Academic journal article Genetics

Evidence for Amino Acid Snorkeling from a High-Resolution, in Vivo Analysis of Fis1 Tail-Anchor Insertion at the Mitochondrial Outer Membrane

Academic journal article Genetics

Evidence for Amino Acid Snorkeling from a High-Resolution, in Vivo Analysis of Fis1 Tail-Anchor Insertion at the Mitochondrial Outer Membrane

Article excerpt

PROTEINS inserted within the mitochondrial outer membrane (OM) by a carboxyl-terminal tail anchor (TA) are important for programmed cell death, mitochondrial protein import, and the control of mitochondrial shape and number (Wattenberg and Lithgow 2001). While ER-directed tail-anchored proteins can take advantage of a conserved set of soluble proteins and membrane-bound receptors (Denic et al. 2013; Johnson et al. 2013; Aviram et al. 2016), the machinery targeting many TAs to mitochondria is yet to be discovered (Lee et al. 2014; Neupert 2015), and genetic and biochemical evidence suggest that spontaneous insertion of TAs at mitochondriamay occur without the need for a translocation machinery (Setoguchi et al. 2006; Kemper et al. 2008; Krumpe et al. 2012). Furthermore, some tail-anchored proteins can be dual localized to mitochondria and other organelles (Borgese and Fasana 2011), but how membrane specificity is controlled is unclear. TA targeting seems to depend, in general, upon incompletely defined structural characteristics of the TA rather than a defined consensus sequence (Beilharz 2003; Rapaport 2003; Borgese et al. 2007).

Genetic selection schemesusingtheorganismSaccharomyces cerevisiae have been of high value in understanding how proteins reach their proper destination within eukaryotic cells. During such studies, a protein required for survival under selective conditions can be mislocalized, and thereby made inactive, by a targeting sequence utilized by the transport process being studied. Next, mutations that allow return of this mistargeted protein to a region of the cell at which it can perform its function are recovered under selective conditions. Trans factors related to protein targeting are identified by standard genetic approaches. Alternatively, cis mutations in the targeting sequence are revealed, typically by Sanger sequencing of individual fusion construct clones. Most prominently, this genetic approach to studying protein targeting and transport has been important in understanding protein transit to and through the endomembrane system (Deshaies and Schekman 1987; Robinson et al. 1988; Stirling et al. 1992). This approach has also been applied to the study of protein import and export at the mitochondrial inner membrane (Jensen et al. 1992; Maarse et al. 1992; He and Fox 1999).

Even with the availability of these powerful genetic strategies, a fine-grained analysis of any single eukaryotic protein targeting signal has been lacking. However, with the advent of next-generation sequencing, more comprehensive studies of protein targeting sequences are possible. In this study, we successfully coupled genetic selection to nextgeneration sequence analysis to define characteristics important for localization of the tail-anchored Fis1 protein to the mitochondrial OM.

Materials and Methods

Yeast strains and plasmids

Details of strains used in this study are provided in Supplemental Material, Table S2. Plasmid acquisition details and associated references, as well as details of plasmid construction, are found in Table S3. Oligonucleotides used in this study are listed in Table S4.

Culture conditions

Synthetic complete (SC) medium contains 0.67% yeast nitrogen base without amino acids, 2% dextrose, 0.1% casamino acids, 50 mg/ml adenine hemisulfate, and either 25 mg/ml uracil (SC 2Trp) or 100 mg/ml tryptophan (SC 2Ura). Supplemented minimal medium (SMM) contains 0.67% yeast nitrogen base without amino acids, 2% dextrose, 20 mg/ml adenine hemisulfate, 20 mg/ml uracil, 20 mg/ml methionine, and 30 mg/ml lysine. SMM also contains, depending on selection needs, 20 mg/ml histidine, 100 mg/ml leucine, and/or 20 mg/ml tryptophan, as indicated. SLac medium lacking histidine contains 0.67% yeast nitrogen basewithout amino acids, 1.2%NaOH, a volume of lactic acid sufficient to subsequently bring the pH to 5.5, 20 mg/ml adenine hemisulfate, 20 mg/ml uracil, 20 mg/ml methionine, 30 mg/ml lysine, 100 mg/ml leucine, and 20 mg/ml tryptophan. …

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