title: Potential use of potassium efflux-deficient yeast for studying trafficking signals and potassium channel functions.
abstract: The activity of potassium (K(+)) channels critically depends on their density on the cell surface membrane, which is regulated by dynamic protein-protein interactions that often involve distinct trafficking signals on the cargo proteins. In this paper we explored the possibility of utilizing the Saccharomyces cerevisiae strain B31 for identification of the signal motifs that regulate surface expression of membrane proteins and for studying structure-function relationships of K(+) channels. B31 cells lack the K(+) efflux system and were reported to show overloaded K(+)-mediated growth inhibition in high K(+) media upon heterologous expression of a mammalian inwardly rectifying K(+) channel (Kir2.1). We show that while the expression of wild-type Kir2.1 channel inhibits the growth of B31 cells in high K(+) media, the human disease-causing mutations of Kir2.1 that abolish K(+) conduction (V302M) or surface trafficking (Δ314/315) fully restores the growth. The expression of two-pore-domain K(+) channel KCNK3 or KCNK9 also inhibited the growth of B31 in high K(+) media while C-terminal mutations that reduce their 14-3-3 protein-dependent cell surface trafficking restored the growth of B31. Finally, the expression of Kir2.1 channels that were C-terminally fused with known sequence motifs including ER retention/retrieval signals and an endocytosis signal allowed the growth of B31 in high K(+) media. These results demonstrate the potential of B31 yeast strain as a unique biological tool to screen the random peptide libraries for novel sequence signals that down-regulate surface expression of membrane proteins, as well as to systematically identify the structural determinants for cell surface trafficking and/or ion conductance of K(+) channels.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Bernstein
authors.forename: Yukari
authors.initials: Y
authors.lastname: Okamoto
authors.forename: Minjee
authors.initials: M
authors.lastname: Kim
authors.forename: Sojin
authors.initials: S
authors.lastname: Shikano
keywords: Ab, antibody; COP, coatomer protein; ER, endoplasmic reticulum; FCM, flow cytometry; HA, hemagglutinine; IP, immunoprecipitation; O/N, overnight; Potassium channel; SD, standard deviation; Screening; Surface membrane; Trafficking; Yeast; aa, amino acids