Identification of Core Components and Transient ... - ACS Publications

Article pubs.acs.org/jpr

Identification of Core Components and Transient Interactors of the Peroxisomal Importomer by Dual-Track Stable Isotope Labeling with Amino Acids in Cell Culture Analysis Silke Oeljeklaus,†,⊥ Benedikt S. Reinartz,‡,⊥ Janina Wolf,§ Sebastian Wiese,† Jason Tonillo,‡ Katharina Podwojski,‡ Katja Kuhlmann,‡ Christian Stephan,‡ Helmut E. Meyer,‡ Wolfgang Schliebs,§ Cécile Brocard,∥ Ralf Erdmann,§ and Bettina Warscheid*,† †

Faculty of Biology and BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany Medizinisches Proteom-Center, Zentrum für klinische Forschung, Ruhr-University Bochum, Universitätsstraße 150, 44780 Bochum, Germany § Institute of Physiological Chemistry, Department of Systems Biochemistry, Ruhr-University Bochum, Universitätsstraße 150, 44780 Bochum, Germany ∥ University of Vienna, Center of Molecular Biology, Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, Dr. Bohrgasse 9, 1030 Vienna, Austria ‡

S Supporting Information *

ABSTRACT: The importomer complex plays an essential role in the biogenesis of peroxisomes by mediating the translocation of matrix proteins across the organellar membrane. A central part of this highly dynamic import machinery is the docking complex consisting of Pex14p, Pex13p, and Pex17p that is linked to the RING finger complex (Pex2p, Pex10p, Pex12p) via Pex8p. To gain detailed knowledge on the molecular players governing peroxisomal matrix protein import and, thus, the integrity and functionality of peroxisomes, we aimed at a most comprehensive investigation of stable and transient interaction partners of Pex14p, the central component of the importomer. To this end, we performed a thorough quantitative proteomics study based on epitope tagging of Pex14p combined with dual-track stable isotope labeling with amino acids in cell culture-mass spectrometry (SILAC-MS) analysis of affinity-purified Pex14p complexes and statistics. The results led to the establishment of the so far most extensive Pex14p interactome, comprising 9 core and further 12 transient components. We confirmed virtually all known Pex14p interaction partners including the core constituents of the importomer as well as Pex5p, Pex11p, Pex15p, and Dyn2p. More importantly, we identified new transient interaction partners (Pex25p, Hrr25p, Esl2p, prohibitin) that provide a valuable resource for future investigations on the functionality, dynamics, and regulation of the peroxisomal importomer. KEYWORDS: SILAC, protein−protein interactions, affinity purification, membrane proteins, importomer, peroxisomes, quantification, mass spectrometry

■

INTRODUCTION Peroxisomes are single membrane-bound organelles lacking DNA, which are present in virtually all eukaryotic cells. They are highly dynamic and fulfill a multitude of essential physiological roles, such as the oxidative degradation of fatty acids and detoxification of reactive oxygen species, that vary depending on cell and tissue type as well as environmental conditions. Their importance for cellular fitness is reflected by the occurrence of a number of lethal human disorders, e.g., Zellweger Syndrome, caused by defects in the biogenesis and metabolic function of peroxisomes.1 A fundamental step in peroxisome biogenesis is the posttranslational peroxisomal import of matrix proteins. Proteins © 2012 American Chemical Society

designated for peroxisomes are synthezised on free ribosomes in the cytosol and recognized by import receptors like Pex5p, which bind the newly synthesized proteins and direct them to the docking complex at the peroxisomal membrane. Protein import into peroxisomes requires the importomer, a large multiprotein complex that spans the peroxisomal membrane. In Saccharomyces cerevisiae, the importomer consists of the docking complex (Pex14p, Pex13p, Pex17p) that is connected to the RING finger complex (Pex2p, Pex10p, Pex12p) via the intraorganellar organizer Pex8p as well as Pex5p.2−4 Pex14p Received: January 11, 2012 Published: February 29, 2012 2567

dx.doi.org/10.1021/pr3000333 | J. Proteome Res. 2012, 11, 2567−2580

Journal of Proteome Research

Article

recombination as described previously,14 and genomic tagging of PEX14 was performed by chromosomal integration according to Knop et al.15 using CCTGACTGGCAAAATGGACAGGTCGAAGACTCCATCCCAtccggttct gctgctag as forward and GTCCATAGTATCTAGGACAGTTACAATTACAATTTCCGcctcga ggccagaagac as the reverse primer. Correct integration and deletion were confirmed by colony PCR. The functionality of peroxisomes in cells expressing TEVcs-PA-tagged Pex14p has been reported previously.4

