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Quantitative profiling for substrates of the mitochondrial presequence processing protease reveals a set of nonsubstrate proteins increased upon proteotoxic stress Julia Burkhart, Asli Aras Taskin, René Peiman Zahedi, and F.-Nora Vogtle J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00327 • Publication Date (Web): 08 Oct 2015 Downloaded from http://pubs.acs.org on October 9, 2015

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Journal of Proteome Research

Article

Quantitative profiling for substrates of the mitochondrial presequence processing protease reveals a set of non-substrate proteins increased upon proteotoxic stress

Julia M. Burkhart1, Asli A. Taskin2,3,4, René P. Zahedi1,* and F.-Nora Vögtle2,*

1

Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund,

Germany; 2Institut für Biochemie und Molekularbiologie, ZBMZ, 3Faculty of Biology, Universität Freiburg, 79104 Freiburg, Germany, 4Spemann Graduate School of Biology and Medicine, University of Freiburg, 79104 Freiburg, Germany

* Corresponding authors: F.-Nora Vögtle (Phone: (49) 761 203-97474. Fax: (49) 761 203-5261. E-mail: [email protected]) or René P. Zahedi (Phone: (49) 231 1392-4143. Fax: (49) 231 1392-4850. Email: [email protected])

Abstract The majority of mitochondrial preproteins are targeted via N-terminal presequences that are cleaved upon import into the organelle. The essential mitochondrial processing protease (MPP) is assumed to cleave the majority of incoming precursors. However, only a small fraction of mitochondrial

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precursors have been experimentally analyzed limiting the information on MPP recognition and substrate specificity. Here, we present the first systematic approach for identification of authentic MPP substrate proteins using a temperature-sensitive mutant of the MPP subunit Mas1. Inactivation of MPP at non-permissive temperature leads to accumulation of immature precursors in mitochondria, which were measured by quantitative N-terminal ChaFRADIC. This led to the identification of 66 novel MPP substrates. Deduction of the cleaved presequences determines arginine in position -2 of the cleavage site as main factor for MPP recognition. Interestingly, a set of non-processed proteins was also increased in mas1 mutant mitochondria. Additionally, mas1 mitochondria respond to temperature elevation with an increase in membrane potential and oxygen consumption. These changes might indicate that mas1 cells exert a response to balance the proteotoxic stress induced by MPP dysfunction.

Keywords Mitochondria, presequence protein import, mitochondrial processing protease, ChaFRADIC, mitochondrial stress response, proteostasis, protein turnover

1. Introduction Mitochondria are essential organelles of eukaryotic cells that fulfill important functions in metabolism, bioenergetics and cell survival. They contain approximately 1000 different proteins in yeast and 1500 in human cells.1 Even though mitochondria still maintain their own genome, approximately 99% of


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their proteins are encoded in the nuclear DNA, translated on cytosolic ribosomes and imported into the organelle. The preproteins carry targeting signals within their amino acid sequences that direct import and sorting into the mitochondrial subcompartments.2-4 About 70% of these preproteins use a positively

charged

N-terminal

presequence

as

import

signal.5

This

presequence is recognized by receptors of the TOM complex, the central entry gate in the mitochondrial outer membrane and directs import across the outer membrane where it is handed over to the TIM23 complex for translocation across the inner membrane.2-4 Upon arrival in the innermost mitochondrial compartment, the matrix, the presequence is cleaved by presequence proteases.6,7 While several proteases possess presequence cleavage activity, the majority of presequences is assumed to be processed by the mitochondrial processing peptidase MPP.8-11 This first processing of the precursor is often followed by a second cleavage event carried out by either the Icp55 or Oct1 protease.

5,7,12

The removal of an additional single

amino acid residue or eight residues from the MPP processed intermediate is required to generate a stable N-terminus of the mature protein.5,12 The cleaved presequence is subsequently degraded by the matrix peptidase Cym1.13,14 It has been shown recently that impaired Cym1 activity leads to accumulating presequences that in turn inhibit precursor processing by MPP.15 This results in preprotein accumulation and global mitochondrial dysfunction due to increased turnover of non-processed mitochondrial proteins.15 MPP is a metalloprotease consisting of the two essential subunits Mas1 and Mas2.16,17 An initial sequence analysis of MPP substrates indicated that



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arginine residues at defined positions (R-2, -3, -10 or R-none) within the presequence are important determinants of MPP cleavage site recognition.18 The structure of yeast MPP revealed that presequences bind to and are cleaved by MPP in an elongated conformation and that arginine in position -2 and an aromatic residue in +1 are recognized in a large polar cavity of the enzyme.19 The discovery of Icp55 revealed that R-3 does not represent an MPP cleavage site, but sequentially processing by MPP followed by the removal of a single amino acid converting R-3 into an R-2 motif.5 However, the characterization of MPP cleavage sites was limited due to the availability of only a small subset of experimentally determined authentic MPP substrate proteins including F1β, Cit1, cytochrome b2, Cox4, Mas1, cytochrome c1, Adh3 and Sod2.9,15,16 A systematic and global approach to identify MPP substrates has been missing so far. While several studies have used 1, 10-Phenanthroline, often in combination with EDTA, to identify MPP substrates, this compound does not only inhibit processing activity of MPP but of metalloproteases in general.20-22 However, mitochondria contain several metalloproteases, among them e.g. the m-AAA protease in the inner membrane that also possesses presequence cleavage capacity and proteases involved in peptide degradation (e.g. Cym1 and Prd1). Inhibition of peptide degradation in the mitochondrial matrix results in impaired MPP processing activity. Furthermore, in recent years several novel mitochondrial metalloproteases, e.g. Icp55, have been identified.5 Therefore general inhibition of metalloprotease activity might be unsuitable to identify solely MPP substrates. A small set of substrates was identified by usage of a MPP temperature-sensitive mutant strain, mas1, with point mutations in the


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MPP subunit Mas1.15,23 Growth at elevated temperature results in inactivation of MPP activity and accumulation of newly imported MPP substrates. In this study we aimed to identify novel MPP substrate proteins and to characterize the MPP cleavage site specificity with a systematic approach using mas1 mutant mitochondria. For this we applied quantitative mass spectrometry to compare the authentic N-termini of mitochondrial proteins in wild-type and mas1 mitochondria after shift to non-permissive temperature in vivo.24

Quantitative

ChaFRADIC

(charge-based

fractional

diagonal

chromatography) analysis identified 66 novel MPP substrate proteins, which allowed refinement of the MPP cleavage site establishing R-2 as major determinant of MPP recognition and processing. Furthermore, an unexpected increase in protein levels of non-processed mitochondrial proteins as well as an elevated membrane potential and increased oxygen consumption in mas1 mitochondria upon growth at nonpermissive temperature was observed. These findings might point towards a response in mas1 cells triggered to counterbalance the proteotoxic stress induced by MPP dysfunction.

