An Yeast‐Based Screening for Signal Motifs That Regulate Membrane Protein Trafficking

Abstract

Cell surface density of membrane proteins are regulated by the interactions that often occur between distinct signal motifs on the cargo proteins and cellular transport machineries. The majority of the trafficking signals have been discovered by studies on individual protein molecules. While effective, a more systematic approach to identify trafficking signals would further facilitate our understanding of membrane trafficking. Here we report development of a new yeast‐based screening system that potentially enables identification of the sorting motifs that down‐regulate surface expression of membrane proteins. B31, a S. cerevisiae strain that lacks potassium (K+) efflux transporters, is unable to grow in high K+ media when expressing the mammalian inward rectifying K+ channel Kir2.1 due to accumulation of excessive intracellular K+. However, B31 cells expressing the disease mutant Kir2.1 deficient in post‐Golgi trafficking or the Kir2.1 that is C‐terminally fused with ER retrieval motif survive in high K+ media due to reduced K+ uptake. This B31 growth complementation in high K+ media allowed a screen for the peptide motifs that would down‐regulate cell surface expression of the reporter Kir2.1 channel. We performed a pilot screening of a C‐terminally‐fused peptide library and identified sequence motifs that reduced surface expression of the channel. Our study provides a technical basis for developing the unique gain‐of‐function screening that can potentially identify novel signal motifs that regulate surface expression of membrane proteins.