Sequential processing of a mitochondrial tandem protein: insights into protein import in Schizosaccharomyces pombe

Abstract

The sequencing of the genome of Schizosaccharomyces pombe revealed the presence of a number of genes encoding tandem proteins, some of which are mitochondrial components. One of these proteins (pre-Rsm22-Cox11) consists of a fusion of Rsm22, a component of the mitochondrial ribosome, and Cox11, a factor required for copper insertion into cytochrome oxidase. Since in Saccharomyces cerevisiae, Cox11 is physically attached to the mitochondrial ribosome, it was suggested that the tandem organization of Rsm22-Cox11 is used to covalently tie the mitochondrial ribosome to Cox11 in S. pombe. We report here that pre-Rsm22-Cox11 is matured in two subsequent processing events. First, the mitochondrial presequence is removed. At a later stage of the import process, the Rsm22 and Cox11 domains are separated by cleavage of the mitochondrial processing peptidase at an internal processing site. In vivo data obtained using a tagged version of pre-Rsm22-Cox11 confirmed the proteolytic separation of Cox11 from the Rsm22 domain. Hence, the tandem organization of pre-Rsm22-Cox11 does not give rise to a persistent fusion protein but rather might be used to increase the import efficiency of Cox11 and/or to coordinate expression levels of Rsm22 and Cox11 in S. pombe.

FIG. 1.

Schematic representation of mitochondrial fusion proteins of S. pombe and of their homologues in S. cerevisiae. The proteins depicted are (A) SPAC1420.04c, (B) SPAC22E12.01c. (C) SPAC22A12.08c, and (D) SPBP4H10.15 (55, 56). Numbers indicate amino acid positions in the proteins. Black boxes depict conserved regions of the proteins and are labeled according to the S. cerevisiae nomenclature. Mitochondrial targeting sequences (pre) were predicted using the TargetP or Mitoprot algorithm (9, 11); positions of the predicted processing sites are indicated. Transmembrane domains (TM) of the various proteins are indicated. S.c., S. cerevisiae; S.p., S. pombe.

FIG. 2.

Pre-Su9DHFR can be imported into isolated mitochondria of S. cerevisiae and of S. pombe. (A) Pre-Su9DHFR was synthesized in the presence of [³⁵S]methionine in reticulocyte lysate and incubated with isolated mitochondria of S. cerevisiae and S. pombe in the presence of NADH and ATP (+Δψ) or the presence of valinomycin to deplete the membrane potential (−Δψ). After the import reaction, the samples were incubated in the absence or presence of proteinase K (PK). Mitochondria were reisolated, washed, and dissolved in sample buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. (B) Mitochondria of S. cerevisiae and S. pombe were incubated for different time periods with radiolabeled pre-Su9DHFR as described for panel A. The amount of matured and protease-protected Su9DHFR was quantified and is expressed in relation to the total amount of added preprotein after correction for the respective methionine contents of the preprotein and the mature species. (C) Pre-Su9DHFR was incubated with isolated mitochondria for 10 min at 20°C in the absence or presence of ATP, malate, succinate, creatine phosphate and creatine kinase (CK+CP), NADH, or valinomycin (Val.). The amount of imported protein was quantified and expressed in relation to that of the total precursor protein.

FIG. 3.

Pre-Rsm22-Cox11 can be imported into isolated mitochondria. (A) Radiolabeled pre-Rsm22-Cox11 was incubated with isolated S. pombe mitochondria for 20 min at 30°C in the absence (lanes 2 and 3) or presence (lanes 4 and 5) of valinomycin. The samples were divided, and proteinase K (PK) was added to half of the samples. After 30 min on ice, mitochondria were reisolated, washed, and dissolved in sample buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. Protease-resistant fragments of the imported precursor protein are indicated by arrows. Lane 1 shows 10% of the precursor protein used for each of the import reactions shown in lanes 2 to 5. Positions of molecular weight standards are indicated. (B) S. pombe wild-type (wt) mitochondria or mitochondria of a strain harboring a plasmid for expression of a C-terminally HA-tagged version of pre-Rsm22-Cox11 were incubated in the absence or presence of proteinase K. In the samples shown in lanes 4 and 5, the mitochondria were lysed by incubation with 1% Triton X-100 (TX) prior to the protease treatment. Proteins were subjected to SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and detected by Western blotting using HA-specific antibodies. Western blot signals of the matrix protein aconitase and the inner membrane protein cytochrome c₁ (Cyt. c₁) are shown as controls.

FIG. 4.

