Yeast AEP3p Is an Accessory Factor in Initiation of Mitochondrial Translation

Changkeun Lee,Anne S Tibbetts,Gisela Kramer,Dean R Appling

doi:10.1074/jbc.m109.055350

Changkeun Lee, Anne S Tibbetts + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.055350

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Abstract

Initiation of protein synthesis in mitochondria and chloroplasts normally uses a formylated initiator methionyl-tRNA (fMet-tRNA(f)(Met)). However, mitochondrial protein synthesis in Saccharomyces cerevisiae can initiate with nonformylated Met-tRNA(f)(Met), as demonstrated in yeast mutants in which the nuclear gene encoding mitochondrial methionyl-tRNA formyltransferase (FMT1) has been deleted. The role of formylation of the initiator tRNA is not known, but in vitro formylation increases binding of Met-tRNA(f)(Met) to translation initiation factor 2 (IF2). We hypothesize the existence of an accessory factor that assists mitochondrial IF2 (mIF2) in utilizing unformylated Met-tRNA(f)(Met). This accessory factor might be unnecessary when formylated Met-tRNA(f)(Met) is present but becomes essential when only the unformylated species are available. Using a synthetic petite genetic screen in yeast, we identified a mutation in the AEP3 gene that caused a synthetic respiratory-defective phenotype together with Delta fmt1. The same aep3 mutation also caused a synthetic respiratory defect in cells lacking formylated Met-tRNA(f)(Met) due to loss of the MIS1 gene that encodes the mitochondrial C(1)-tetrahydrofolate synthase. The AEP3 gene encodes a peripheral mitochondrial inner membrane protein that stabilizes mitochondrially encoded ATP6/8 mRNA. Here we show that the AEP3 protein (Aep3p) physically interacts with yeast mIF2 both in vitro and in vivo and promotes the binding of unformylated initiator tRNA to yeast mIF2. We propose that Aep3p functions as an accessory initiation factor in mitochondrial protein synthesis.

Highlights

Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9040. 2 To whom correspondence should be addressed: Dept. of Chemistry and eukaryotes [3]
We show that the AEP3 protein (Aep3p) physically interacts with Yeast mIF2 (ymIF2) and promotes the binding of unformylated initiator tRNA to ymIF2
We demonstrate here a functional interaction between Aep3p and mitochondrial translation initiation factor 2, revealed when only unformylated initiator tRNA is available

Summary

EXPERIMENTAL PROCEDURES

Reagents, and Strains—Escherichia coli tRNAs (tRNAfMet and tRNALys) and cycloheximide were purchased from Sigma. Other AEP3 constructs could be transformed into DLY2 on pRS415 and studied after eviction of the wild-type pRS416AEP3 plasmid by growth on 5-FOA. Cloning Yeast AEP3 for Protein Expression in E. coli—The wild-type AEP3 ORF (YPL005W) was amplified from genomic DAY4 DNA using primers AP3-pMAL-For (5Ј-CCGGGATCCATGAATACATTAAGG-3Ј; the BamHI site is underlined, and the start codon is in bold) and AP3-pMAL-Rev (5Ј-CCGAAGCTTTCAAACCTCCCCAAC-3Ј; the HindIII site is underlined, and the stop codon is in bold). Different amounts of each protein were incubated for 20 min on ice in 50 ␮l of total volume containing 50 mM Tris-HCl, pH 7.0, 2 mM dithiothreitol, and 10 ␮g of bovine serum albumin with 4 pmol of 35S-labeled initiator tRNA (fMet-tRNAfMet or Met-tRNAfMet). Dium, and colonies were covered with agar containing 0.1% 2,3,5triphenyltetrazolium chloride to determine the number of respiratory-defective (petite; white) and respiratory-competent (red) colonies

Isolation of Mutants That Are

Yeast strains

DISCUSSION