Poster: Membrane Transport

Abs # 1208: Chimeras between the two yeast TRK proteins demonstrate powerful roles for the long hydrophilic loop in transport regulation and affinity control

Presenter:	Zeng, GeFei , gefei.zeng@cmp.yale.edu
Authors	Zeng, GeFei  (A)   Zhang, LiGuo  (A)   Slayman, Clifford  (A)
Affiliations:	(A): Cellular & Molecular Physiology, Yale School of Medicine

Trk1p and Trk2p, the major proteins for potassium uptake in Saccharomyces cerevisiae, belong to a class of transporters which display carrier-type kinetics but channel-like folding. In a single polypeptide, they possess 4 mimic K⁺-channel “MPM” domains, with absolute conservation of a characteristic glycine residue (G) and 4 easily recognized variants of the diagnostic GYG motif. They also possess a very long hydrophilic loop, the M2a-M1b linker: 329 amino acids in Trk2p and 645 amino acids in Trk1p. Recent epitope tagging (Zeng et al., MS in prep.) has shown this domain to be intracellular, consistent with 4-MPM folding. Because it contains many potential phosphorylation sites, this loop is probably the protein’s key regulatory element. Using the ends of the M2a-M1b linker as break points, we have constructed the entire family of chimeric genes between TRK1 and TRK2, inserted these into a centromeric plasmid (pYCpGAL0) behind a GAL1 promoter, and expressed them in TRK-negative yeast (trk1 trk2) grown on galactose. [Chimeras are named ### according to the origin, in Trk1p or Trk2p, of each segment: reconstructed wild-types are 111 & 222, and molecules simply deleted of the M2a-M1b linker are 1-1 & 2-2.] Resultant expression was tested for K⁺-supported growth, by conventional agar-plate drop tests and by quantitative liquid-growth measurements on microtiter plates. In both tests, the relative efficacy of K⁺ transport was as follows for the different constructs: Strong: 212, 111,112 > Weak: 122, 121, 222 > Zero: 211, 221, 1-1, 2-2. Thus, the M2a-M1b linker is essential for protein function, and the long linker from Trk1p supports faster growth on low K⁺--and therefore probably higher-affinity K⁺ transport—than the short linker from Trk2p.