Novel Highly Sensitive, Specific, and Straightforward Strategy for Comprehensive N‑Terminal Proteomics Reveals Unknown Substrates of the Mitochondrial Peptidase Icp55

We present a novel straightforward method for enrichment of N-terminal peptides, utilizing charge-based fractional diagonal chromatography (ChaFRADIC). Our method is robust, easy to operate, fast, specific, and more sensitive than existing methods, enabling the differential quantitation of 1459 nonredundant N-terminal peptides between two <i>S. cerevisiae</i> samples within 10 h of LC–MS, starting from only 50 μg of protein per condition and analyzing only 40% of the obtained fractions. Using ChaFRADIC we compared mitochondrial proteins from wild-type and icp55Δ yeast (30 μg each). Icp55 is an intermediate cleaving peptidase, which, following mitochondrial processing peptidase (MPP)-dependent cleavage of signal sequences, removes a single amino acid from a specific set of proteins according to the N-end rule. Using ChaFRADIC we identified 36 icp55 substrates, 14 of which were previously unknown, expanding the set of known icp55 substrates to a total of 52 proteins. Interestingly, a novel substrate, Isa2, is likely processed by Icp55 in two consecutive steps and thus might represent the first example of a triple processing event in a mitochondrial precursor protein. Thus, ChaFRADIC is a powerful and practicable tool for protease and peptidase research, providing the sensitivity to characterize even samples that can be obtained only in small quantities

TAC42 and TAC40 contain C-terminal β-signals.

<p>(A) Amino acid sequence of the C-termini of the indicated β-barrel proteins in different trypanosomatids. Tb, <i>T</i>. <i>brucei</i>; Tv, <i>T</i>. <i>viva</i>x; Tc, <i>T</i>. <i>cruzi</i>; Lm, <i>L</i>. <i>major</i> and Li, <i>L</i>. <i>infantum</i>. The residues contributing to the predicted β-signal are indicated by the grey bars. The β-signal consenus sequence and the sequence of the two mutated variants (1mut, 4mut) of the β-signal consensus sequence are indicated. Changed residues are shown in red. P_O, polar amino acid; Hy, hydrophobic amino acid. (B) TAC42 top panel: immunoblot analysis of whole cells (Tot), soluble (Cyt) and digitonin-extracted mitochondria-enriched pellet (Mit) fractions of cell lines expressing C-terminally Myc-tagged full length (wt), 1mut and 4mut variants of TAC42, respectively. Lipoamide dehydrogenase (LipDH) and EF1a serve as mitochondrial and cytosolic markers, respectively. TAC42 bottom panel: carbonate extraction at pH 11.5 of the mitochondria-enriched pellet fraction (Mit) of the cell lines depicted above. The pellet (Pe) and the supernatant (Sup) fractions correspond integral membrane and soluble proteins, respectively. ATOM40 and Cyt C serve as markers for integral and peripheral membrane proteins, respectively. TAC40 top and bottom panels, same experiments as for TAC42 and its variants were performed for TAC40 and its two mutated variants. (C) Graphs showing the mean and the standard errors of a quantification of three biological replicates of the experiment shown in (B).</p

Biogenesis of the mitochondrial DNA inheritance machinery in the mitochondrial outer membrane of <i>Trypanosoma brucei</i>

<div><p>Mitochondria cannot form de novo but require mechanisms that mediate their inheritance to daughter cells. The parasitic protozoan <i>Trypanosoma brucei</i> has a single mitochondrion with a single-unit genome that is physically connected across the two mitochondrial membranes with the basal body of the flagellum. This connection, termed the tripartite attachment complex (TAC), is essential for the segregation of the replicated mitochondrial genomes prior to cytokinesis. Here we identify a protein complex consisting of three integral mitochondrial outer membrane proteins—TAC60, TAC42 and TAC40—which are essential subunits of the TAC. TAC60 contains separable mitochondrial import and TAC-sorting signals and its biogenesis depends on the main outer membrane protein translocase. TAC40 is a member of the mitochondrial porin family, whereas TAC42 represents a novel class of mitochondrial outer membrane β-barrel proteins. Consequently TAC40 and TAC42 contain C-terminal β-signals. Thus in trypanosomes the highly conserved β-barrel protein assembly machinery plays a major role in the biogenesis of its unique mitochondrial genome segregation system.</p></div

Recombinant ncTom40 has a β-barrel structure. (a) Far UV CD spectra of ncTom40 in decylmaltoside. (b) Superposition of ¹³C-¹³C proton driven spin diffusion spectra of ncTom40 (red) and hVDAC1 (green; reproduced from [4]), both in DMPC liposomes. The mixing time was 15 ms.

<p>Recombinant ncTom40 has a β-barrel structure. (a) Far UV CD spectra of ncTom40 in decylmaltoside. (b) Superposition of ¹³C-¹³C proton driven spin diffusion spectra of ncTom40 (red) and hVDAC1 (green; reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112374#pone.0112374-Schneider1" target="_blank">[4]</a>), both in DMPC liposomes. The mixing time was 15 ms.</p

Trypanosomal β-signals are functional in yeast and knock-down of trypanosomal Sam50 causes a kDNA segregation defect.

