)) directions. This preparation has allowed us to examine, in detail, the properties of mutations D778A and Q783A in transmembrane segment 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 the 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 insensitive to inhibition by Ca2+ or Mn2+ ions, which is evidence for a specific inability to bind cations in this mutant. We also show that Ca2+ can activate ATP hydrolysis in the purified Q783A mutant, with a half-maximal concentration of 0.06 μM, essentially identical to that of wild type (0.07 μM). Mn2+ activation of ATP hydrolysis was half-maximal at 0.02 μM in wild type, establishing a normal selectivity profile of Mn2+ > Ca2+. Strikingly, Mn2+-ATPase in the Q783A mutant was nearly abolished, even at concentrations of up to 10 μM. These results were confirmed in assays of phosphoenzyme intermediates. Molecular modeling of the packing between helices M4 and M6 suggests that residue Gln783 in M6 may form a critical hydrophobic interaction with Val335 in M4, such that the Ala substitution modifies the packing or tilt of the helices and thus the ion pore. The data emphasize the critical role of transmembrane segment M6 in defining the cation binding pocket of P-type ATPases.

Original languageEnglish (US)
Pages (from-to)23933-23938
Number of pages6
JournalJournal of Biological Chemistry
Volume275
Issue number31
DOIs
StatePublished - Aug 4 2000

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Ion Pumps
Secretory Pathway
Manganese
Yeast
Cations
Adenosine Triphosphate
Yeasts
Hydrolysis
Adenosine Triphosphatases
Ions
Affinity chromatography
Phosphorylation
Molecular modeling
Chelating Agents
Nickel
Hydrophobic and Hydrophilic Interactions
Affinity Chromatography
Histidine
Toxicity
Assays

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{21d05866664846d4bbd6889921e49b84,
title = "Manganese selectivity of Pmr1, the yeast secretory pathway ion pump, is defined by residue Gln783 in transmembrane segment 6: Residue Asp778 is essential for cation transport",
abstract = "We have solubilized and purified the histidine-tagged yeast secretory pathway/Golgi ion pump Pmr1 to near homogeneity in one step, using nickel affinity chromatography. The purified pump demonstrates both Ca2+-and Mn2+-dependent ATP hydrolysis and phosphoenzyme intermediate formation in forward (ATP) and reverse (P(i)) directions. This preparation has allowed us to examine, in detail, the properties of mutations D778A and Q783A in transmembrane segment 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 the 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 insensitive to inhibition by Ca2+ or Mn2+ ions, which is evidence for a specific inability to bind cations in this mutant. We also show that Ca2+ can activate ATP hydrolysis in the purified Q783A mutant, with a half-maximal concentration of 0.06 μM, essentially identical to that of wild type (0.07 μM). Mn2+ activation of ATP hydrolysis was half-maximal at 0.02 μM in wild type, establishing a normal selectivity profile of Mn2+ > Ca2+. Strikingly, Mn2+-ATPase in the Q783A mutant was nearly abolished, even at concentrations of up to 10 μM. These results were confirmed in assays of phosphoenzyme intermediates. Molecular modeling of the packing between helices M4 and M6 suggests that residue Gln783 in M6 may form a critical hydrophobic interaction with Val335 in M4, such that the Ala substitution modifies the packing or tilt of the helices and thus the ion pore. The data emphasize the critical role of transmembrane segment M6 in defining the cation binding pocket of P-type ATPases.",
author = "Debjani Mandal and Woolf, {Thomas B} and Rajini Rao",
year = "2000",
month = "8",
day = "4",
doi = "10.1074/jbc.M002619200",
language = "English (US)",
volume = "275",
pages = "23933--23938",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
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TY - JOUR

T1 - Manganese selectivity of Pmr1, the yeast secretory pathway ion pump, is defined by residue Gln783 in transmembrane segment 6

T2 - Residue Asp778 is essential for cation transport

AU - Mandal, Debjani

AU - Woolf, Thomas B

AU - Rao, Rajini

PY - 2000/8/4

Y1 - 2000/8/4

N2 - We have solubilized and purified the histidine-tagged yeast secretory pathway/Golgi ion pump Pmr1 to near homogeneity in one step, using nickel affinity chromatography. The purified pump demonstrates both Ca2+-and Mn2+-dependent ATP hydrolysis and phosphoenzyme intermediate formation in forward (ATP) and reverse (P(i)) directions. This preparation has allowed us to examine, in detail, the properties of mutations D778A and Q783A in transmembrane segment 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 the 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 insensitive to inhibition by Ca2+ or Mn2+ ions, which is evidence for a specific inability to bind cations in this mutant. We also show that Ca2+ can activate ATP hydrolysis in the purified Q783A mutant, with a half-maximal concentration of 0.06 μM, essentially identical to that of wild type (0.07 μM). Mn2+ activation of ATP hydrolysis was half-maximal at 0.02 μM in wild type, establishing a normal selectivity profile of Mn2+ > Ca2+. Strikingly, Mn2+-ATPase in the Q783A mutant was nearly abolished, even at concentrations of up to 10 μM. These results were confirmed in assays of phosphoenzyme intermediates. Molecular modeling of the packing between helices M4 and M6 suggests that residue Gln783 in M6 may form a critical hydrophobic interaction with Val335 in M4, such that the Ala substitution modifies the packing or tilt of the helices and thus the ion pore. The data emphasize the critical role of transmembrane segment M6 in defining the cation binding pocket of P-type ATPases.

AB - We have solubilized and purified the histidine-tagged yeast secretory pathway/Golgi ion pump Pmr1 to near homogeneity in one step, using nickel affinity chromatography. The purified pump demonstrates both Ca2+-and Mn2+-dependent ATP hydrolysis and phosphoenzyme intermediate formation in forward (ATP) and reverse (P(i)) directions. This preparation has allowed us to examine, in detail, the properties of mutations D778A and Q783A in transmembrane segment 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 the 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 insensitive to inhibition by Ca2+ or Mn2+ ions, which is evidence for a specific inability to bind cations in this mutant. We also show that Ca2+ can activate ATP hydrolysis in the purified Q783A mutant, with a half-maximal concentration of 0.06 μM, essentially identical to that of wild type (0.07 μM). Mn2+ activation of ATP hydrolysis was half-maximal at 0.02 μM in wild type, establishing a normal selectivity profile of Mn2+ > Ca2+. Strikingly, Mn2+-ATPase in the Q783A mutant was nearly abolished, even at concentrations of up to 10 μM. These results were confirmed in assays of phosphoenzyme intermediates. Molecular modeling of the packing between helices M4 and M6 suggests that residue Gln783 in M6 may form a critical hydrophobic interaction with Val335 in M4, such that the Ala substitution modifies the packing or tilt of the helices and thus the ion pore. The data emphasize the critical role of transmembrane segment M6 in defining the cation binding pocket of P-type ATPases.

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