The yeast Trk1 polypeptide, like other members of the Superfamily of K Transporters (SKT proteins) consists of four Membrane-Pore-Membrane motifs (MPMs A-D) each of which is homologous to a single K-channel subunit. SKT proteins are thought to have evolved from ancestral K-channels via two gene duplications and thus single MPMs might be able to assemble when located on different polypeptides. To test this hypothesis experimentally we generated a set of partial gene deletions to create alleles encoding one, two, or three MPMs, and analysed the cellular localisation and interactions of these Trk1 fragments using GFP tags and Bimolecular Fluorescence Complementation (BiFC). The function of these partial Trk1 proteins either alone or in combinations was assessed by expressing the encoding genes in a K+-uptake deficient strain lacking also the K-channel Tok1 (trk1,trk2,tok1Δ) and (i) analysing their ability to promote growth in low [K+] media and (ii) by ion flux measurements using "microelectrode based ion flux estimation" (MIFE). We found that proteins containing only one or two MPM motifs can interact with each other and assemble with a polypeptide consisting of the rest of the Trk system to form a functional K+-translocation system.

Center for Nanobiology and Structural Biology Institute of Microbiology of the Czech Academy of Sciences Zamek 136 373 33 Nove Hrady Czech Republic

Center for Nanobiology and Structural Biology Institute of Microbiology of the Czech Academy of Sciences Zamek 136 373 33 Nove Hrady Czech Republic; University of South Bohemia in Ceske Budejovice Faculty of Science Branisovska 1760 370 05 Ceske Budejovice Czech Republic

Centre of New Technologies University of Warsaw Stefana Banacha 2c 02 097 Warsaw Poland

Citace poskytuje Crossref.org

Dimerisation of the Yeast K+ Translocation Protein Trk1 Depends on the K+ Concentration