Journal
GENETICS
Volume 205, Issue 2, Pages 691-705Publisher
GENETICS SOCIETY AMERICA
DOI: 10.1534/genetics.116.196428
Keywords
mitochondrial protein targeting; mitochondrial division; membrane insertion; amino acid snorkeling; deep mutational scanning
Categories
Funding
- European Molecular Biology Organization Installation Grant [2138]
- European Research Council [637649-RevMito]
- Koc University
Ask authors/readers for more resources
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 may be important for mitochondrial targeting, we focused our attention upon the TA of the Saccharomyces cerevisiae Fis1 protein. Specifically, we generated a library of Fis1p TA variants fused to the Gal4 transcription factor, then, using next-generation sequencing, revealed which Fis1p TA mutations inhibited membrane insertion and allowed Gal4p activity in the nucleus. Prompted by our global analysis, we subsequently analyzed the ability of individual Fis1p TA mutants to localize to mitochondria. Our findings suggest that the membrane-associated domain of the Fis1p TA may be bipartite in nature, and we encountered evidence that the positively charged patch at the carboxyl terminus of Fis1p is required for both membrane insertion and organelle specificity. Furthermore, lengthening or shortening of the Fis1p TA by up to three amino acids did not inhibit mitochondrial targeting, arguing against a model in which TA length directs insertion of TAs to distinct organelles. Most importantly, positively charged residues were more acceptable at several positions within the membrane-associated domain of the Fis1p TA than negatively charged residues. These findings, emerging from the first high-resolution analysis of an organelle targeting sequence by deep mutational scanning, provide strong, in vivo evidence that lysine and arginine can snorkel, or become stably incorporated within a lipid bilayer by placing terminal charges of their side chains at the membrane interface.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available