Comparative Analysis of Mitochondrial N-Termini from Mouse, Human, and Yeast

Abstract

The majority of mitochondrial proteins are encoded in the nuclear genome, translated in the cytoplasm, and directed to the mitochondria by an N-terminal presequence that is cleaved upon import. Recently, N-proteome catalogs have been generated for mitochondria from yeast and from human U937 cells. Here, we applied the subtiligase method to determine N-termini for 327 proteins in mitochondria isolated from mouse liver and kidney. Comparative analysis between mitochondrial N-termini from mouse, human, and yeast proteins shows that whereas presequences are poorly conserved at the sequence level, other presequence properties are extremely conserved, including a length of ∼20-60 amino acids, a net charge between +3 to +6, and the presence of stabilizing amino acids at the N-terminus of mature proteins that follow the N-end rule from bacteria. As in yeast, ∼80% of mouse presequence cleavage sites match canonical motifs for three mitochondrial peptidases (MPP, Icp55, and Oct1), whereas the remainder do not match any known peptidase motifs. We show that mature mitochondrial proteins often exist with a spectrum of N-termini, consistent with a model of multiple cleavage events by MPP and Icp55. In addition to analysis of canonical targeting presequences, our N-terminal dataset allows the exploration of other cleavage events and provides support for polypeptide cleavage into two distinct enzymes (Hsd17b4), protein cleavages key for signaling (Oma1, Opa1, Htra2, Mavs, and Bcs2l13), and in several cases suggests novel protein isoforms (Scp2, Acadm, Adck3, Hsdl2, Dlst, and Ogdh). We present an integrated catalog of mammalian mitochondrial N-termini that can be used as a community resource to investigate individual proteins, to elucidate mechanisms of mammalian mitochondrial processing, and to allow researchers to engineer tags distally to the presequence cleavage.

Fig. 1.

Identification of cleavage sites in mouse mitochondrial proteins and comparison with other high throughput datasets. A, schematic overview of N-terminal mapping of mitochondrial proteins from mouse liver and kidney. B, comparison between mouse cleavage sites and published high throughput N-proteome datasets from human (6) and yeast (4) mitochondria, out of all proteins detected in both studies. Overlaid numbers represent percent matches within 2 aa to account for potential alignment errors. C, comparison of mouse cleavage sites determined by subtiligase enrichment versus a re-analysis of existing mass spectrometry data from mitochondria isolated from 14 mouse tissues in MitoCarta2.0 (1, 2), out of all proteins detected in both studies.

Fig. 2.

Integration of complementary data sources to support N-terminal cleavages. For four selected genes, the partial N-terminal sequence for the full-length protein is shown (black text) along with all observed N-terminal peptides from the subtiligase dataset in mouse (gray text) as well as a table showing experimental support from (i) this study; (ii) MS/MS of mitochondria isolated from 14 mouse tissues (1, 2); and (iii) homologous cleavage site in the human ortholog in the Vaca Jacome et al. (6) dataset, as well as computational predictions from TargetP (18), TPpred3 (19), and MitoFates (20). Red shading indicates strength of evidence normalized for each column separately. Dashed red line indicates most common cleavage, based on number of mouse subtiligase spectra. Based on published canonical cleavage sites (4), MPP indicates that the observed cleavage site has an arginine at position −2 (P₂), and MPP+Icp55 indicates that the observed cleavage site has an arginine at position −3 (P₃).

Fig. 3.

Comparative analysis of mitochondrial presequences. A, accuracy of three computational programs (TargetP (18), TPpred3 (19), and MitoFates (20)) at predicting the correct cleavage site for 327 mouse genes, based on the subtiligase N-proteome master set. B, evolutionary conservation of mouse presequences based on precomputed multiple alignments (EggNOG version 4.0 (21)). Histogram of presequence length (aa) (C) and histogram of presequence net charge (D) show similar results between the three independent studies are shown. Histogram of frequency of each amino acid in the first position after presequence cleavage show an enrichment for small residues (E) compared with the histogram of each amino acid in all mitochondrial full-length proteins (F). Note comparative analyses refer to N-terminal master sets derived from this study (mouse) and published data in human from Vaca Jacome et al. (6) and in yeast from Vogtle et al. (4). N-end rule categorization as in Vogtle et al. (4).

Fig. 4.

Comparative analysis of cleavage site motifs. A, histogram of position of the first arginine relative to the reported cleavage site from the stringent reference set of N-termini from this study (mouse, n = 119), Vaca Jacome et al. (6) (human, n = 47), and Vogtle et al. (4) (yeast, n = 94). B, barplot shows percent of all reference set N-termini that have an arginine at position −2, −3, −10, or none. Note: a cleavage site may be counted in multiple categories. C, cleavage site motifs generated by WebLogo (24) are shown for all reference set N-termini (1st row), the subset with the first arginine at position −2 (2nd row), the subset with first arginine at position −3 and not −2 (3rd row), the subset excluding R-2 and R-3 (4th row), and the subset with arginine at position −10 (5th row). D, shown are all known targets of Icp55 in yeast (3) that have orthologous mouse N-termini detected in this study. Gene names are shown with the 10 aa preceding the cleavage site in yeast (red text highlights known motifs for MPP and Icp55). E, shown are all known targets of Oct1 in yeast (4) that have orthologous mouse N-termini detected in this study. Gene names are shown with the 10 aa preceding the cleavage site (residues in red highlights the canonical motifs for Oct1 in yeast and for MPP and Icp55 in mouse).

Fig. 5.

Outlier cleavages >100 aa from annotated protein start. 17 proteins with outlier cleavages are displayed along with their PFAM protein domains and transmembrane helices (light and dark gray rectangles, respectively). Location of observed N-terminal peptides are indicated by tick marks, with red color highlighting the peptide with the greatest spectral count. Asterisk indicates N-terminal peptide detected at orthologous site in human (6) as well as in mouse. Red rectangles indicate supporting literature evidence of cleavage in a mammalian ortholog.