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. 2020 Nov 15;31(24):2657-2668.
doi: 10.1091/mbc.E20-08-0524. Epub 2020 Sep 30.

More than just a ticket canceller: the mitochondrial processing peptidase tailors complex precursor proteins at internal cleavage sites

Jana Friedl  1 , Michael R Knopp  2 , Carina Groh  1 , Eyal Paz  3 , Sven B Gould  2 , Johannes M Herrmann  1 , Felix Boos  1
Affiliations

More than just a ticket canceller: the mitochondrial processing peptidase tailors complex precursor proteins at internal cleavage sites

Jana Friedl et al. Mol Biol Cell. .

Abstract

Most mitochondrial proteins are synthesized as precursors that carry N-terminal presequences. After they are imported into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase (MPP). Using the mitochondrial tandem protein Arg5,6 as a model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into mitochondria and subsequently separated into two distinct enzymes. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor at its N-terminus and at an internal site. The peculiar organization of Arg5,6 is conserved across fungi and reflects the polycistronic arginine operon in prokaryotes. MPP cleavage sites are also present in other mitochondrial fusion proteins from fungi, plants, and animals. Hence, besides its role as a "ticket canceller" for removal of presequences, MPP exhibits a second conserved activity as an internal processing peptidase for complex mitochondrial precursor proteins.