2. Materials and Methods Yeast strains and growth conditions The following yeast strains were used in this study: YPH499 (Mata, ade2-101, his3-∆200, leu2-∆1, ura3-52, trp1-∆63, lys2-801), mas1 (Matα; ura3-52; trp11; leu2-3; leu2-112; his3-11; his3-15) and corresponding wild-type.5 Cells were grown on non-fermentable carbon source (1% (w/v) yeast extract, 2% (w/v) bacto-peptone, 3% (w/v) glycerol) at 24 °C. Mas1 and corresponding



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wild-type strain were shifted to non-permissive temperature (37 °C) for 6 or 12 h prior to isolation of mitochondria.

Isolation and purification of mitochondria Yeast cells were grown to an OD600 of 1.0. Cells were harvested and incubated for 20 min in DTT buffer (0.1 M Tris-H2SO4, pH 9.4). Cells were reisolated followed by incubation in zymolyase buffer (1.2 M sorbitol, 20 mM K2HPO4-HCl, pH 7.4) containing 3 mg zymolyase per g wet weight of the cells and incubated for 45 min resulting in spheroblast formation. Spheroblasts were washed and resuspended in homogenization buffer (0.6 M sorbitol, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.2% (w/v) BSA, 1 mM PMSF) and cells broken by applying 20 strokes in a glass homogenizer, all following steps were carried out on ice. Cell debris and unbroken cells were separated by centrifugation for 5 min at 1500g. Crude mitochondria were isolated by centrifugation at 16 000g for 15 min. Mitochondria were adjusted to a concentration of 10 mg/ml in SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM MOPS-KOH, pH 7.2), snap-frozen in liquid nitrogen and stored at -80 °C. To obtain highly purified mitochondrial organelles for ChaFRADIC analysis, crude mitochondria were loaded on a sucrose gradient (1.5 ml 60%, 4 ml 32%, 1.5 ml 15% sucrose in EM buffer) according to Meisinger et al.25

In organello protein import Radiolabeled precursor proteins were synthesized using rabbit reticulocyte lysate (Promega) in the presence of [35S]methionine. For in organello import isolated mitochondria (60 µg) and radiolabeled precursor proteins were


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incubated at 25 °C in import buffer (10 mM MOPS-KOH, pH 7.2, 3% (w/v) BSA, 250 mM sucrose, 5 mM MgCl2, 80 mM KCl, 5 mM KPi) supplied with 2 mM ATP and 2 mM NADH. For import in mas1 mutant mitochondria, mitochondria were incubated at 37 °C for 15 min prior to addition of radiolabeled precursor proteins, followed by further incubation at 37 °C for 45 min. Where indicated, the membrane potential was abolished with 1% (v/v) AVO (1 µM valinomycin, 20 µM oligomycin and 8 µM antimycin A) prior to the import reaction. Reactions were stopped by addition of 1% (v/v) AVO and transfer of the samples on ice. Samples were treated with 50 µg/ml proteinase K (Prot. K) for 10 min on ice. Proteinase K was inhibited by addition of 2 mM PMSF (phenylmethylsulfonyl fluoride, in isopropanol). Mitochondria were washed with SEM buffer and analyzed on SDS-PAGE or tris-tricine SDSPAGE followed by digital autoradiography.

Mitochondrial fractionation For hypotonic swelling, mitochondria were suspended in 400 µl EM buffer (1 mM EDTA, 10 mM MOPS-KOH, pH 7.2). Samples were mixed and incubated on ice for 30 min with occasional vortexing. Treatment with Triton X-100 was carried out by lysis of mitochondria in 100 µl SEM buffer containing 1% (v/v) Triton X-100. After mixing and incubation on ice for 10 min, samples were centrifuged for 10 min at 20 000g and analyzed by SDS-PAGE and immunodecoration. For carbonate extraction, mitochondria were incubated in 400 µl freshly prepared 0.1 M sodium carbonate (pH 11.5). After incubation on ice for 30 min, samples were centrifuged at 100 000g for 45 min at 4 °C separating integral membrane (pellet) and soluble or peripheral membrane



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attached proteins (supernatant). Supernatant was precipitated with 10% (w/v) trichloroacetic acid. For sonication, mitochondria were suspended in SEM buffer in the presence of 50 or 500 mM NaCl and sonicated for 3 x 30 s on ice, followed by centrifugation for 45 min at 100 000g. Samples were solubilized in Lämmli buffer, separated by SDS-PAGE and analyzed by Western blotting. Antibodies were generated by immunization of rabbits with synthetic peptides with the following sequences: CNKLVGNEPADIDKYIIQRK for Fmp46/Ipm16; CSKDILDISASLEKIAT for Fmp52/Ipm25. The peptides were coupled to keyhole

limpet

hemocyanin

(KLH)

via

N-terminal

cysteines.

Immunodecoration was performed according to standard protocols.

Membrane potential measurement The membrane potential across the inner mitochondrial membrane (∆ψ) was measured in isolated mitochondria by fluorescence quenching. Crude mitochondria (50 µg) were suspended in 3 ml membrane potential buffer (0.6 M sorbitol, 0.1% (w/v) BSA, 10 mM MgCl2, 0.5 mM EDTA, 20 mM KPi, pH 7.2) containing 3 µl DiSC3 (3,3’-dipropylthiadicarbocyanine iodide, 2 mM in ethanol). After addition of mitochondria the absorption (excitation 622 nm, emission 670 nm) was measured in a luminescence spectrometer (Aminco Bowman2, Thermo Electron Corporation) until a distribution equilibrium was reached. Addition of 1 µM valinomycin dissipated the membrane potential. Data were analysed with FL WinLab (Perkin Elmer).

High resolution respirometry


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Mitochondrial respiration was measured using the Oxygraph 2-k (Oroboros Instruments, Austria). Measurements were performed by addition of isolated mitochondria (100 µg) and 2 ml respiration buffer (10 mM MOPS-KOH, pH 7.2, 250 mM sucrose, 5 mM MgCl2, 80 mM KCl, 5 mM KPi) into a 2 ml chamber maintained at a temperature of 30 °C. Samples were supplemented with 1 mM NADH and 1 mM ADP and the basic respiration rate was measured for 5 min. Data were analyzed with the DATLAB software and the wild-type values were set to 100%.

Blue native-PAGE and mitochondrial ATPase activity staining Native protein complexes were analyzed by solubilization of 50 µg mitochondria in either digitonin buffer (1% (w/v) digitonin, 20 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 10% (v/v) glycerol, 50 mM NaCl) or Dodecylmaltoside (DDM) buffer (0.3% (w/v) DDM instead of digitonin). Samples were incubated on ice for 15 min followed by centrifugation at 20 000g for 5 min at 4 °C. The supernatant was analyzed on a 4-13% gradient BN-PAGE followed by immunodecoration. For mitochondrial ATPase activity staining, mitochondria were lysed in 1% (w/v) digitonin buffer and the complexes separated by blue native electrophoresis. Gels were washed in distilled water for 20 min and subsequently incubated for 20 min in buffer containing ATP (5 mM MgCl2, 50 mM glycine, 20 mM ATP, pH 8.4). Gels were transferred into a 10% (w/v) CaCl2 solution and further incubated until calcium phosphate precipitation was visible. Gels were washed in distilled H2O and documented using the LAS4000 camera system (Fujifilm).