Pre-Rsm22-Cox11 is processed in two steps. (A) Pre-Rsm22-Cox11 was imported at 30°C for the time periods indicated. The mitochondria were reisolated and divided into three aliquots. One was mock treated (lanes 2 to 7), one was incubated with proteinase K (PK) (lanes 8 to 13), and one was diluted 10-fold in 20 mM HEPES (pH 7.4) and treated with protease (lanes 14 to 19). The mitochondria were further treated as described for Fig. 3. The inset at the bottom shows an enlargement of a section of the upper panel. (B) The radioactive signal of the matured Rsm22-Cox11 species for each time point was quantified by densitometry. The values are depicted as percentages of the signals of the total precursor protein used for the import experiments following correction for the specific methionine contents of the precursor and the Rsm22-Cox11 protein. Thus, after 5 to 10 min of import, about 6% of the imported protein was present as N-terminally processed Rsm22-Cox11 intermediate. At later stages of the import process, this species was decreased and instead the fully matured Rsm22 and Cox11 species accumulated. (C) The removal of the presequence of pre-Rsm22-Cox11 depends on the membrane potential. Pre-Rsm22-Cox11 was imported in the absence or presence of a membrane potential for 2 or 5 min as indicated. The generation of the Rsm22-Cox11 intermediate was analyzed as for panel A. (D) Pre-Rsm22-Cox11 and pre-Rsm22-Cox11ΔC were synthesized in vitro and incubated with S. pombe mitochondria. Each import reaction mixture was divided into three aliquots. One was mock treated, and one was incubated with proteinase K. In the third sample, the outer membrane of the mitochondria was ruptured by hypotonic swelling (sw) and the sample was treated with protease. The generated fragments were analyzed as for panel A. (E) Schematic representation of the protein fragments which are produced from pre-Rsm22-Cox11 following import into mitochondria. ATG codons allowing internal starts of the translation products are depicted. The fragments observed in the in vitro import experiments are indicated and labeled as in panel A. See text for details.

FIG. 5.

Pre-Rsm22-Cox11 can be imported and correctly processed in S. cerevisiae mitochondria. (A) The import of radiolabeled pre-Rsm22-Cox11 into mitochondria of S. cerevisiae was assessed as described for Fig. 4D. PK, proteinase K; sw, rupture of outer membrane of mitochondria by hypotonic swelling. (B) The presequence of pre-Rsm22-Cox11 is removed upon import into isolated S. cerevisiae mitochondria. Pre-Rsm22-Cox11 was incubated for 5 min with S. cerevisiae mitochondria, and the generation of the Rsm22-Cox11 intermediate was analyzed as described for Fig. 4. (C) Mitochondria were purified from wild-type cells (wt), a Tim23-depleted strain (Tim23↓), or an ssc1-3 mutant. The mitochondria were resuspended in import buffer and incubated for 10 min at 37°C to induce the temperature-sensitive phenotype of the ssc1-3 mutant. To assess the kinetics of the import reaction, radiolabeled pre-Rsm22-Cox11 was added and left for various time periods. Nonimported material was removed by protease treatment, and the amount of imported Rsm22 fragment was quantified. Following correction of the specific methionine content of the Rsm22 fragment and the pre-Rsm22-Cox11 precursor, the percentage of imported protein was calculated for each time point.

FIG. 6.

MPP can proteolytically separate the Rsm22 and Cox11 domains of pre-Rsm22-Cox11. (A) The plasmid for in vitro synthesis of pre-Rsm22-Cox11 was digested at unique restriction sites in order to produce truncated versions of the radiolabeled preprotein. The upper panel shows the positions of the restriction sites in the corresponding protein sequence of pre-Rsm22-Cox11. The truncated variants of the preprotein were imported into S. pombe mitochondria. Nonimported protein was removed by protease treatment, and the size of the processed Rsm22 fragment was assessed by SDS-polyacrylamide gel electrophoresis and autoradiography. Black arrowheads depict the Rsm22 fragment resulting from maturation of the undigested plasmid. White arrowheads show the positions of the fragments which resulted from the truncated preproteins. (B) Amino acid sequence of the region around the internal processing of pre-Rsm22-Cox11. Hydroxylated residues are highlighted by black boxes, and charged residues are depicted. The hydrophobic transmembrane (TM) domain of the Cox11 part is indicated. The arrows point to the C termini of the truncated versions of the precursor protein. (C) Radiolabeled pre-Oxa1, pre-Rsm22-Cox11, and pre-Rsm22-Cox11ΔC were incubated in 10 mM NaCl and 20 mM Tris (pH 7.4) in the absence or presence of 5 μg purified MPP (33) for 3 h at 30°C. The proteins were resolved by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. The positions of the mature Oxa1 (Oxa1) and the Oxa1 presequence (pre) are indicated. Signals 1, 2, and 3 depict the three translation products obtained by in vitro synthesis with the plasmids for expression of the tandem proteins. The resulting fragments are indicated. See text for details. (D) Schematic representation of pre-Rsm22-Cox11 and its processing sites.

FIG. 7.

Model for the topogenesis of Cox11 in S. cerevisiae (left) and S. pombe (right). In S. cerevisiae, as in most organisms, Cox11 is expressed in the cytosol as precursor protein with a mitochondrial presequence (pre-Cox11). Proteins of this type typically follow a stop-transfer pathway via the TOM and TIM23 translocases. During or following membrane insertion, the presequences are removed by MPP. The pre-Rsm22-Cox11 tandem protein of S. pombe is matured by at least two subsequent processing events. First, the presequence is removed by MPP early during the import process. Then, the Rsm22-Cox11 intermediate is further imported in a reaction that is driven by mtHsp70 until the internal processing site reaches the matrix. There, the Rsm22 and Cox11 domains are proteolytically separated by MPP. Presumably this second cleavage by MPP is followed by a subsequent processing step, as the endogenous Cox11 protein is smaller than the in vitro processing product of MPP.