Mitochondrial translation of Saccharomyces cerevisiae COX2 mRNA is controlled by the nucleotide sequence specifying the pre-Cox2p leader peptide

Abstract

The mitochondrial gene encoding yeast cytochrome oxidase subunit II (Cox2p) specifies a precursor protein with a 15-amino-acid leader peptide. Deletion of the entire leader peptide coding region is known to block Cox2p accumulation posttranscriptionally. Here, we examined in vivo the role of the pre-Cox2p leader peptide and the mRNA sequence that encodes it in the expression of a mitochondrial reporter gene, ARG8m, fused to the 91st codon of COX2. We found within the coding sequence antagonistic elements that control translation: the positive element includes sequences in the first 14 codons specifying the leader peptide, while the negative element appears to be within codons 15 to 91. Partial deletions, point mutations, and local frameshifts within the leader peptide coding region were placed in both the cox2::ARG8m reporter and in COX2 itself. Surprisingly, the mRNA sequence of the first six codons specifying the leader peptide plays an important role in positively controlling translation, while the amino acid sequence of the leader peptide itself is relatively unconstrained. Two mutations that partially block translation can be suppressed by nearby sequence substitutions that weaken a predicted stem structure and by overproduction of either the COX2 mRNA-specific translational activator Pet111p or the large-subunit mitochondrial ribosomal protein MrpL36p. We propose that regulatory elements embedded in the translated COX2 mRNA sequence could play a role, together with trans-acting factors, in coupling regulated synthesis of nascent pre-Cox2p to its insertion in the mitochondrial inner membrane.

FIG. 1

The cox2-20 mutation prevents expression of the ARG8^m reporter fused to COX2 codon 91. (A) The COX2 leader peptide coding region is in black, other COX2 codons are in white, and the COX2 mRNA 5′-UTL is indicated by the thin line. ARG8^mcodons are in grey. Triangles, processing sites of the pre-Cox2p leader peptide (black) and the pre-Arg8p matrix targeting signal (grey); ×, the mutated pre-Cox2p cleavage site of the cox2-24 mutants, in which COX2 codons 15 and 16 were changed from AAT GAT (ND) to AAG CTT (KT) (creating an HindIII site). Strains were patched on complete glucose medium, replica plated to nonfermentable medium (YPEG) and minimal medium lacking arginine (−Arg) or containing arginine (+Arg), and incubated for 2 days at 28°C. In descending order, the strains used were DL2, NB80, HMD22 (this strain contains the only chimeric gene used here entirely lacking COX2 codons), NB43, NB65, NB72, NB58, and NB120 (Table 1). (B) Mitochondrial translation products were radioactively pulse labeled for 10 min in cells treated with cycloheximide (see Materials and Methods). Protein aliquots normalized for radioactivity were analyzed by electrophoresis on sodium dodecyl sulfate–15% polyacrylamide gels and subsequently by phosphorimaging. The positions of the Cox2p-Arg8p fusion protein and products of endogenous mitochondrial genes are indicated. The fusion protein coded by cox2-24::ARG8^m migrated slightly slower than the cox2::ARG8^m fusion protein on this gel and, more dramatically, on others (not shown) due to the lack of leader peptide processing, and it appears to have yielded novel degradation products. Mature Arg8p comigrates with the upper part of the Cox1p band and is difficult to detect after pulse labeling. Strains were cox2::ARG8^m (NB43), cox2-60::ARG8^m (NB54; this strain lacks COX2 nucleotides −63 to +66), cox2-24::ARG8^m (NB72), and cox2-20::ARG8^m (NB120).