Dimethyl labeling and proteolytic digestion 9 Environment ACS Paragon Plus


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All LC solvents were ULC/MS grade (Biosolve). Mitochondria were lysed in 6 M GuHCl, 50 mM Na2HPO4, pH 7.8. Protein concentrations were determined using the bicinchoninic acid (BCA) assay (Thermo Scientific). Cysteines were reduced with 10 mM DTT for 30 min at 56 °C and subsequently free sulfhydryles were carbamidomethylated by adding 20 mM IAA for 30 min at room temperature, in the dark. For dimethylation, protein samples were incubated for 2 h at 37 °C in either (i) light labeling buffer (wild-type): 20 mM CH2O, 40 mM NaBH3CN and 200 mM HEPES, pH 8.0, or (ii) heavy labeling buffer (mas1): 20 mM CD2O, 40 mM NaBD3CN and 200 mM HEPES, pH 8.0. Excess formaldehyde was quenched by addition of 60 mM glycine. After 10 min samples were treated with 130 mM hydroxylamine for 15 min at room temperature. Next, the differentially labeled samples were pooled in a 1:1 ratio, and proteins were precipitated using ethanol. For this, samples were diluted 10-fold with ice-cold ethanol and stored at -40 °C for 1 h, followed by centrifugation at 4 °C at 16 000g for 30 min. After careful removal of the supernatant protein pellets were resolubilized in 2 M GuHCl, 50 mM Na2HPO4, pH 7.8, followed by 10-fold dilution with 50 mM NH4HCO3, 5% ACN and 1 mM CaCl2. Trypsin was added in a 1:30 (wt/wt) ratio and samples were incubated for 12 h at 37 °C and digest efficiency was controlled as described previously.26 Peptides were desalted using C18 solid phase Tips (SPEC C18 AR, 4 mg bed, Agilent Technologies, Böblingen, Germany) according to the manufacturer´s instructions and dried to completeness under vacuum. Peptides were resolubilized in 52 µL of SCX buffer A (10 mM KH2PO4, 20% ACN, pH 2.7). A 1 µg aliquot was analyzed by nano-LC-MS/MS, and labeling efficiency was determined by comparing the total number of lysines in all


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identified PSM to the number of labeled lysines at 1% FDR, and was above 95%.

Enrichment of N-terminal peptides using ChaFRADIC SCX was performed on a U3000 HPLC system (Thermo Scientific) using a 150 x 1 mm POLYSULFOETHYL A column (PolyLC, Columbia, US, 5 µm particle size, 200 Å pore size) and a tertiary buffer system: SCX buffer A (10 mM KH2PO4, 20% ACN, pH 2.7), SCX buffer B (10 mM KH2PO4, 188 mM KCl, 20% ACN, pH 2.7), SCX buffer C (10 mM KH2PO4, 800 mM NaCl, 20% ACN, pH 2.7). The gradient for separating peptides in charge state fractions was as follows: 100% of A for 10 min, followed by a linear ramp to 15% of B in 9 min. After 9 min at 15%, B was increased to 30% in 8 min. After 11 min at 30%, B was increased to 100% in 5 min. After 5 min, C was increased to 100% in 1 min. After 5 min at 100% of C, A was increased to 100% in 1 min and the column was equilibrated for 20 min. Flow-throughs of the first SCX dimension were dried under vacuum, desalted using Poros Oligo R3 (Applied Biosystems). Samples resolubilized in 50 µl of 0.1% TFA were loaded two times onto the R3 material, washed two times with 0.1% of FA and afterwards eluted with 50 µl of 70% (v/v) ACN, 0.1% (w/v) FA. Collected fractions were reduced to ~40 µL under vacuum and brought to 300 µL with 200 mM Na2HPO4, pH 8.0 to a final pH of ~7.0. Next, free N-termini of internal peptides were derivatized with NHS-trideutero acetate in two steps, modified from Staes et al.27 NHS-trideutero acetate was added to a final concentration of 20 mM, samples were incubated at 37 °C for 1 h, and another 10 mM NHS-trideutero acetate was added. After 1 h the reaction was



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quenched and peptides were desalted as described above. Dried charge state fractions were resolubilized in 50 µL SCX buffer A and fractionated in a 2nd SCX run under exactly the same conditions as above. Collected fractions were desalted using Poros R3 and resolubilized in 0.1% TFA.

nano-LC-MS/MS 1/3 of each fraction was analyzed by nano-LC-MS/MS using a Q-Exactive Plus (QE+) or an Orbitap Fusion mass spectrometer, both online-coupled to nano RSLC HPLC systems (all Thermo Scientific). Peptides were separated using a linear gradient ranging from 3-42% of B in 90 min. Flow-through fractions from the 1st SCX dimension were analyzed using a shorter 50 min gradient. All samples were measured in data-dependent acquisition using a dynamic exclusion of 15 s. In the QE+ full MS scans were acquired at a resolution of 70,000, and the 15 most abundant ions (Top15) were selected for MS/MS at a resolution of 17,500. Target values were set to 1 x 106 for MS and 1 x 105 for MS/MS. Maximum injection times were 120 ms and 250 ms, respectively. A normalized collision energy of 29 was used and the first fixed mass was set to 120 m/z. In the Fusion, full MS scans were acquired at a resolution of 120,000 and the 20 most abundant ions were fragmented using a charge state decision tree either by HCD (+3) and MS/MS were acquired in the ion trap. Target values were set to 4 x 105 for MS, 1 x 104 for HCD and 3 x 104 for ETD. Maximum injection times were 120 ms and 50 ms, respectively. For HCD a normalized collision energy of 30 was used and the first fixed mass was set to 120 m/z.


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For SCX charge state fractions +3, +4 and +5, 10% ammonia solution was placed in front of the ion source to compensate for increased charge states, as described by Thingholm et al.28 For flow-through and +1 fractions fragmentation of singly charged peptides was allowed, excluding m/z values of singly charged ions occurring in previous blank runs. Generated raw data were searched against an SGD database (September 1st, 2011; 6,717 target sequences). For peptide identification the Proteome Discoverer software version 1.4 (Thermo Scientific) was used with Mascot 2.4 (Matrix Science) as search engine, precursor ion quantifier and percolator nodes. Enzyme specificity was set to semi-ArgC with two allowed missed cleavage sites. To enable the quantification of both classes of N-terminal peptides, those with Nterminal dimethyl label and those with endogenous N-acetylation, we performed a two-step search strategy: First, data were searched with dimethyl light/heavy (+28.0313 Da / +34.0689 Da) as variable modifications at Lys and peptide N-termini; second dimethyl light/heavy of Lys as variable and Nterminal acetylation (+42.0105 Da) as fixed modifications. In both cases, carbamidomethylation of Cys (+57.0214 Da) was selected as fixed and oxidation (+15.9949 Da) of methionine as variable modification. Mass tolerances were set to 10 ppm for MS and 0.02 (QE+)/0.5 (Fusion) Da for MS/MS.

