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J. Biol. Chem., Vol. 279, Issue 4, 3003-3013, January 23, 2004

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Epitope Tagging of the Yeast K⁺ Carrier Trk2p Demonstrates Folding That Is Consistent with a Channel-like Structure^*

Ge-Fei Zeng, Marc Pypaert, and Clifford L. Slayman¶

From the Department of Cellular and Molecular Physiology and the Center for Cell and Molecular Imaging, Yale University School of Medicine, New Haven, Connecticut 06520

TRK family proteins, which mediate the concentrative uptake of potassium by plant cells, fungi, and bacteria, resemble primitive potassium channels in sequence and have recently been proposed actually to fold like potassium channels in a 4-MPM motif (Durell, S. R., and Guy, H. R. (1999) Biophys. J. 77, 789 - 807), instead of like conventional substrate porters in the 12-TM motif (Gaber, R. F., Styles, C. A., and Fink, G. R. (1988) Mol. Cell. Biol. 8, 2848-2859). The known fungal members of this family possess a very long hydrophilic loop, positioned intracellularly in the K⁺-channel model and extracellularly in the substrate porter model. This and two shorter hydrophilic segments have been tested as topological markers for the true folding pattern of TRK proteins using Saccharomyces cerevisiae Trk2p. Hemagglutinin epitope tags were inserted into all three segments, and the enhanced green fluorescent protein (EGFP) was fused to the C terminus of Trk2p. The gene constructs were expressed from a high copy plasmid, and sidedness of the tags was determined by native fluorescence (EGFP), indirect immunofluorescence, and immunoelectron microscopy. Both the long-loop tag and the C-terminal EGFP fusion allowed abundant protein to reach the plasma membrane and support normal yeast growth. In all determinations, the long-loop tag was localized to the inner surface of the yeast cell plasma membrane, thus strongly supporting the channel-like folding model. Additional observations showed (i) membrane-associated Trk2p to lie in proteolipid rafts; (ii) significant tagged protein, expressed from the plasmid, to be sequestered in cytoplasmic vesicular-tubular clusters; and (iii) suppression of such clusters by yeast growth in 5-10% glycerol. This chaperone-like effect may assist other membrane proteins (overexpressed or heterologously expressed) to function within the yeast plasma membrane.

Received for publication, September 3, 2003 , and in revised form, October 20, 2003.

^* This work was supported by National Institutes of Health Research Grant GM-60696 (to C. L. S.) and a Yale Medical School James Hudson Brown-Alexander Brown Coxe fellowship (to G.-F. Z.). 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: Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520. Tel.: 203-785-4478; Fax: 203-785-5535; E-mail: clifford.slayman{at}yale.edu.

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