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Originally published In Press as doi:10.1074/jbc.M002618200 on May 5, 2000

J. Biol. Chem., Vol. 275, Issue 31, 23927-23932, August 4, 2000
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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, and Rajini RaoDagger

From the Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore Maryland 21205

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 trafficking 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.

* This work was supported by American Cancer Society Grants IRG11-33 and JFRA 538, American Heart Association Grant-in-aid 95012290, and National Institutes of Health Grant GM52414 (to R. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Physiology, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore MD 21205. Tel.: 410-955-4732; Fax: 410-955-0461; E-mail: rrao@jhmi.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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