Identified

peptides

were

filtered

for

medium

confidence

corresponding to an FDR < 5% at the PSM level, and a search engine rank of 1. The precursor ion quantifier node was adjusted according to the search settings. To compensate for deuterium-induced retention time shifts, the maximum window for corresponding peptides was set to 1 minute. The datasets can be accessed via ProteomeXchange (PXD002063).29 To



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generate the final list of N-terminal peptides (supplemental table S1), only sequences of at least six amino acids, representing the first 100 amino acids of the respective protein’s sequence were considered.

Data processing Prediction of mitochondrial processing sites was performed with MitoFates using a prediction cut-off of 0.5.30 Sequence logos showing the relative abundance of amino acids at certain positions within the presequence were generated using the weblogo program.31 The IMP substrate Cyt1 was omitted from analysis for Figure 3D. For ChaFRADIC analysis only mitochondrial annotated proteins and their first 100 N-terminal amino acids were evaluated. Uncharacterized ORFs with changes in the mas1/WT ratio (see below) were included in the analysis. Nterminal peptides corresponding to position 1-8 of the full-length annotated proteins were considered as non-processed and therefore accumulating unprocessed precursors. Proteins were considered as potential MPP substrates if corresponding N-terminal peptides quantified using ChaFRADIC were at least 15-fold upregulated in mas1 mitochondria. In case several Nterminal peptides were identified and led to the calculation of different ratios for the same protein the following ratios are given: for non-processed Ntermini starting with amino acid 1-8 the lowest ratio was considered, whereas for the mature, processed protein the highest ratio is shown.

3. Results 3.1 Identification of MPP substrates by quantitative ChaFRADIC


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Mitochondria import the majority of their proteins along the presequence import pathway that uses a positively charged N-terminal presequence as targeting signal. This signal sequence is recognized by the TOM and TIM23 import machineries, which direct translocation across the outer and inner membrane. The driving force of this process is provided by the PAM complex and the membrane potential across the inner membrane (Figure 1A). The presequence is cleaved upon import into the matrix and it is assumed that the majority of presequences are cleaved by the essential mitochondrial processing peptidase (MPP). To analyze the cleavage site specificity of MPP we aimed to identify novel MPP substrate proteins. Therefore, we isolated mitochondria from a mas1 temperature-sensitive yeast strain, which renders MPP inactive after growth at non-permissive temperature (37 °C) for 12 h, and from the corresponding wild-type. To test for a decline in MPP activity we assessed presequence processing of previously identified MPP substrate proteins by SDS-PAGE and immunodecoration.5,16,32 The precursor proteins of the co-chaperone Mge1, the citric acid cycle enzymes Mdh1 and Cit1 and the respiratory chain proteins Atp2, Cox4 and Rip1 were all efficiently processed to their mature form in wild-type mitochondria (Figure 1B). In contrast, in the mas1 mutant their non-processed precursor forms and intermediates accumulated in mitochondria. In addition, due to impaired processing decreased levels of the mature forms were observed for some of the MPP substrates. Non-processed mitochondrial proteins that are not imported via the presequence import pathway remained unchanged (e.g. Om45, Figure 1B).



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We analyzed highly purified mitochondria isolated from mas1 and wild-type yeast by quantitative N-terminal ChaFRADIC.24 The experimental strategy is shown in Figure 2. Mitochondria were dimethylated, N-terminal peptides enriched and mas1/WT ratios determined by quantitative mass spectrometry (Table S1). To retrieve proteins specifically accumulating as preproteins in mas1 mutant mitochondria a threshold of mas1/WT >15 was set. This led to the identification of 80 proteins, among them the known MPP substrates alcohol dehydrogenase Adh3, cytochrome c1, the cysteine desulfurase Nfs1 and superoxide dismutase Sod2.5,15,16 To validate the identified candidates as bona fide MPP substrate proteins it was required to show that these proteins in fact possess cleavable presequences. We chose two approaches: First we searched the literature for experimentally determined mature N-termini of processed mitochondrial proteins.5,24,33-36 Second, we employed the novel, reliable mitochondrial presequence prediction program MitoFates,30 which was trained on several hundred authentic mature N-termini from S. cerevisiae, A. thaliana and O. sativa. Taken together, 70 of the proteins identified in the ChaFRADIC analysis are processed upon import or have a high probability for cleavable presequences (Table 1) and were therefore considered as candidates for MPP processing.

3.2 In vivo and in organello validation of novel MPP substrates For validation of the novel MPP substrate candidates identified by ChaFRADIC two different methods were applied: Wild-type and mas1 mitochondria isolated from cells after growth at non-permissive temperature


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were either analyzed by SDS-PAGE and immunodecoration or used for in organello import of radiolabeled precursor proteins. Decoration with various antibodies recognizing the potential MPP substrate proteins showed an accumulation of higher molecular weight species in mas1 mitochondria (Figure 3A). Judged from the molecular weight they represent the nonprocessed precursor forms of these proteins. In addition, for several of the tested substrates a decrease in the mature processed protein levels was observed in mas1 compared to WT (e.g. Atp16, Cox6, Jac1, Mcx1). The levels of the non-processed proteins Por1 and Tim23 were not changed. For in organello import radiolabeled precursor proteins of the MPP substrate candidates were synthesized and incubated with isolated mitochondria from wild-type and mas1 strains after growth at 37 °C for 6 hours to inactivate MPP function (Figure 3B). The samples were treated with proteinase K to digest non-imported preproteins and analyzed on SDS-PAGE. The precursors were processed upon import into WT mitochondria in a membrane-potential dependent manner (Figure 3B, lanes 2, 3 and 7, 8). Upon import in mas1 mitochondria the precursors reached a proteinase K protected location but did not undergo proteolytic removal of the presequence (Figure 3B, lanes 4, 5 and 9, 10). Hence, while import into the organelle occurred processing in the mas1 mutant was impaired confirming these proteins as MPP substrates. Taken together, all of the 16 tested candidates identified by ChaFRADIC analysis were indeed MPP substrate proteins. A first global approach to uncover the MPP cleavage site motif analyzing yeast mitochondria by N-terminal COFRADIC indicated R-2 rather than R-3 as main MPP recognition site motif.5 However, this study identified the mature



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N-termini of mitochondrial proteins within the whole organelle and contained therefore also proteins processed by other presequence proteases, reducing the accuracy of the MPP motif. Therefore, we used the systematically identified genuine MPP substrates (Table 1) to characterize the C-terminal part of the presequence and the first five amino acids following the MPP cleavage site to refine the MPP cleavage site motif (Figure 3C and D). Deduction of the presequence from the start of the mature N-terminus and analysis with a sequence logo program

31

showed an increased abundance of

arginine in positions -2, -3, -10 and -11 (Figure 3C). The presence of arginine at four different positions can be explained with the frequent secondary processing by Oct1 and Icp55 after initial MPP cleavage. We analyzed the 70 MPP substrates for Oct1/Icp55 processing motifs (Table 1) and shifted the MPP cleavage site by 8 amino acids for Oct1 and a single amino acid for Icp55 substrates in respect to the mature N-terminus. When this second processing event was integrated into the sequence logo analysis, arginine in position -2 remained as significantly preferred amino acid within the presequence (Figure 3D). Other positions within the presequence showed no preference for a particular residue.

3.4 MPP-dependent increase of a subset of non-processed N-terminal peptides The proteins identified in the ChaFRADIC analysis can be divided in two different groups. On the one hand 70 of the 80 proteins were presequencecontaining precursors that are processed upon import by MPP (Table 1). On the other hand the remaining 10 proteins were either experimentally shown or


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predicted not to contain a cleavable presequence (Table S2). We imported a subset of these proteins into isolated wild-type mitochondria (Figure 4A). Indeed, none of the radiolabeled precursors showed presequence processing upon import (unlike the novel MPP substrates that underwent proteolytic processing upon import shown in Figure 3B). Hence, although the N-terminal peptides of these proteins are enriched in mas1 mutant mitochondria these proteins seem to be no substrates of MPP and therefore do not accumulate in their precursor form due to impaired MPP processing. Rather the mature protein amount seems to be increased in mas1. We tested the proteins Din7, Mrx8 and Yhb1 for in organello import in mas1 and wild-type mitochondria. All three proteins showed enhanced import in mas1 compared to WT (Figure 4B). For analysis of a potential up-regulation of non-processed proteins in mas1 in vivo we raised antibodies against the uncharacterized proteins Fmp46/Ipm16 and Fmp52/Ipm25 and analyzed their protein levels in mitochondria isolated from wild-type and mas1 strains after shift to non-permissive temperature for 12 hours. Fmp46/Ipm16 and Fmp52/Ipm25 increased significantly in mas1 compared to wild-type mitochondria, while other non-processed proteins like the ADP/ATP carrier (AAC) did not change (Figure 4C). Therefore, mature non-processed nonMPP substrate proteins increase in vivo upon inhibition of the major mitochondrial presequence protease. This is in contrast to MPP substrates that accumulate as precursors and show a typical decrease in the mature processed form in mas1.



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3.5 Elevated membrane potential and oxygen consumption in mas1 at non-permissive temperature Interestingly, in organello import in wild-type and mas1 mutant mitochondria revealed an increased import signal of the non-processed proteins Yhb1, Din7 and Mrx8 in mas1 compared to WT (Figure 4B). To distinguish if this is a special characteristic of these particular non-processed proteins or a general feature of mas1 mitochondria we chose the control proteins Hsp10, which is not processed upon import into the mitochondrial matrix, and Mrpl32, that is processed by the m-AAA protease but not by MPP.15,37 Both, Hsp10 and Mrpl32 showed an accelerated import rate into mas1 mitochondria that had been isolated after cell growth at 37 °C for 12 hours (Figure 5A and B). Increased import rates might be due to an elevated membrane potential. Therefore, we measured the membrane potential in mitochondria isolated from wild-type and mas1 cells, that had been grown under the permissive temperature 24 °C, or shifted to 37 °C for 6 and 12 hours, respectively (Figure 5C). While the membrane potential was unaltered in mas1 after growth at permissive temperature, it increased gradually with the duration of the temperature shift i.e. the level of MPP inactivation. This elevated membrane potential can explain the increased import rates into mas1 mitochondria. We next asked if the increase in membrane potential is accompanied by an elevation in oxygen consumption and measured mitochondrial respiration in organelles isolated after the same growth conditions (Figure 5D). Oxygen consumption was indeed elevated in mas1 upon shift to 37°C, reflecting the results of the membrane potential measurements. Furthermore, using blue native gel electrophoresis we analyzed the organization of the respiratory


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chain complexes and supercomplexes in wild-type and mas1 after shift to non-permissive

temperature

(Figure

5E).

Both

wild-type

and

mas1

mitochondria showed subtle changes in complex abundance and distribution upon shift to 37°C. Interestingly, upon the 6 hours shift to non-permissive temperature the supercomplexes formed of two dimers of complex III and complex IV and the dimer of the ATP synthase (complex V) increased in mas1 mitochondria and the mitochondrial ATPase activity was enhanced in mas1 mitochondria compared to wild-type (Figure S1). Taken together, the increase in membrane potential and oxygen consumption does not seem to be caused by impaired respiratory chain complex formation or ATPase activity and might therefore indicate a potential adjustment of mitochondrial functions triggered by the proteotoxic stress caused upon MPP dysfunction.

3.6 Novel mitochondrial proteins increased upon proteotoxic stress in mas1 The observation that the mitochondrial membrane potential and respiration rate is significantly increased after 6 hours growth at non-permissive temperature prompted us to analyze the levels of mitochondrial proteins present in the different subcompartments including the two non-processed proteins Fmp46/Ipm16 and Fmp52/Ipm25, that had been identified in our ChaFRADIC analysis and showed increased steady state protein levels after a 12 hours shift to non-permissive temperature, in more detail. While nonprocessed proteins like Por1, Om14 and AAC were not changed, Fmp46/Ipm16 and Fmp52/Ipm25 exhibited a dramatic increase in protein levels (Figure 6A – C). In contrast, MPP substrates (e.g. Cym1, Atp2, Ssq1)



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Page 22 of 48

showed no or only very moderate accumulation of precursor proteins reflecting the shorter growth at non-permissive temperature. This could further point towards the existence of a response in mas1 cells upon shift to nonpermissive temperature. Due to their specific increase in mas1 after short induction of MPP dysfunction we annotated Fmp46 as Ipm16 and Fmp52 as Ipm25 for Increased upon proteotoxic stress in mitochondria, respectively. Fmp46/Ipm16 and Fmp52/Ipm25 have been found in proteomic analysis of highly pure mitochondria.38,39 We wanted to determine their mitochondrial sublocalization and therefore treated mitochondria with proteinase K in combination with or without osmotic swelling that leads to a rupture of the outer membrane (Figure 6D, lanes 1-4). Fmp46/Ipm16 was only accessible to Proteinase K after lysis of both membranes with Triton X-100 (Figure 6D, lanes 5 and 6), while Fmp52/Ipm25 was already digested upon disruption of the outer membrane (Figure 6D, lane 4). To assess their potential membrane association, carbonate extraction, which separates integral membrane proteins from soluble and peripheral membrane proteins, and sonication in the presence of different salt concentrations, was performed. While upon carbonate extraction both Fmp46/Ipm16 and Fmp52/Ipm25 were released in the supernatant, the proteins were retained largely in the pellet fraction upon sonication in the presence of low salt concentration indicating a predominant peripheral membrane association for both proteins (Figure 6E). Due to the identification

of

Fmp52/Ipm25

in

the

outer

membrane

proteome,39

Fmp52/Ipm25 is likely peripherally attached to the outer membrane from the intermembrane space site, whereas Fmp46/Ipm16 is localized in the matrix peripherally attached to the IM.


