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Journal of Bacteriology, April 1999, p. 2527-2534, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The Extracellular Domain of the Saccharomyces cerevisiae Sln1p Membrane Osmolarity Sensor Is Necessary for Kinase Activity

Darin B. Ostranderdagger and Jessica A. Gorman*

Department of Leads Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000

Received 26 October 1998/Accepted 8 February 1999

The function of the extracellular domain (ECD) of Sln1p, a plasma membrane two-transmembrane domain (TMD) sensor of the high-osmolarity glycerol (HOG) response pathway, has been studied in the yeast Saccharomyces cerevisiae. Truncations of SLN1 that retain an intact kinase domain are capable of complementing the lethality of an sln1Delta strain. By observing levels of Hog1p phosphorylation as well as the phosphorylation state of Sln1p, the kinase activities of various SLN1 constructions were determined. In derivatives that do not contain the first TMD, Sln1p activity was no longer dependent on medium osmolarity but appeared to be constitutively active even under conditions of high osmolarity. Removal of the first TMD (Delta TMD1 construct) gave a protein that was strongly phosphorylated whereas Hog1p was largely dephosphorylated, as expected if the active form of Sln1p is phosphorylated. When both TMDs as well as the ECD were deleted, so that the kinase domain is cytosolic, Sln1p was not phosphorylated whereas Hog1p became constitutively hyperphosphorylated. Surprisingly, this hyperactivity of the HOG mitogen-activated protein kinase signaling pathway was not sufficient to result in cell lethality. When the ECD of the Delta TMD1 construct was replaced with a leucine zipper motif, Sln1p was hyperactive, so that Hog1p became mostly unphosphorylated. In contrast, when the Sln1p/leucine zipper construct was crippled by a mutation of one of the internal leucines, the Sln1 kinase was inactive. These experiments are consistent with the hypothesis that the ECD of Sln1p functions as a dimerization and activation domain but that osmotic regulation of activity requires the presence of the first TMD.

* Corresponding author. Mailing address: Mail Stop H24-02, Bristol-Myers Squibb Pharmaceutical Research Institute, Rt. 206 and Province Line Rd., Princeton, NJ 08543-4000. Phone: (609) 252-4456. Fax: (609) 252-6813. E-mail: gormanj{at}bms.com.

dagger Present address: Department of Biochemistry and Molecular Biology, University of Texas Houston Medical School, Houston, TX 77225.

Journal of Bacteriology, April 1999, p. 2527-2534, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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