FIG. 2

Deletion of the leader peptide coding region has no effect on cox2::ARG8^m mRNA levels but prevents accumulation of reporter protein Arg8p. Northern blot analysis (top two panels) was carried out on total cellular RNAs from the indicated strains: COX2 Δarg8 (NB80), COX2 ARG8 (DL2), cox2::ARG8^m (NB43), Δcox2 (NB40-3C), cox2-20::ARG8^m (NB120), and cox2-21::ARG8^m (NB121) (all strains, except DL2, contain a nuclear arg8::hisG mutation). The COX2 mRNA (0.85 kb) and cox2::ARG8^m mRNA (1.7 kb) were detected simultaneously with the COX2 and cox2::ARG8^m probes (see Materials and Methods), and 15S rRNA hybridization served as a loading control. Western blot analysis was carried out with 100 μg of total protein from the indicated strains (as above) by using anti-Arg8p antibody (see Materials and Methods). Both the mature Arg8p protein (lower band) and the unprocessed Cox2p-Arg8p fusion protein (upper band) are indicated. The middle band corresponds to a cross-reacting protein present in strains lacking a functional ARG8 gene. For unknown reasons, the strength of this signal is variable (Fig. 5 and 9).

FIG. 3

Effect of mutations within the leader peptide coding region on expression of COX2 and the cox2::ARG8^m reporter gene. (A) The sequence of the first 18 codons of the wild-type COX2 open reading frame and the amino acids they specify are shown for reference. The arrow indicates the leader peptide cleavage site. Strains carrying the indicated alleles affecting the COX2 leader peptide coding region in either COX2 or cox2::ARG8^m were patched on complete glucose medium and replica plated to nonfermentable medium (YPEG) for alleles in COX2 and to minimal medium lacking arginine (−Arg) or containing arginine (+Arg) for alleles in cox2::ARG8^m. Incubation was for 2 days at 28°C. Periods indicate deleted residues, and the boxed “R” in bold indicates a mutation of codon 6 from AGA (R) to CGT (R). The arrow marks the leader peptide cleavage site. The indicated alleles of COX2 and cox2::ARG8^m, respectively, correspond to the following strains: COX2 (NB80 and NB43), cox2-27 (NB117 and NB127), cox2-23 (NB100 and NB123), cox2-28 (NB118 and NB128), cox2-35 (NB172 and NB135), cox2-22 (NB64 and NB122), cox2-29 (NB119 and NB129), and cox2-20 (NB58 and NB120). (B) Northern blot analysis was used to detect the COX2 mRNA and 15S rRNA in total RNA (see Materials and Methods) prepared from strains with the following alleles: COX2 (NB80), Δcox2 (NB40-3C), cox2-22 (NB64), and cox2-27 (NB117). (C). Western blot analysis probing with the anti-Cox2p monoclonal antibody CCO6 (38) was used to detect Cox2p in 100 μg of total protein (see Materials and Methods) from strains with the following alleles: cox2-22 (NB64), cox2-24 (NB65), COX2 (HMD21), Δcox2 (NB73), and cox2-27 (NB140). A faint band of slightly lower mobility than the wild type, corresponding to unprocessed pre-Cox2p, was detected in the cox2-24 lane after overexposure (not shown).

FIG. 4

Suppression of the cox2-22 and cox2-27 mutations in transformants containing multiple copies of the nuclear genes MRPL36 and PET111. Strains carrying the cox2-22 or cox2-27 alleles (NB64 or NB117) as well as the cox2-22::ARG8^m or cox2-27::ARG8^m alleles (NB122 or NB127) were transformed with the empty vector (YEp352) and plasmids carrying MRPL36 (pNB107), PET111 (pJM20), or PET111-20 (pJM57), as was indicated (see Materials and Methods). Transformants were grown as patches on minimal medium containing arginine (+Arg) and then were replica plated to nonfermentable medium (YPEG) and minimal medium lacking arginine (−Arg) and incubated at 28°C for 2 or 4 days, respectively.

