The Saccharomyces cerevisiae SOP1 and SOP2 Genes, Which Act in Cation Homeostasis, Can Be Functionally Substituted by the Drosophila lethal(2)giant larvae Tumor Suppressor Gene* (Received for publication, July 29, 1998, and in revised form, September 24, 1998) Katrin Larsson‡, Florian Bo ¨ hl§, Ingrid Sjo ¨ stro ¨ m‡, Noreen Akhtar‡, Dieter Strand§, Bernard M. Mechler§, Reiner Grabowski‡, and Lennart Adler‡¶ From the ‡Department of Cell and Molecular Biology, Microbiology, Go ¨teborg University, Box 462, SE 40530 Go ¨teborg, Sweden and the §Department of Developmental Genetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany By complementation of a salt-sensitive mutant of Sac- charomyces cerevisiae, we cloned the SOP1 gene, encod- ing a 114.5-kDa protein of 1033 amino acids. Cells de- leted for SOP1 exhibited sensitivity to sodium stress, but showed no sensitivity to general osmotic stress. Fol- lowing exposure of sop1 cells to NaCl stress, the intra- cellular Na  level and the Na  /K  ratio rose to values significantly higher than in wild type cells. Deletion of SOP2, encoding a protein sharing 54% amino acid iden- tity with Sop1p, produced only slight Na  sensitivity. Cells carrying a sop1sop2 double deletion became, however, hypersensitive to Na  and exhibited increased sensitivity also to Li  and K  , suggesting involvement of both SOP1 and SOP2 in cation homeostasis. The pre- dicted amino acid sequences of Sop1p and Sop2p show significant homologies with the cytoskeletal-associated protein encoded by the Drosophila lethal(2)giant larvae tumor suppressor gene. Immunolocalization of Sop1p revealed a cytoplasmic distribution and cell fraction- ation studies showed that a significant fraction of Sop1p was recovered in a sedimentable fraction of the cytoso- lic material. Expression of a Drosophila l(2)gl cDNA in the sop1sop2 strain partially restored the Na  toler- ance of the cells, indicating a functional relationship between the Sop proteins and the tumor suppressor pro- tein, and a novel function in cell homeostasis for this family of proteins extending from yeast to human. Ions are continuously transported across the cell membrane, the net flux being adjusted to satisfy the requirement for a cytosol rich in potassium and scarce in sodium. Control of the intracellular concentration of these major monovalent cations is crucial to generate a biochemically-functional intracellular milieu. Since in natural environments Na + is generally abun- dant and K + scarce, transport must occur against concentra- tion gradients. Genetic analysis of salt tolerance in Saccharo- myces cerevisiae has identified a number of cation transporters which interact with multiple regulatory components in a largely unidentified fashion (1). In particular, a major system involved in K + uptake is constituted by the TRK1- and TRK2- encoded membrane proteins (2– 4), which appear to contribute to the uptake of K + in symport with protons (1). The proton gradient providing the driving force for secondary transport is generated by the PMA1-encoded plasma membrane ATPase, a major membrane protein whose activity shows little sensitivity to high extracellular NaCl concentration (5). The TRK1/TRK2- dependent transport system also permits influx of Na + , while under NaCl stress, the uptake system has the capacity of increasing its selectivity for K + over Na + (6). In yeast cells, influx of Na + is counteracted by Na + efflux, the primary pathway being mediated by the P-type ATPase encoded by the PMR2A gene (also known as ENA1) (6, 7). The PMR2A gene is part of a gene cluster, containing tandem repeats of 2–5 nearly identical genes (8). However, only PMR2A appears to be significantly expressed (7, 8), and transcription of this gene is induced in cells subjected to Na + or Li + stress or cells exposed to alkaline pH (7). An additional sodium trans- porter encoded by the NHA1 gene and acting as a putative Na + /H + antiporter was recently identified in S. cerevisiae (9). Disruption of the NHA1 gene displays only minor effects in wild type cells but elicits increased Na + sensitivity in S. cer- evisiae cells lacking the PMR2 genes. To identify components that are crucial for salt tolerance, the isolation of recessive, salt-sensitive mutations is an obvious approach. However, the only S. cerevisiae mutant character- ized so far by this procedure is the calcineurin-defective strain isolated by Mendoza et al. (10). These authors demonstrated that the protein phosphatase calcineurin, is involved in Na + tolerance and is required for (i) induced expression of the PMR2A gene and (ii) modulating the K + uptake system to display increased K + versus Na + discrimination. Further evi- dence that protein phosphorylation and dephosphorylation reg- ulate Na + tolerance in S. cerevisiae is provided by the increased cellular tolerance to sodium ions following inactivation of the PPZ1 and PPZ2 encoded serine-threonine phosphatases (11). In addition, increased dosage of the YCK1 or YCK2 gene, en- coding yeast homologues of casein kinase I, enhances sodium tolerance (12), while cells defective in either of the a or b subunits of the yeast casein kinase II homologue become spe- cifically sensitive to high concentrations of Na + (13). By isolation and functional complementation a NaCl-sensi- tive mutant, we cloned the SOP1 gene. Here we report the initial characterization of the gene product and show that the predicted sequence of Sop1p lacks apparent membrane span- ning regions or other characteristics of previously isolated de- terminants for Na + tolerance, and displays significant homol- ogy with the Drosophila p127 protein encoded by the lethal(2)giant larvae (l(2)gl) tumor suppressor gene and its homologues in mouse and man. Our results demonstrate that * This work was supported by grants from the Swedish National Science Research Council, the Swedish Council for Forestry and Agri- cultural Research, the Swedish Research Council for Engineering Sci- ences, and by EU Programs BIOL-CT 950161, ERB4061 PL95-0014, and BMH1-CT94-1572. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ To whom correspondence should be addressed. Tel.: 46-31-7732500; Fax: 46-31-7732599; E-mail: Lennart.Adler@gmm.gu.se. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 50, Issue of December 11, pp. 33610 –33618, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 33610 by guest on February 26, 2018 http://www.jbc.org/ Downloaded from