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Figures

FIGURE 1:
FIGURE 1:
Arg5,6 is a composite mitochondrial precursor that is processed twice by MPP in the mitochondrial matrix. (A) Schematic representation of arginine biosynthesis in S. cerevisiae. Shown in bold are the enzymes that catalyze the respective steps as well as their E. coli orthologues. (B) When Arg5,6 was C-terminally HA-tagged, immunoblotting revealed a single band at 40 kDa, indicating proteolytic cleavage of the 90-kDa precursor protein. (C) Radiolabeled Arg5,6 precursor was incubated with isolated mitochondria for the indicated times and analyzed by SDS–PAGE and autoradiography. Nonimported material was digested with proteinase K (left half). Twenty percent of the total lysate used per import lane was loaded for control. The membrane potential (Δψ) was dissipated with VAO (valinomycin, antimycin, oligomycin). p, precursor, i, intermediate. (D) His-tagged MPP was expressed and purified from E. coli. Radiolabeled Arg5,6 precursor was incubated with isolated mitochondria for 15 min or purified MPP for 90 min. The processing of Arg5,6 was analyzed by SDS–PAGE, Western blotting, and autoradiography. (E) C-terminally HA-tagged Arg5,6 was expressed in wild-type and mas1ts cells. Cultures were grown at 37°C overnight to impair MPP activity. The processing of Arg5,6 was analyzed by SDS–PAGE and immunoblotting. Ilv5 is a presequence-containing matrix protein. Sod1 is localized to the cytosol and the intermembrane space and carries no presequence. (F) Arg5,6 is imported into the mitochondrial matrix and cleaved twice by MPP: once at the N-terminus to remove the presequence, and once internally at an iMTS-L to separate Arg6 and Arg5.
FIGURE 2:
FIGURE 2:
The internal MPP cleavage site in Arg5,6 is flanked by an iMTS-L and adheres to the R-2 motif. (A) Arg5,6 was subjected to TargetP profiling. High values indicate regions within the protein that structurally resemble mitochondrial presequences. (B) Putative cleavage sites for mitochondrial processing proteases in the second iMTS-L region of Arg5,6 were predicted with MitoFates. Mutations in the predicted recognition motif were introduced by site-directed mutagenesis. (C) Arg5,6 variants with and without the mutations displayed in B were synthesized as radiolabeled precursors and incubated with isolated mitochondria, followed by removal of nonimported material by PK treatment. Processing of the imported proteins was analyzed by SDS–PAGE, Western blotting, and autoradiography.
FIGURE 3:
FIGURE 3:
Arg5 and Arg6 can be imported separately in vitro and in vivo. (A-C) Radiolabeled precursor proteins of Arg61–502, Arg5503–862, and Su9-Arg5503–862 were incubated with isolated mitochondria for the indicated times and analyzed by SDS–PAGE and autoradiography. Nonimported material was digested with proteinase K (left half). Twenty percent of the total lysate used per import lane is loaded for control. The membrane potential (Δψ) was depleted with VAO. Red arrowheads indicate processing sites. p, precursor, m, mature. (D, E) Yeast cells that lack endogenous Arg5,6 (Δarg5,6) were transformed with plasmids for expression of the indicated Arg5,6 variants and streaked out on plates containing minimal growth medium without arginine.
FIGURE 4:
FIGURE 4:
MPP requires a strong N-terminal MTS for internal processing of precursor proteins. (A, B) Radiolabeled precursor proteins of Arg5344–862 and Su9-Arg5344–862 were incubated with isolated mitochondria for the indicated times and analyzed by SDS–PAGE and autoradiography. Nonimported material is digested with proteinase K (left half). Twenty percent of the total lysate used per import lane is loaded for control. The membrane potential (Δψ) was depleted with VAO. Red arrowheads indicate processing sites. p, precursor, i, intermediate, m, mature. (C) Overview of truncated Arg5,6 variants and their import competence. Su9, presequence of N. crassa subunit 9. (D) Yeast cells expressing indicated variants of Arg5,6, all carrying a C-terminal HA tag, were lysed and protein extracts were analyzed by SDS–PAGE and immunoblotting directed against the HA epitope or Sod1 as a loading control. ev, empty vector. (E) MPP cleaves the Arg5,6 precursors at its internal processing site only if they carry a bona fide N-terminal presequence. Presumably, MPP recognizes its substrates primarily at their N-terminus and then scans the downstream polypeptide for internal cleavage sites.
FIGURE 5:
FIGURE 5:
Tandem organization and mitochondrial localization of Arg5,6 is conserved in fungi, whereas algae synthesize two separate proteins that localize to their plastids. (A) A database of 150 eukaryotes was searched for homologues of full-length Arg5,6 and the separate Arg5 and Arg6 proteins of S. cerevisiae. Clades in which Arg5,6 homologues were encoded as a single fusion protein are colored in blue; clades in which two separate Arg6 and Arg5 genes were found are colored in green. Gray, no Arg5,6 homologues were found in these clades. (B) In species that encode Arg5,6 as fusion protein, TargetP predicts the protein to be localized to mitochondria. In species with separate genes for Arg5 and Arg6, localization is predicted to be plastidal. (C) In fungi, Arg5,6 is a fusion protein that is matured by MPP into two separate enzymes in the mitochondrial matrix. In contrast, algae encode two separate proteins that localize to their plastids. Gamma-proteobacteria encode Arg5 and Arg6 polycistronically.
FIGURE 6:
FIGURE 6:
MPP not only functions as a presequence peptidase, but also has a conserved processing activity at internal cleavage sites of composite precursor proteins. (A) The iMTS-L at which MPP cleaves the Arg5,6 precursor in S. cerevisiae is conserved among species that encode Arg5,6 as fusion protein. Shown are iMTS-L propensity profiles along the sequence of Arg5,6 fusion protein homologues. (B) Previously described mitochondrial polyproteins from different species harbor an iMTS-L at the position of the junction between the fused polypeptides. Arrowheads indicate potential cleavage sites for MPP (red) and the downstream processing peptidases Icp55 (green) or Oct1 (yellow) as predicted by MitoFates. Sdha is no polyprotein, but an alternative N-terminus (dashed line) was recently identified, presumably the result of a posttranslational cleavage (Calvo et al., 2017). (C) In addition to its canonical role in presequence removal from mitochondrial precursor proteins, MPP has a second conserved function. It recognizes internal cleavage sites and processes complex precursor proteins into separate polypeptides.
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