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4. Discussion N-terminal proteomics has been successfully used as tool to study the processing sites and substrate specificity of different proteases in several biological systems.5,40-44 Deletion of proteases in different organisms for in vivo analysis or purified proteases incubated with cell lysates allowed specific substrate identification of an individual protease.5,15,42,45 However, the in vivo analysis of essential proteases was not feasible so far due to the lack of a model system possessing a specifically impaired protease activity. Here we apply quantitative ChaFRADIC to analyze the substrate specificity of the essential mitochondrial protease MPP using a temperature-sensitive mas1 mutant strain. In this mutant MPP is inactive when the cells are shifted to growth at non-permissive temperature (37 °C). The duration of the shift has implications on the analysis: Short growth at non-permissive temperature can result in a low identification rate, whereas prolonged growth can have severe side effects due to the inhibition of an essential mitochondrial function. Here, we have chosen a 12 hours shift to induce the mas1 phenotype, which led to the identification of the so far largest number of novel MPP substrate proteins. Together with the global determination of the mature mitochondrial N-termini (N-proteome)5 this allowed for the first time a detailed analysis of the MPP cleavage site. The corresponding presequences, deduced from the mature Ntermini validated experimentally or by high-confidence prediction, resulted in refinement of the MPP recognition site, which identifies R-2 as the most frequent cleavage motif of MPP. In addition, the identification of a high number of genuine MPP substrates revealed that the amino acids



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phenylalanine, tyrosine and leucine are enriched in position +1 of the MPP cleavage site. All three residues are destabilizing according to the N-end rule and result in a decreased protein half-life and accelerated turn-over.5,12,46,47 MPP-processed proteins bearing these N-terminal residues are subjected to secondary processing by Oct1 or Icp55. The predominance of F, Y and L at position +1 in the identified MPP substrates might indicate that far more proteins than previously anticipated undergo secondary cleavage underlining the importance of intermediate processing by Oct1 and Icp55. Indeed, when we screened the cleavage sites of processed proteins identified in the Nproteome for the re-fined cleavage site motif 127 proteins showed a potential consecutive MPP-Icp55 or MPP-Oct1 cleavage pattern. It is expected that MPP is processing more mitochondrial precursors than the 70 proteins identified in our ChaFRADIC analysis. Notably, in this study only proteins that have been imported to a certain extent during the temperature shift (i.e. 12 h) produce significantly differential signals in the quantitative Nterminal proteome analysis. Thus, owing to the long half-life of mitochondrial proteins

5,12

only a subset of MPP substrates could be identified. Additionally,

only proteins, which can fold as precursors and adopt a protease resistant conformation can be expected to be found by this type of analysis. Extended shifts to non-permissive growth temperature will likely result in severe mitochondrial defects accompanied with increased turnover of mitochondrial proteins. Degradation of accumulating precursors will lead to many additional cleavage products that would hamper the identification of authentic N-terminal peptides. Therefore, different growth conditions that influence the turnover of the mitochondrial proteome or lead to a change in a subset of mitochondrial


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proteins could be an interesting approach for the identification of further MPP substrates. Surprisingly, our ChaFRADIC analysis also revealed changes in a set of proteins that do not possess a presequence and are therefore not MPP substrate proteins. By using antibodies directed against two of these identified proteins, Fmp46/Ipm16 and Fmp52/Ipm25, we could confirm an increased protein level in mas1 upon shift to non-permissive temperature. Fmp52/Imp25 is reported to be induced upon DNA damage48 and Fmp46/Imp16 is a redox protein, likely involved in the reduction of small toxic molecules.49 Our sublocalization studies identified Fmp52/Ipm25 as an outer membrane associated protein and Fmp46/Ipm16 as attached to the inner membrane, demonstrating that a dysfunctional essential matrix-processing enzyme can influence the protein composition of the entire organelle. Further analysis of the mas1 mutant revealed an unexpectedly increased import rate in mas1 compared to WT mitochondria demonstrated by import of the established non-processed preprotein Hsp10 and Mrpl32, which is processed by the m-AAA protease.15,37 The observed increase in membrane potential correlated with the duration of the shift to non-permissive temperature. Furthermore, in line with the increased membrane potential mas1 mitochondria also showed increased respiration upon shift to nonpermissive temperature, while the organization of the respiratory chain complexes did not depend on mas1 inactivation. The increase of the non-processed mitochondrial proteins Fmp52/Ipm25 and Fmp46/Ipm16 and the increase in membrane potential and respiration were observed already after a short exposure to non-permissive temperature at



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Page 26 of 48

which only a very minor amount of precursor proteins accumulated. This could imply that mitochondria exert a stress response triggered by impairment of the essential mitochondrial presequence processing function due to mutations in Mas1. In this potential stress response the increased membrane potential and oxygen consumption could be a means for the cell to compensate the processing defect in mas1 by overriding the impaired maturation of preproteins with an accelerated import of novel proteins. It will be interesting to analyze this potential rescue mechanism in detail in the future. Taken together, the changes in mitochondrial proteins localized in various mitochondrial

compartments

and

mitochondrial

functions

upon

MPP

dysfunction underline the importance of the major mitochondrial presequence protease in maintaining the integrity of the essential mitochondrial organelle.

Supporting information Figure S1: F1FO-ATPase activity staining of wild-type and mas1 mitochondria. Table S1: List of N-terminal peptides identified by ChaFRADIC of wild-type (L, light) and mas1 (H, heavy) mitochondria isolated after growth at nonpermissive temperature. Table S2: List of non-processed mitochondrial proteins with increase in N-terminal peptides in mas1 mitochondria after growth for 12 hours at non-permissive temperature. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements We thank Drs. Chris Meisinger and Nils Wiedemann for helpful discussions and comments. This work was supported by the Baden-Württemberg Stiftung


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(F.N.V.) and by the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen (J.M.B., R.P.Z.).

Author contributions J.M.B., A.A.T. and F.N.V. performed experiments, designed together with R.P.Z.; F.N.V. and R.P.Z. developed the project and wrote the manuscript. All authors approved the final version of the manuscript.

Conflict of interest The authors declare that they have no conflict of interest.

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(43) Aivaliotis, M.; Gevaert, K.; Falb, M.; Tebbe, A.; Konstantinidis, K.; Bisle, B.; Klein, C.; Martens, L.; Staes, A.; Timmerman, E.; Van Damme, J.; Siedler, F.; Pfeiffer, F.; Vandekerckhove, J.; Oesterhelt, D. Large-scale identification of N-terminal peptides in the halophilic archaea Halobacterium salinarum and Natronomonas pharaonis. J. Proteome Res. 2007, 6 (6), 2195-204.

(44) Huang, S.; Taylor, N.L.; Whelan, J.; Millar, A.H. Refining the definition of plant mitochondrial presequences through analysis of sorting signals, Nterminal modifications, and cleavage motifs. Plant Physiol. 2009, 150 (3), 1272-85.

(45) Carrie, C.; Venne, A.S.; Zahedi, R.P.; Soll, J. Identification of cleavage sites and substrate proteins for two mitochondrial intermediate peptidases in Arabidopsis thaliana. J. Exp. Bot. 2015, pii: erv064.

(46) Sriram, S.M.; Kim, B.Y.; Kwon, Y.T. The N-end rule pathway: emerging functions and molecular principles of substrate recognition. Nat. Rev. Mol. Cell. Biol. 2011, 12 (11), 735-47.


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(47) Varshavsky, A. The N-end rule pathway and regulation by proteolysis. Protein Sci. 2011, 20, 1298-345.

(48) Dardalhon, M.; Lin, W.; Nicolas, A.; Averbeck, D. Specific transcriptional responses induced by 8-methoxypsoralen and UVA in yeast. FEMS Yeast Res. 2007, 7 (6), 866-78.

(49) Jung, J.W.; Yee, A.; Wu, B.; Arrowsmith, C.H.; Lee, W. Solution structure of YKR049C, a putative redox protein from Saccharomyces cerevisiae. J. Biochem. Mol. Biol. 2005, 38 (5), 550-4.

Figure legends Figure 1. Analysis of mas1 mutant mitochondria after shift to non-permissive temperature. (A) Schematic overview of the mitochondrial presequence protein import pathway and proteolytic processing by MPP. TOM, translocase of the mitochondrial outer membrane; TIM, translocase of the mitochondrial inner membrane; PAM, presequence translocase-associated import motor; MPP, mitochondrial processing peptidase; ∆ψ, membrane potential across the inner mitochondrial membrane. (B) Mitochondria were isolated from wild-type (WT) and mas1 mutant mitochondria after growth at non-permissive temperature

for

12

hours

and

analyzed

by

SDS-PAGE

and

immunodecoration. p, precursor; i, intermediate; m, mature.

Figure 2. Workflow for identification of N-termini of mitochondrial proteins from wild-type and mas1 yeast strains using ChaFRADIC analysis.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Mitochondria were isolated from yeast cells after growth at 37 °C for 12 h. Highly pure organelles were obtained by sucrose gradient centrifugation. Mitochondria were lysed and dimethylated. Samples were mixed, digested with Trypsin, N-terminal peptides enriched and the mas1/WT ratios determined by quantitative mass spectrometry.

Figure 3. Validation of novel MPP substrate proteins identified by ChaFRADIC. (A) Mitochondria from wild-type (WT) and mas1 yeast cells grown at 37°C for 12 hours were separated on SDS-PAGE and analyzed by western blotting. p, precursor; i, intermediate; m, mature. (B) Mitochondria isolated from WT and mas1 cells grown at non-permissive temperature for 6 hours were incubated with radiolabeled precursors in the presence or absence of the membrane potential (∆ψ), treated with proteinase K and analyzed by SDS- or tris-tricine-PAGE and autoradiography. Prec., precursor. (C) The relative frequency of amino acids (15 residues of the C-terminal segment of the presequence and 5 amino acids of the mature protein) in the identified MPP substrates is shown. (D) Analysis as in (C) with secondary cleavage by Oct1 (8 amino acids) or Icp55 (one amino acid) in relation to the mature N-terminus resulting in the authentic presequence cleaved by MPP.

Figure 4.

Characterization of non-processed precursor proteins in mas1

mitochondria. (A) Radiolabeled precursors were imported into mitochondria isolated from wild-type yeast cells. Where indicated, the membrane potential (∆ψ) was dissipated prior to the import reaction and samples were digested with proteinase K (Prot. K). Prec., precursor; mock, no mitochondria were


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added to the import reaction. (B) Radiolabeled precursor proteins were incubated with isolated mitochondria from wild-type (WT) and mas1 mitochondria isolated after cell growth at non-permissive temperature for 12 hours. Samples were treated with Proteinase K and analyzed via SDS-PAGE and autoradiography. (C) Mitochondria isolated from WT and mas1 mutant cells after growth at non-permissive temperature for 12 hours were analyzed by SDS-PAGE and immunodecoration.

Figure 5. Increased membrane potential in mas1 results in elevated import rate. (A) [35S]Hsp10 and [35S]Mrpl32 were imported for various time points into wild-type (WT) and mas1 mitochondria isolated after growth at 37 °C for 12 h. Where indicated the membrane potential (∆ψ) was dissipated prior to the import reaction. Samples were treated with proteinase K followed by analysis on SDS-PAGE and autoradiography. p, precursor; m, mature. (B) Quantification of the import reactions shown in (A). The WT 20 min value was set to 100%, mean ± SEM (n=3). (C) Analysis of the membrane potential in wild-type (WT, gray bars) and mas1 (black bars) mitochondria isolated from cells grown at permissive temperature (24 °C) or an additional shift to nonpermissive temperature (37 °C) for 6 or 12 hours. WT was set to 100%, mean ± SEM (0 h shift n=3; 6 h shift n=6; 12 h shift n=4). (D) Oxygen consumption measurements in wild-type (WT, gray bars) and mas1 (black bars) mitochondria isolated after cell growth at 24°C or after an additional in vivo shift for 6 or 12 hours to 37°C. WT values were set to 100%, mean ± SEM (n=3). (E) WT and mas1 mitochondria isolated after cell growth at either 24°C or 37°C (for 6 and 12 hours) were solubilized in digitonin or dodecylmaltoside



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Page 38 of 48

buffer (lower two panels) and analyzed by blue native electrophoresis and immunoblotting. III2, dimer of respiratory chain complex III; IV2, dimer of respiratory chain complex IV; V2, dimer of ATPase (complex V of respiratory chain).

Figure 6. Submitochondrial localization of the proteins Fmp46/Ipm16 and Fmp52/Ipm25 (increased upon proteotoxic stress in mitochondria). (A) Mitochondria isolated from wild-type (WT) and mas1 yeast cells after growth at 24°C or 6 hours shift at 37°C were analyzed by SDS-PAGE and immunoblotting.

(B)

and

(C)

Quantifications

of

Fmp46/Ipm16

and

Fmp52/Ipm25 protein levels and of representative proteins from various submitochondrial compartments from (A). Quantifications represent mean ± SEM (n≥3). (D) Wild-type mitochondria were subjected to treatment in hypotonic buffer (swell.) or lysed in Triton X-100 (TX-100). Samples were digested with proteinase K and analyzed by SDS-PAGE and immunoblotting. (E) Mitochondria were treated with sodium carbonate (lanes 1 and 2) or sonicated in the presence of 50 or 500 mM salt (lanes 3-6). Samples were analyzed by SDS-PAGE and western blotting.

Figure S1. Mitochondrial ATPase activity staining of wild-type and mas1 mitochondria. Mitochondria isolated from wild-type (WT) and mas1 cells after growth at 24°C or 6 or 12 hours shift to 37°C were lysed in digitonin buffer and separated on BN-PAGE. The activity of the mitochondrial ATPase was visualized by in-gel calcium phosphate precipitation upon ATP hydrolysis. V2,


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F1FO-ATP synthase dimer; V, F1FO-ATP synthase monomer; F1, F1 part of the ATP synthase.

Table 1 List of MPP substrate proteins identified by ChaFRADIC in this study.

Graphic



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


A

B

Preprotein Cytosol

Mito. (μg)

Outer membrane

mas1 20 40

α-Mge1

+

Inner membrane

PAM

MPP

Mas2 Presequence

mas1 20 40

α-Cit1

α-Mas1

p m

α-Rip1

-p -i -m 1

2

3

4

Figure 1, Burkhart et al.

