Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 284 (49), 34116-25

Yeast AEP3p Is an Accessory Factor in Initiation of Mitochondrial Translation

Affiliations

Yeast AEP3p Is an Accessory Factor in Initiation of Mitochondrial Translation

Changkeun Lee et al. J Biol Chem.

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.

Figures

FIGURE 1.
FIGURE 1.
Point mutation in AEP3 causes a synthetic respiratory defect in strains lacking formylated Met-tRNAfMet. A, synthetic petite mutant strain 175 (Δfmt1 aep3-Y305N), harboring full-length FMT1 on a URA3 plasmid, was transformed with either wild-type pRS415-AEP3 (top) or empty plasmid (pRS415, bottom) as a negative control, streaked onto a 5-FOA/YPEG plate, and incubated at 30 °C for 5 days. B (top), strain DLY2 (Δaep3 FMT1+), harboring wild-type AEP3 on a URA3 plasmid (pRS416), was transformed to leucine prototrophy with either pRS415-aep3-Y305N, wild-type pRS415-AEP3, or empty plasmid (pRS415) as a negative control. The pRS416-AEP3 plasmid was evicted by growth on 5-FOA, and then the transformants were streaked onto a YPEG plate and incubated at 30 °C for 5 days. Bottom, 10-fold serial dilutions of DLY2 harboring either the wild-type or mutant aep3 plasmid were spotted on YPD or YPEG plates and incubated at 30 °C for 3 or 6 days, respectively. C, the original synthetic petite 175 strain (Δfmt1 aep3-Y305N leu2 MIS1/pRS416-FMT1) and a Leu+ integrant of this strain were streaked onto YPEG plates and incubated at 30 °C for 5 days to test for respiratory growth. MIS1, wild-type MIS1 locus, Δmis1::LEU2, disrupted mis1 locus. See “Experimental Procedures” for details of MIS1 gene disruption.
FIGURE 2.
FIGURE 2.
SDS-PAGE analysis of purification of MBP-Aep3p. Lane 1, soluble extract from induced cells. Lane 2, flow-through fraction from Ni2+ column. Lane 3, purified MBP-Aep3p (63 pmol). Sizes of molecular mass markers (M) in kDa are shown on the left. The 10% gel was stained with Coomassie Blue.
FIGURE 3.
FIGURE 3.
Aep3p exhibits no significant tRNA binding. Each reaction mixture contained the indicated amount of protein and 4 pmol of a labeled tRNA in the filter binding assay described under “Experimental Procedures.” MBP-Aep3p fusion protein was incubated with [35S]fMet-tRNAfMet (○), [35S]Met-tRNAfMet (▼), or [14C]Lys-tRNALys (□). ymIF2 + [35S]Met-tRNAfMet (●) served as a positive control. MBP alone + [35S]Met-tRNAfMet (♦) is a negative control. Nonspecific binding to the filters in the absence of protein was subtracted. Results shown are the means of two separate determinations ± S.E. (error bars are included for all data points but are obscured by the data symbol when the scatter is small).
FIGURE 4.
FIGURE 4.
Aep3p binds ymIF2 in vitro. Purified proteins were incubated for 2 h at room temperature and treated with amylose beads as described under “Experimental Procedures.” Unbound and bound fractions were analyzed by SDS-PAGE and immunoblotting (IB) with antibodies against ymIF2 (lanes 1–6) or cytoplasmic C1-THF synthase (lanes 7–8). Lane 1, unbound fraction from MBP-Aep3p + ymIF2 incubation (40 pmol each); lane 2, bound fraction; lane 3, unbound fraction from MBP + ymIF2 incubation (20 pmol each); lane 4, bound fraction. Lane 5, unbound fraction from MBP-Aep3p + ymIF2 C2 domain incubation (20 pmol each); lane 6, bound fraction; lane 7, unbound fraction from MBP-Aep3p + cytosolic C1-THF synthase incubation (20 pmol each); lane 8, bound fraction. Locations and size (kDa) of molecular mass markers are shown on the left of the gels.
FIGURE 5.
FIGURE 5.
Aep3p binds ymIF2 in vivo. Yeast strain LOY1 (Δifm1 AEP3) was grown in minimal medium containing glucose to A600 = 3, lysed, immunoprecipitated with anti-HA antibodies, and analyzed by SDS-PAGE and immunoblotting with antibodies against ymIF2 as described under “Experimental Procedures.” Lane 1, control immunoprecipitate from cells harboring only wild-type IFM1 multicopy plasmid pVT101U-ymIF2; lane 2, immunoprecipitate from cells harboring pVT101U-ymIF2 and HA-tagged AEP3 on plasmid pRS415-AEP3-HA; lane 3, immunoprecipitate from cells harboring only pRS415-AEP3-HA (no ymIF2). Locations and size (kDa) of molecular mass markers are shown on the left. ymIF2 migrates at ∼70 kDa.
FIGURE 6.
FIGURE 6.
Aep3p stimulates the binding of Met-tRNAfMet, but not fMet-tRNAfMet, to ymIF2. Each filter binding assay contained 7 pmol of ymIF2 and increasing amounts of MBP-Aep3p fusion protein with either 5 pmol of [35S]fMet-tRNAfMet (○) or 4 pmol of [35S]Met-tRNAfMet (●). Nonspecific binding to the filters in the absence of protein was subtracted. Results shown are the means of two separate determinations ± S.E. (error bars are included for all data points but are obscured by the data symbol when the scatter is small). At 0 MBP-Aep3p added, ymIF2 bound 0.15 ± 0.02 pmol of unformylated and 0.4 ± 0.07 pmol of formylated Met-tRNAfMet.
FIGURE 7.
FIGURE 7.
Aep3p-Y305N causes mitochondrial protein synthesis defect in combination with Δfmt1. Mitochondrially synthesized proteins were labeled in vivo with [35S]methionine in the presence of cycloheximide, extracted, and analyzed by SDS-PAGE and phosphorimaging as described under “Experimental Procedures.” Lanes 1 and 2, strain DLY2 (Δaep3 FMT1) harboring pRS415 plasmids with either wild-type AEP3 (lane 1) or point mutant aep3-Y305N (lane 2). Lanes 3 and 4, synthetic petite mutant strain 175 (Δfmt1 aep3-Y305N) harboring full-length FMT1 on URA3 plasmid (lane 3) or after eviction of FMT1 plasmid (lane 4). Lane 5, strain DLY2 (Δaep3 FMT1) maintained with wild-type pRS416-AEP3, which was evicted by 5-FOA before metabolic labeling.
FIGURE 8.
FIGURE 8.
Predicted domain structure of Aep3p. The 606-amino acid AEP3 ORF was analyzed for PPR motifs using TPRpred (Bioinformatics Toolkit). The four highest scoring PPR motifs are indicated along with the residue numbers at their boundaries. The two point mutations discussed in the “Discussion” (Y305N, Q320P) are located in the region of highest sequence identity with Aep3p homologs in other related Saccharomyces species (46). MTS, mitochondrial targeting sequence.

Similar articles

See all similar articles

Cited by 11 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback