Phenotypic screening of mutations in Pmr1, the yeast secretory pathway Ca2+/Mn2+-ATPase, reveals residues critical for ion selectivity and transport.
Thirty-five mutations were generated in the yeast secretory pathway/Golgi ion pump, Pmr1, targeting oxygen-containing side chains within the predicted transmembrane segments M4, M5, M6, M7, and M8, likely to be involved in coordination of Ca(2+) and Mn(2+) ions. Mutants were expressed in low copy number in a yeast strain devoid of endogenous Ca(2+) pumps and screened for loss of Ca(2+) and Mn(2+) transport on the basis of hypersensitivity to 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and Mn(2+) toxicity, respectively. Three classes of mutants were found: mutants indistinguishable from wild type (Class 1), mutants indistinguishable from the pmr1 null strain (Class 2), and mutants with differential sensitivity to BAPTA and Mn(2+) toxicity (Class 3). We show that Class 1 mutants retain normal/near normal properties, including (45)Ca transport, Golgi localization, and polypeptide conformation. In contrast, Class 2 mutants lacked any detectable (45)Ca transport; of these, a subset also showed defects in trafficking and protein folding, indicative of structural problems. Two residues identified as Class 2 mutants in this screen, Asn(774) and Asp(778) in M6, also play critical roles in related ion pumps and are therefore likely to be common architectural components of the cation-binding site. Class 3 mutants appear to have altered selectivity for Ca(2+) and Mn(2+) ions, as exemplified by mutant Q783A in M6. These results demonstrate the utility of phenotypic screening in the identification of residues critical for ion transport and selectivity in cation pumps.[1]References
- Phenotypic screening of mutations in Pmr1, the yeast secretory pathway Ca2+/Mn2+-ATPase, reveals residues critical for ion selectivity and transport. Wei, Y., Chen, J., Rosas, G., Tompkins, D.A., Holt, P.A., Rao, R. J. Biol. Chem. (2000) [Pubmed]
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