ACS Paragon Plus Environment

p m -p -i -m p m -p -i -m

-m

Δψ

TIM23

WT 20 40

α-Cox4

Mas1 Processed protein

Mito. (μg) -p

TOM

Intermembrane space

Matrix

WT 20 40

α-Atp2 α-Mdh1 α-Om45 5

6

7

8

S. cerevisiae mas1

Growth 12 h 37°C

S. cerevisiae Wild-type

Page 44 of 48

Differential centrifugation

crude mitochondria

crude mitochondria Sucrose gradient

purified mitochondria

purified mitochondria Lysis Carbamidomethylation

“heavy“ C2D6

K K COOH Dimethylation of

K

N

K

R N-terminal peptides

N

+2

+3 N

K

R

R

>+4

N

R

R

K

N-terminal

N

-100

Æ internal peptides shift, N-terminal peptides retain their charge states

R

K

FT +1

+2 K

internal

K 5.0

10.0

15.0

20.0

25.0

30.0

35.0

Time in min

40.0

45.0

50.0

55.0

60.1

R

+3 N

R

K

+4

R

>+4

internal

100

0.0

R

2nd SCX-HPLC Charge-based enrichment of N-terminal peptides

100 0

K

internal peptides

Derivatization of free primary amines

+4 K

“light“ C2H6

with ArgC specificity

K

Æ each fraction contains both internal and N-terminal peptides

FT +1

K

N

R

K

1st SCX-HPLC Charge-based fractionation of sample

250

K K COOH

K

free amino groups

K

N

Digestion

Absorbance 214nm [mAU]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


N-terminal

0

K

R



-100

2nd SCX of charge state +3 fraction 0.0

5.0

10.0

15.0

20.0

25.0

30.0

Time in min

35.0

40.0

45.0

50.0

55.0

60.1

Journal of Proteome Research WT mas1

mas1 20 40

WT Mito. (μg) 20 40

Mito. (μg)

20 40

-p

-p α-Atp16

-m 2

3

B

p m p m

α-Ssc1 α-Ssq1

4

5

6

7

8

mas1 + -

+

Mito. Δψ

m p

[35S]Mrpl16

[35S]Glr1 4

11

WT -

+

mas1 + -

12

Mito. Δψ

[35S]Mrpl36

p m p m

m

[35S]Rex2

p

[35S]Mrpl24

3

10

p

m

2

9

[35S]Acn9

p

[35S]Mgm101

1

α-Tim23

Prec.

Prec.

-

α-Por1

[35S]precursor

[35S]precursor WT

p m

α-Mcx1

-m

-p 1

-p -m

α-Jac1

α-Cox6

-m α-Mdj1

mas1 20 40

WT 20 40

Mito. (μg)

20 40

m

[35S]Dia4

p m

p m

[35S]Aim1

p m

5

6

C

7

8

9

10

D Presequence

MPP 3

Mature

1

Presequence

Intermediate/ Mature

bits

2

bits

+5

+4

+3

+2

Position (relative to mature N-termini)



+5

+4

+3

+2

+1

-1

-2

-3

-4

-5

-6

-7

-8

-9

-10

-11

-12

-15

0 -13

-1

Position (relative to mature N-termini)

+1

-2

-3

-4

-5

-6

-7

-8

-9

-10

-11

-12

-13

0

-14

1 -15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

A

-14

Page 45 of 48


[35S]precursor

B

-

+ +

-

+

mock

[35S]precursor -

WT +

Prec.

A Prec.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 48

[35S]Fmp46/ Ipm16

Prot. K Δψ

mas1 + -

[35S]Din7 [35S]Mrx8 [35S]Yhb1

[35S]Fmp52/ Ipm25

1

C

[35S]Din7

Mito. (μg)

[35S]Mrx8

2

3

4

5

WT 20 40

mas1 20 40

1

3

α-Fmp46/ Ipm16

[35S]Yhb1

α-Fmp52/ Ipm25 1

2

3

4

5

6

α-AAC



2

4

Mito. Δψ

Page 47 of 48

B

WT + + + 5 10 20 20

mas1 + + + 5 10 20 20

mas1

Mito. Δψ Time (min)

[35S]Hsp10 -p [35S]Mrpl32 -m 1

C

2

3

4

5

7

8

D Oxygen consumption (% of WT)

140 120 100 80 60 40 20 0

6

0h

6h Growth at 37°C

12h

Imported [35S]Mrpl32 (% of WT)

Prec.

[35S]precursor

Imported [35S]Hsp10 (% of WT)

A

Membrane potential (% of WT)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


140 120 100

WT

80 60 40 20 5

9

10

15

mas1

140 120

WT

100 80 60 40 20 5

20

Time (min)

E

140

160

WT 0 6 12

120 100

III2 IV2 III2 IV

80

V2

10

mas1 0 6 12

20

Mito. Time (hours) α-Cox4 α-Atp2

V

60

15

Time (min)

II

α-Sdh1

20

IV

α-Cox1

0

III2

α-Rip1

40

0h

6h

12h

Growth at 37°C

Figure 5, Burkhart et al. ACS Paragon Plus Environment

1

2

3

4

5

6


6 h 37°C WT mas1 20 40 20 40

B

D

WT 24°C mas1

swell. Prot. K α-Fmp46/ Ipm16

125

% of control (WT)

24°C WT mas1 20 40 20 40

100 75 50 25

Fmp46/ Fmp52/ Por1 Ipm16 Ipm25

C

200 175

% of control (WT)

1 2 3 4 5 6A 7 8 9Mito. (μg) 10 α-Fmp46/ 11Ipm16 12 13 α-Fmp52/ 14Ipm25 15 16 α-Por1 17 18 19α-Om14 20 21 α-AAC 22 23 α-Aco1 24 25 26α-Hsp78 27 28 α-Cym1 29 30 31 α-Sdh4 32 33 α-Atp2 34 35 36 α-Ssq1 37 38 39 40 41 42 43 44 45 46 47

AAC

Aco1

WT 6 h 37°C mas1

3

4

5

6

7

8

-

100 75

+

AAC

Aco1

Figure 6, Burkhart et al. ACS Paragon Plus Environment

TX-100 +

+

α-Tom70

α-Tom70

α-Tim21

α-Tim21

α-Mdh1

α-Mdh1

E

2

pH 11.5

3

5

4

NaCl [mM]

SN

α-Fmp46/ Ipm16 α-Fmp52/ Ipm25 α-Por1

α-Fmp46/ Ipm16 α-Fmp52/ Ipm25

α-Tim44

α-Tim44

50 P SN

6

500 P SN

α-Por1

α-Aco1 Fmp46/ Fmp52/ Por1 Ipm16 Ipm25

-

α-Fmp52/ Ipm25

P

125

+

α-Fmp46/ Ipm16 α-Fmp52/ Ipm25

150

25

2

-

1

50

1

Page 48 of 48

1

2

α-Aco1 3

4

5

6

Quantitative Profiling for Substrates of the ... - ACS Publications

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