Phenotypic screening of mutations in Pmr1, the yeast secretory pathway Ca2+/Mn2+-ATPase, reveals residues critical for ion selectivity and transport

Ying Wei, Jun Chen, Gisele Rosas, D. Andrew Tompkins, P. Andrew Holt, Rajini Rao

Research output: Contribution to journalArticle

Abstract

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 Ca2+ and Mn2+ ions. Mutants were expressed in low copy number in a yeast strain devoid of endogenous Ca2+ pumps and screened for loss of Ca2+ and Mn2+ transport on the basis of hypersensitivity to 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and Mn2+ 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 Mn2+ toxicity (Class 3). We show that Class 1 mutants retain normal/near normal properties, including 45Ca transport, Golgi localization, and polypeptide conformation. In contrast, Class 2 mutants lacked any detectable 45Ca transport; of these, a subset also showed defects in traf-ticking and protein folding, indicative of structural problems. Two residues identified as Class 2 mutants in this screen, Asn774 and Asp778 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 Ca2+ and Mn2+ 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.

Original languageEnglish (US)
Pages (from-to)23927-23932
Number of pages6
JournalJournal of Biological Chemistry
Volume275
Issue number31
DOIs
StatePublished - Aug 4 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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