<p>(A) In vitro translated ³⁵S-labeled TAC42 and TAC42-mut4 were incubated for the indicated time with mitochondria either isolated from wildtype (WT) yeast or from a yeast strain lacking the SAM complex subunit sam37 (Δ<i>sam37</i>). Subsequently all samples were analyzed by BN-PAGE and analyzed by autoradiography. A mock reaction lacking organelles was also analyzed. The putative TAC42/Sam50 complex is indicated. (B) Left graph: growth and loss of kDNA in the procyclic <i>T</i>. <i>brucei</i> Sam50-RNAi cell line. Red lines depict percentage of cells still having the kDNA. Right graph: fluorescent intensities of kDNA networks were measured after Sam50 knock down. Red lines mark the median.</p

TAC42 is an essential TAC subunit.

<p>(A) Left panels: IF analysis of procyclic <i>T</i>. <i>brucei</i> cells expressing HA-tagged TAC42 (red). DNA is stained with DAPI (blue). Bar, 5 μm. Inset: magnification of the kDNA region. Bar inset, 1 μm. Right panels: IF analysis of isolated flagella of <i>T</i>. <i>brucei</i> cells expressing HA-tagged TAC42. (B) Left graph: growth and loss of kDNA of the procyclic TAC42-RNAi cell line. Red lines depict percentage of cells still having the kDNA. Right graph: fluorescent intensities of kDNA networks were measured after TAC42 knock down. Red lines mark the median. (C) Left graph: growth and loss of kDNA in the bloodstream form TAC42-RNAi cell line. Right graph: growth and loss of kDNA of a TAC42-RNAi cell line from a bloodstream form <i>T</i>. <i>brucei</i> strain that contains a compensatory nuclear mutation (ATPase γ, L262P) that allows it to grow in the absence of kDNA (bloodstream-L262P) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006808#ppat.1006808.ref027" target="_blank">27</a>]. Insets: Northern blots confirming ablation of the TAC42 mRNA in the different RNAi cell lines. Ethidium bromide-stained gel showing the rRNA region is used as a loading control. (D) Transmission electron micrographs showing the kDNA region of uninduced (-Tet) and 2 days induced TAC42-RNAi cells (+Tet). Bar: 200 nm.</p

APSY experiments recorded at different temperatures and assignments obtained for denatured ncTom40 (339 non-proline residues) by MARS [31].

<p>Assignments classified by MARS as low are not reliable and were excluded from further analysis.</p><p>APSY experiments recorded at different temperatures and assignments obtained for denatured ncTom40 (339 non-proline residues) by MARS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112374#pone.0112374-Panchal1" target="_blank">[31]</a>.</p

TAC42 is an OM protein whose biogenesis depends on Sam50.

<p>(A) Top panel: immunoblot analysis of whole cells (Tot), digitonin-extracted mitochondria-enriched pellet (Mit) and soluble (Cyt) fractions of cells expressing C-terminally HA-tagged TAC42. ATOM40 and EF1a served as mitochondrial and cytosolic markers, respectively. Middle panel: carbonate extraction at pH 11.5 of the mitochondria-enriched pellet fraction (Mit). The pellet (Pe) and the supernatant (Sup) fractions correspond integral membrane and soluble proteins, respectively. ATOM40 and Cyt C serve as markers for integral and peripheral membrane proteins, respectively. Bottom panel: normalized abundance profile of TAC42 over six subcellular fractions, produced in a previous proteomic analysis [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006808#ppat.1006808.ref035" target="_blank">35</a>]. (B) Immunoblots of total cellular extracts from procyclic Sam50-RNAi cells that constitutively express HA-tagged TAC42. Time of induction is indicated. The mitochondrial β-barrel proteins VDAC and ATOM40, and the α-helically anchored OM protein ATOM69 serve as positive and negative controls, respectively. Cytosolic EF1a was used as a loading control.</p

Trypanosomal β-signals are functional in yeast and knock-down of trypanosomal Sam50 causes a kDNA segregation defect.

<p>(A) In vitro translated ³⁵S-labeled TAC42 and TAC42-mut4 were incubated for the indicated time with mitochondria either isolated from wildtype (WT) yeast or from a yeast strain lacking the SAM complex subunit sam37 (Δ<i>sam37</i>). Subsequently all samples were analyzed by BN-PAGE and analyzed by autoradiography. A mock reaction lacking organelles was also analyzed. The putative TAC42/Sam50 complex is indicated. (B) Left graph: growth and loss of kDNA in the procyclic <i>T</i>. <i>brucei</i> Sam50-RNAi cell line. Red lines depict percentage of cells still having the kDNA. Right graph: fluorescent intensities of kDNA networks were measured after Sam50 knock down. Red lines mark the median.</p

TAC40, TAC60 and TAC42 form a complex.

<p>(A) SILAC-IP of C-terminally HA-tagged TAC40 from digitonin-solubilized whole cell lysates. Mean log₁₀ ratios (TAC40-HA/wt) of proteins detected by quantitative MS in at least two of three independent biological replicates are plotted against the corresponding log₁₀ <i>P</i> values (one-sided t-test). Horizontal dashed line indicates a t-test significance level of 0.05, while vertical dashed lines mark a fivefold enrichment. The bait protein TAC40 is marked in green. The co-precipitated proteins are marked in red. (B) and (C) Cell lines expressing Myc- and HA-tagged versions of the TAC40 interactors, TAC60-Myc and TAC42-HA, respectively, were used for reciprocal SILAC-IPs. For complete lists of proteins for all three IPs, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006808#ppat.1006808.s003" target="_blank">S1</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006808#ppat.1006808.s005" target="_blank">S3</a> Tables. (D) Table indicating the enrichment factors of TAC40, TAC60 and TAC42 in the reciprocal IPs.</p