Mitochondrial protein turnover: role of the precursor intermediate peptidase Oct1 in protein stabilization

Abstract

Most mitochondrial proteins are encoded in the nucleus as precursor proteins and carry N-terminal presequences for import into the organelle. The vast majority of presequences are proteolytically removed by the mitochondrial processing peptidase (MPP) localized in the matrix. A subset of precursors with a characteristic amino acid motif is additionally processed by the mitochondrial intermediate peptidase (MIP) octapeptidyl aminopeptidase 1 (Oct1), which removes an octapeptide from the N-terminus of the precursor intermediate. However, the function of this second cleavage step is elusive. In this paper, we report the identification of a novel Oct1 substrate protein with an unusual cleavage motif. Inspection of the Oct1 substrates revealed that the N-termini of the intermediates typically carry a destabilizing amino acid residue according to the N-end rule of protein degradation, whereas mature proteins carry stabilizing N-terminal residues. We compared the stability of intermediate and mature forms of Oct1 substrate proteins in organello and in vivo and found that Oct1 cleavage increases the half-life of its substrate proteins, most likely by removing destabilizing amino acids at the intermediate's N-terminus. Thus Oct1 converts unstable precursor intermediates generated by MPP into stable mature proteins.

FIGURE 1:

Ygr031w/Imo32 is a novel Oct1 substrate with an unusual cleavage site motif. (A) Radiolabeled Imo32 precursor was incubated with mitochondria isolated from wild-type (WT) or oct1Δ yeast cells, treated with proteinase K, and analyzed by SDS–PAGE and digital autoradiography. Where indicated, the membrane potential (Δψ) was dissipated prior to the import reaction. prec. and p, precursor; i, intermediate; m, mature. (B) Import of [³⁵S]Imo32 into wild-type and mas1 mutant mitochondria. Samples were treated as in (A). (C) Radiolabeled Imo32 preprotein was incubated with isolated yeast mitochondria, and then treated with proteinase K. The samples, as well as ³⁵S-labeled truncated forms of Imo32, were analyzed by SDS–PAGE and digital autoradiography. (D) Import of [³⁵S]Imo32 with altered presequences into wild-type mitochondria. Cys-37 (corresponding to position −2 with regard to the proposed MPP cleavage site) was replaced by either alanine/A or aspartate/D. (E) Alignment of the cleavage motif of the 14 Oct1 substrate proteins from yeast. Characteristic three amino acid motif is highlighted in dark gray. Light gray denotes first amino acid of mature protein. Arrows indicate cleavage by MPP and Oct1, respectively. (F) Amino acid blot of relative frequency of the presequences (10 residues of the C-terminal segment) and first mature amino acids of the Oct1 substrate proteins identified in yeast (see Figure 1E). Arrows show cleavage sites of MPP and Oct1.

FIGURE 2:

Oct1-processing intermediates possess destabilizing N-terminal amino acids. (A) N-terminal amino acids of the 14 Oct1 substrate proteins in oct1Δ (left panel) and wild-type (WT; right panel) mitochondria. Amino acids are classified according to the N-end rule for Escherichia coli. (B) Immunoprecipitation of Rip1 from wild-type (WT) and oct1Δ mitochondria. Samples were analyzed by SDS–PAGE and immunoblotting (left panel) or protein bands of the elution fraction were visualized by Coomassie Blue staining (right panel). L, load (5%); UB, unbound (5%); W, wash (5%); E, elution (100%). The table shows the amino acid sequence of the Rip1 N-terminus from wild-type (WT) and oct1Δ mitochondria obtained by Edman degradation. (C) Immunoprecipitation of Mdh1 from wild-type (WT) and icp55Δ mitochondria ( Vögtle et al., 2009). Analysis was performed as in (B).

FIGURE 3:

Reexpression of Oct1 in oct1Δ yeast cells. (A) Radiolabeled Atp2 precursor was incubated with mitochondria from WT^Rho+, oct1Δ, or WT^Rho0 cells over increasing periods of time (3, 6, 12 min). Samples were treated with proteinase K and analyzed by SDS–PAGE and digital autoradiography. For the longest time point, the membrane potential (Δψ) was dissipated prior to the import reaction. p, precursor; m, mature. (B) Mitochondria isolated from oct1Δ yeast cells transformed with the empty control plasmid and oct1Δ OCT1 yeast reexpressing Oct1 from its endogenous promoter were incubated with ³⁵S-Atp2 or ³⁵S-Cox4. Samples were analyzed as described in (A) and Materials and Methods. i, intermediate.

FIGURE 4:

The half-life of Oct1 substrate proteins is prolonged upon full processing of the intermediate forms in organello. (A) Mitochondria from oct1Δ and oct1Δ OCT1 (reexpressing the OCT1 gene) yeast cells were incubated at 37°C for the indicated time. Mitochondria were reisolated and analyzed by SDS–PAGE and immunoblotting. N-terminal amino acids of Oct1 intermediate (Int.) and mature (Mat.) forms of the proteins tested are indicated. (B) Quantification of protein turnover: graphs indicate averaged protein levels of the last incubation time points (166, 76 h) in comparison to the level at 0 h. Values of oct1Δ OCT1 mitochondria were set to 100% (control). Error bars represent SD from three different experiments.

FIGURE 5:

Oct1 processing leads to stabilization of its substrate proteins in vivo. (A) and (B) In vivo degradation of proteins analyzed in oct1Δ and oct1Δ OCT1 yeast cells. Protein translation was blocked by addition of cycloheximide. Samples were obtained at various time points, subjected to postalkaline protein extraction, and analyzed by SDS–PAGE and immunoblotting. N-terminal amino acids of Oct1 intermediate (Int.) and mature (Mat.) forms of the proteins tested are indicated. Quantification was performed as in Figure 4.