FIG. 5

Analysis of intragenic suppressors of cox2-22 and cox2-27. (A) Nucleotide sequences of mtDNA from the cox2-22 and cox2-27 mutants, as well as the indicated pseudorevertants, were determined (see Materials and Methods). They are aligned with the sequence of the first 18 codons of the wild-type COX2 open reading frame. The names of the wild-type, mutant, and suppressor alleles are shown with their deduced amino acid sequences. Only the codons modified by the intragenic suppressor mutations are shown for those alleles. Deleted nucleotides are indicated by dots, and nucleotide and/or amino acid substitutions are in boldface. The SnaBI site created by the cox2-22 mutation is underlined. Identical suppressor mutations selected from both the cox2-22 and cox2-27 mutants are boxed. Nucleotide substitutions that do not change the encoded amino acid are circled. Multiple independent isolates of identical suppressor mutations are indicated by names on the same line. The arrow indicates the leader peptide cleavage site. (B) Relative steady-state levels of mRNAs and proteins in cox2-22 and a pseudorevertant strain. For the top two panels, the COX2 mRNA and 15S rRNA were detected on the same blot containing RNA from COX2 (NB80), cox2Δ (NB40-3C), cox2-22 (NB64), and cox2-22S8a (NB64S8A) strains by successive hybridization to COX2 and 15S probes (see Materials and Methods). In the third panel, 100 μg of total proteins from the same strains used in the Northern analysis were probed simultaneously with anti-Cox2p and anti-porin antibodies (both from Molecular Probes, Inc.), as indicated. For the bottom panel, the strains named above were crossed with the cox2-60::ARG8^m strain NB66 and diploids were selected on minimal medium supplemented with leucine and arginine. Samples containing 50 μg of total proteins were prepared from mitochondrial recombinants and analyzed by Western blotting using anti-Arg8p antibody.

FIG. 6

Arg8p synthesis is blocked by cox2-27, it was restored in its pseudorevertants, and it was unaffected by intragenic suppressor mutations in an otherwise wild-type context. Mitochondrial translation products were radioactively pulse labeled and analyzed as described for Fig. 1B. The position of mature Arg8p is indicated. (A) Arg8p synthesis in the cox2-27 mutant and pseudorevertants cox2::ARG8^m (NB43), cox2-60::ARG8^m (NB54), cox2-27::ARG8^m (NB127), cox2-27,S1::ARG8^m (NAB3), cox2-27,S2::ARG8^m (NAB4), cox2-27,S4::ARG8^m (NAB5), cox2-27,S6::ARG8^m (NAB6), cox2-27,S7::ARG8^m (NAB7), cox2-27,S12::ARG8^m (NAB8), and cox2-27,S3::ARG8^m (NAB9). (B) Arg8p synthesis in strains bearing only intragenic suppressor mutations: cox2::ARG8^m (NB43), cox2-60::ARG8^m (NB54), cox2-27::ARG8^m (NB127), cox2-S1::ARG8^m (NAB41), cox2-S2::ARG8^m (NAB46), cox2-S3::ARG8^m (NAB45), and cox2-S4::ARG8^m (NAB84).

FIG. 7

Evidence for a stem-loop structure in the leader peptide coding region that affects translation. (A) Predicted secondary structure of the 18 first codons of the wild-type COX2 mRNA and the corresponding codons of the cox2-27 and cox2-22 alleles. Structures were generated for 28°C by using the program mfold 3.0 (http://mfold2.wustl.edu/∼mfold/rna/form1-2.3.cgi) with default parameters (29, 59). Similar structures of this region were obtained when folding extended mRNA fragments including the 18 first codons. The initiation codon in each sequence is boxed. Arrows indicate the pre-Cox2p cleavage site position in the encoded polypeptide. Black dots indicate G:C base pairs and grey dots indicate weaker A:U or G:U base pairs. The nucleotide substitutions found in the various cox2-27 and cox2-22 pseudorevertants (Fig. 5) are marked. The cox2-23 allele is also shown on the cox2-22 structure since it can be viewed as an intragenic suppressor of cox2-22 with two substitutions. (B) Phenotypic effects of mutations in the stem structure of the cox2-27 mRNAs. Diploid recombinant cells carrying the indicated alleles were replicated on minimal SD medium lacking arginine (−Arg) and nonfermentable medium (YPEG) and were incubated at 28°C for 2 and 3 days, respectively. The diploid cells were selected for complementing nutritional markers on minimal SD medium containing arginine after mating strains containing the following alleles with the cox2-60::ARG8^m strain NB54 (lacking nucleotides −63 to +66): cox2-27, NB140; cox2-27, S7, NB140S7; cox2-27, S12, NB140S12; cox2-27, S7, S12, NB241 for growth on YPEG medium and NB242 for growth on medium lacking arginine.

