TY - JOUR T1 - Deep mutational scanning reveals tail anchor characteristics important for mitochondrial targeting JF - bioRxiv DO - 10.1101/045351 SP - 045351 AU - Abdurrahman Keskin AU - Emel Akdoğan AU - Cory D. Dunn Y1 - 2016/01/01 UR - http://biorxiv.org/content/early/2016/03/25/045351.abstract N2 - Proteins localized to mitochondria by a carboxyl-terminal tail anchor (TA) play roles in apoptosis, mitochondrial dynamics, and mitochondrial protein import. To reveal characteristics of TAs that are important for mitochondrial targeting, we examined the TA of the Saccharomyces cerevisiae Fis1 protein. We generated a library of Fis1p TA variants fused to the Gal4 transcription factor, then selected for mutations allowing Gal4 activity in the nucleus. Next-generation sequencing allowed quantification of TA variants in our mutant library both before and after selection for reduced TA targeting. High-throughput results were confirmed by further analysis of individual Fis1p TA mutants. Intriguingly, positively charged residues were more acceptable at several positions within the membrane-associated domain of the Fis1p TA than negatively charged residues. These findings provide strong, in vivo evidence that lysine and arginine can “snorkel” when associated with a lipid bilayer by placing their terminal charges at the membrane interface. Our work provides the first high-resolution analysis of an organelle targeting sequence by deep mutational scanning. ER -