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. 2005 Feb 1;385(Pt 3):755-61.
doi: 10.1042/BJ20041396.

Recognition and processing of a nuclear-encoded polyprotein precursor by mitochondrial processing peptidase

Tsutomu Oshima  1 , Eiki Yamasaki, Tadashi Ogishima, Koh-ichi Kadowaki, Akio Ito, Sakae Kitada
Affiliations

Recognition and processing of a nuclear-encoded polyprotein precursor by mitochondrial processing peptidase

Tsutomu Oshima et al. Biochem J. .

Abstract

The nuclear-encoded protein RPS14 (ribosomal protein S14) of rice mitochondria is synthesized in the cytosol as a polyprotein consisting of a large N-terminal domain comprising preSDHB (succinate dehydrogenase B precursor) and the C-terminal RPS14. After the preSDHB-RPS14 polyprotein is transported into the mitochondrial matrix, the protein is processed into three peptides: the N-terminal prepeptide, the SDHB domain and the C-terminal mature RPS14. Here we report that the general MPP (mitochondrial processing peptidase) plays an essential role in processing of the polyprotein. Purified yeast MPP cleaved both the N-terminal presequence and the connector region between SDHB and RPS14. Moreover, the connector region was processed more rapidly than the presequence. When the site of cleavage between SDHB and RPS14 was determined, it was located in an MPP processing motif that has also been shown to be present in the N-terminal presequence. Mutational analyses around the cleavage site in the connector region suggested that MPP interacts with multiple sites in the region, possibly in a similar manner to the interaction with the N-terminal presequence. In addition, MPP preferentially recognized the unfolded structure of preSDHB-RPS14. In mitochondria, MPP may recognize the stretched polyprotein during passage of the precursor through the translocational apparatus in the inner membrane, and cleave the connecting region between the SDHB and RPS14 domains even before processing of the presequence.

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Figures

Figure 1
Figure 1. Gene and protein structures of rice SDHB and RPS14, and of the polyprotein precursor
(A) Schematic representation of the rice nuclear rps14 and sdhB genes, and the translated proteins. Grey boxes indicate the SDHB region, and black boxes show the RPS14 region. White boxes represent the presequences and connector regions. The destinations of the mature proteins after processing of the preproteins are indicated. (B) Domain structure of preSDHB–RPS14. Grey and black boxes indicate SDHB and RPS14 domains respectively. The amino acid sequence of the connector region between the SDHB and RPS14 domains is shown. The calculated molecular masses of the respective regions are indicated.
Figure 2
Figure 2. Import of preSDHB–RPS14 into isolated yeast mitochondria
(A) Polyprotein import into isolated mitochondria. The import assay was performed as described in the Experimental section. Isolated yeast mitochondria were pre-incubated for 5 min at 30 °C in import buffer without (lanes 2 and 4) or with (lane 3) valinomycin, which inhibits protein import into mitochondria. 1,10-Phenanthroline (o-Phe) and EDTA (lane 4) were used as inhibitors of metal-dependent proteases. Radiolabelled polyprotein precursor was added to the import mixture, and the incubation was continued for 15 min at 30 °C. The mitochondria were then re-isolated by centrifugation, and the mitochondrial pellet was treated with proteinase K at 0 °C for 30 min. The samples were subjected to SDS/PAGE and analysed using an imaging analyser. Lane 1 contains radiolabelled polyprotein precursor without mitochondria. The arrowheads indicate the polyprotein precursor and the processing products in the mitochondria. (B) Kinetic analysis of polyprotein import. Radiolabelled preSDHB–RPS14 was incubated with isolated yeast mitochondria in import mixture at 30 °C for the times indicated, and the imported proteins were analysed as described above.
Figure 3
Figure 3. Processing of preSDHB–RPS14 by MPP
(A) Dose-dependent processing of preSDHB–RPS14 by MPP. Radiolabelled preSDHB–RPS14 was incubated with purified yeast MPP (0, 25, 50, 100 and 200 ng of MPP in lanes 1–5 respectively) at 30 °C for 30 min. SDS/PAGE was performed on the cleaved polypeptides and they were visualized using an imaging analyser. The arrows indicate the precursor protein and the processing products with the calculated molecular masses. (B) Kinetic analysis of polyprotein processing. Radiolabelled preSDHB–RPS14 was incubated with purified yeast MPP (50 ng) in processing assay buffer at 30 °C for the times indicated. The rate of cleavage of each polypeptide region is shown.
Figure 4
Figure 4. Determination of the site of cleavage between SDHB and RPS14 by MPP
(A) Cleavage of the GST fusion polyprotein by MPP. PreSDHB–RPS14 fused to GST at the N-terminus and with a hexahistidine tag at the C-terminus (GST–SDHB–RPS14) was mostly produced as inclusion bodies in E. coli extract. The polyprotein was then denatured and purified as described in the Experimental section. The unfolded GST fusion polyprotein was incubated with purified yeast MPP in processing assay buffer at 30 °C for 90 min. The processed products were separated by SDS/PAGE and stained with Coomassie Brilliant Blue R-250. Arrows indicate the processing products (GST–SDHB and RPS14). The N-terminal amino acid sequences of the two bands were determined. (B) Site of cleavage between the SDHB and RPS14 regions of the polyprotein. The N-terminal amino acid sequence of GST–SDHB–RPS14 is shown with the processing site that was determined by direct protein sequencing. Actual protein sequence is denoted by an underline.
Figure 5
Figure 5. Effects of mutations in the connector region of the polyprotein on cleavage between the SDHB and RPS14 domains by MPP
Radiolabelled polyprotein precursor was incubated with purified yeast MPP (50 ng) for 4 min at 30 °C. Processed products were subjected to SDS/PAGE and visualized using an imaging analyser. Processing efficiency was determined by quantifying the band density of the cleaved polyprotein precursor. The graphs show the processing efficiency of various polyproteins mutated at positions on the N-terminal side (A) and the C-terminal side (B) of the cleavage site, taking the processing efficiency of MPP for wild-type (WT) polyprotein precursor as 100%. The processing efficiencies were calculated from three independent experiments. The amino acid sequences of the various mutants are given in Table 1.
Figure 6
Figure 6. Unfolding requirement for processing of preSDHB–RPS14 by MPP
(A) Trypsin sensitivity of purified polyproteins. Purified protein (100 ng) was incubated with various concentrations of trypsin (0, 0.01, 0.1, 0.5, 1, 2.5, 5, 10 or 50 ng of trypsin in lanes 1–9 respectively) for 15 min at 30 °C. The polyprotein precursors and the degraded C-terminal polypeptides were analysed by immunoblotting with anti-hexahistidine antibodies and peroxidasecoupled anti-mouse secondary antibodies. (B) Processing of purified polyproteins by MPP. SDHB–RPS14 and GST–SDHB–RPS14 polyprotein precursor were expressed in E. coli, and purified from the soluble fraction. Purified proteins (100 ng) were incubated with various concentrations of MPP (0, 1, 10 or 100 ng of MPP in lanes 1–4 respectively) in processing assay buffer for 30 min at 30 °C. All samples were subjected to SDS/PAGE and gels were analysed by silver staining.
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