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

• Published in: The Journal of biological chemistry Vol. 279; no. 4; pp. 3003 - 3013
• Main Authors: Zeng, Ge-Fei, Pypaert, Marc, Slayman, Clifford L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 23-01-2004; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Cation Transport Proteins - chemistry; Cation Transport Proteins - genetics; Cation Transport Proteins - immunology; Epitope Mapping; Immunohistochemistry; Ion Channels - chemistry; Ion Channels - genetics; Ion Channels - immunology; Molecular Sequence Data; Protein Folding; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - immunology; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - immunology; Sequence Alignment
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M309760200

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.