A Novel Yeast‐Based Screening System Identifies Signal Motifs that Regulate Membrane Protein Trafficking

• Published in: The FASEB journal 2017-04, Vol.31 (S1)
• Main Authors: Bernstein, Joshua, Okamoto, Yukari, Shikano, Sojin
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.31.1_supplement.lb233

Abstract only The cell surface density of membrane proteins dictates their corresponding cellular activity. Many diseases, such as cystic fibrosis, arise from defects in cell surface trafficking of membrane proteins. Intracellular trafficking of membrane proteins is often controlled by the distinct signal motifs on the cargo proteins. A systematic approach to identify trafficking signals would facilitate our understanding of membrane protein trafficking. Here we report development of a new yeast‐based screening system that enables identification of the sequence motifs that down‐regulate surface expression of membrane proteins. We show that B31, a mutant S. cerevisiae strain lacking potassium (K + ) efflux transporters, is unable to grow in high K + media when expressing the mammalian K + uptake channel Kir2.1 due to overloaded K + . However, B31 expressing the Kir2.1 channel defective in post‐Golgi trafficking or those fused with intracellular targeting motifs were rescued in high K + media. Thus a growth rescue of B31 cells in high K + media represents the reduction in the surface expression of the reporter Kir2.1 protein. Using this system, we have developed a method of screening a random C‐terminal peptide library fused to Kir2.1 channel for signals which reduce the levels of Kir2.1 on the cell surface. We have conducted a pilot screening, and identified sequences that down‐regulated cell surface expression of Kir2.1. We demonstrate that the identified sequences are functionally transplantable into different protein contexts such as CD4 and CD8. Furthermore, we have characterized the molecular mechanism of transport caused by these sequences and show that the screening system can identify signal sequences that operate through multiple distinct trafficking pathways such as ER retrieval and endocytosis. Thus our system offers a unique tool to discover potentially novel sequence motifs that regulate cell surface expression of membrane proteins Support or Funding Information NIH R01GM099974