FIG. 8

Shifting the reading frame to +1 in the leader peptide coding region has no effect on respiratory growth or arginine prototrophy. Control strains and strains carrying the indicated frameshift alleles in the COX2 leader peptide coding region were patched on complete glucose medium and replica plated on nonfermentable medium (YPEG) for alleles inserted in the plain COX2 gene and on minimal medium lacking arginine (−Arg) or containing arginine (+Arg) for alleles inserted in the reporter construct. Photographs were taken after a 2-day incubation at 28°C. +, addition of an A nucleotide; for cox2-36, cox2-38, and cox2-39, this is immediately following the initiation codon, and for cox2-37, it is immediately following the sixth codon. −, deletion of the G nucleotide immediately before wild-type codon 15 in cox2-36 and cox2-37 and of the A nucleotide immediately before wild-type codon 7 in cox2-38 and cox2-39. Deleted amino acids are shown by dots, and modified amino acids are in bold letters. The arrow marks the leader peptide cleavage site. Alleles and strains (for COX2 and for cox2::ARG8^m, respectively) were as follows: COX2 (NB80 and NB43), cox2-20 (NB58 and NB120), cox2-36 (NB173 and NB136), cox2-37 (NB174 and NB137), cox2-38 (NB175 and NB138), and cox2-39 (NB176 and NB139).

FIG. 9

Effects of silent substitutions and shifting the reading frame to −1 in the first six codons of a leader peptide coding region lacking codons 7 to 14. (A) Allele names, their nucleotide sequences and predicted amino acid sequences are shown on the left. All are derived from the functional allele cox2-35, which carries a deletion of codons 7 to 14 indicated by the kinked line (Fig. 3). Altered nucleotides and amino acids are shown in bold letters. −, deletion of two T nucleotides and addition of an A nucleotide in the DNA sequence; +, addition of a T nucleotide in cox2-43. The two codons further deleted in cox2-43R1 are indicated by dots. The long arrow indicates the origin of cox43R1 as a spontaneous pseudorevertant of cox2-43. The short arrow indicates the leader peptide cleavage site. On the right, strains carrying the indicated versions of the COX2 leader peptide coding region were patched on complete glucose medium and replica plated on nonfermentable medium (YPEG) or glucose medium (YPD) for alleles inserted in the COX2 gene (left panel) and on minimal medium lacking arginine (−Arg) or containing arginine (+Arg) for alleles inserted in the cox2::ARG8^m construct (right panel). Plates were incubated at 16, 28, or 36°C as indicated and were photographed after 1- to 10-day incubations, depending on the medium and temperature. Strains were (for COX2 and for cox2::ARG8^m, respectively) COX2, NB80 and NB43; cox2-35, NB172 and NB135; Δcox2, NB97 and NB54; cox2-41, NB177 and NB179; cox2-43, NB178 and NB181; cox2-43R1, NB178R1 and a diploid recombinant from the cross of NB178R1 × NB66. (B) Relative steady-state levels of mutant mRNAs were determined by Northern blot analysis of total RNAs from the indicated strains (as for panel A), which were hybridized simultaneously to the COX2 and cox2::ARG8^m probes and were reprobed with the 15S rRNA probe (see Materials and Methods). (C) Relative steady-state levels of Cox2p and Arg8p in cells grown at 28 or 36°C were determined by Western blot analysis. Samples with 200 μg of total proteins from the indicated strains (as in panel A), which were grown at the indicated temperatures, were probed with either anti-Arg8p or anti-Cox2p and anti-porin antibodies (all from Molecular Probes, Inc.) as indicated. The COX2 panel was overexposed to reveal low levels of Cox2p in the cox2-43 mutant grown at 36°C.