and Pex5p, the cytosolic receptor for most peroxisomal matrix proteins in yeast, have recently been proposed to form a large and highly dynamic pore facilitating the translocation of folded matrix proteins across the peroxisomal membrane.5 However, information about the molecular mechanisms underlying formation, function, and regulation of the importomer are still scarce. The recent discovery of an association of Pex14p and Dyn2p, a cytoplasmic light chain dynein in S. cerevisiae,6 suggests the existence of further proteins interacting with Pex14p, the central component of the peroxisomal importomer, that have not yet been identified. In recent years, affinity purification techniques combined with mass spectrometry (AP-MS) have proven to be most effective methodologies to dissect protein complexes (reviewed by Gingras et al.7). In general, a major challenge in AP-MSbased interaction studies is the reliable discrimination between true interaction partners and copurified contaminants. This could be greatly alleviated through the establishment of quantitative MS methods8 often employing stable isotope labeling with amino acids in cell culture (SILAC).9−11 The latest advancements in SILAC-based AP-MS approaches further allow for the mapping of transiently interacting proteins, e.g., dynamic interactors of protein complexes that typically elude identification.12,13 However, despite these recent refinements in AP-MS methodologies, the characterization of large membrane protein complexes such as the Pex14p complex still remains a major challenge.8 The strategy employed in this work includes epitope tagging of Pex14p and affinity purification of native multiprotein complexes from crude membranes of SILAC-labeled cells followed by high-resolution MS for relative protein quantification and two independent approaches for statistical data evaluation. Previous information on Pex14p and its binding partners4 provided a basic knowledge scaffold and allowed for efficient evaluation of our quantitative SILAC-AP-MS approach. The application of two complementary experimental tracks, affinity purification after mixing (AP-AM) and prior to mixing (AP-PM), facilitated the recognition of 9 core components as well as 12 additional transient binding partners. This led to the establishment of the so far most comprehensive interactome of Pex14p comprising virtually all known bona f ide binding partners as well as various novel interaction partners. The association of new transient components previously described to be of mainly cytosolic/nuclear (Hrr25p), mitochondrial (prohibitin), or unknown origin (Esl2p) with peroxisomes was validated by colocalization studies using Optiprep density gradient centrifugation. Our data provide new insight into the intrinsic molecular network associated with the peroxisomal importomer and are expected to prompt new studies on the functionality, dynamics, and regulation of this macromolecular membrane protein complex.

■

Culture Conditions and Metabolic Labeling Using SILAC

Minimal liquid medium [0.17% (w/v) yeast nitrogen base (YNB) without amino acids, 0.5% (w/v) ammonium sulfate, and 0.3% (w/v) glucose; adjusted to pH 6.0 with KOH] supplemented with selected amino acids and nucleobases (Lhistidine, L-tryptophan, L-methionine, adenine, and uracil, 20 mg/L each; L-isoleucine and L-tyrosine, 30 mg/L each; Lphenylalanine, 50 mg/L; L-leucine, 100 mg/L; L-valine, 150 mg/L; L-threonine, 200 mg/L; L-lysine and L-arginine, 50 mg/L each) was inoculated with yeast cells from a liquid culture at the stationary phase to reach an OD600 of 0.2. Following incubation at 30 °C and shaking for approximately 8 h, peroxisome proliferation was induced by adding 1/4 of the cell culture volume of oleate medium containing 0.85% (w/v) YNB without amino acids, 2.5% (w/v) ammonium sulfate, 0.5% (v/v) oleic acid, and 0.25% (v/v) Tween 40, adjusted to pH 6.0, and supplemented with the 5-fold concentration of the amino acids and nucleobases listed above. Cultures were grown for further 16 h under the same conditions. Yeast populations expressing the untagged wild-type form of Pex14p were metabolically labeled by growth in minimal and oleate medium in which normal lysine and arginine were replaced by 13C6lysine and -arginine (Eurisotop, Saarbrücken, Germany). Virtually complete incorporation (>98%) of 13C6-coded amino acids was verified by liquid chromatography−mass spectrometry (LC−MS) analysis of proteins extracted from CB199 yeast cells grown in the presence of heavy lysine and arginine. Affinity Purification of Native Membrane Protein Complexes

Yeast cells were harvested and washed twice with deionized water. Protein extracts were prepared as described previously4 with slight modifications. Membrane fractions were resuspended in lysis buffer (20 mM Tris, 80 mM NaCl, pH 7.5) containing protease and phosphatase inhibitors (PMSF, 174 μg/mL; aprotinin, 2 μg/mL; bestatin, 0.35 μg/mL; pepstatin, 1 μg/mL; leupeptin, 2.5 μg/mL; benzamidine, 160 μg/mL; antipain, 5 μg/mL; chymostatin, 6 μg/mL; NaF, 420 μg/mL) and homogenized using a Potter-Elvehjem. Homogenates were adjusted to 5 mg protein/mL with lysis buffer supplemented with 10% (v/v) glycerol and 1% (w/v) digitonin (Calbiochem). Solubilization of membrane proteins, affinity purification using IgG sepharose, and subsequent cleavage of bound protein complexes with TEV protease (Invitrogen; 100 units/100 μL IgG sepharose) were performed as described.4 The TEV protease was removed via affinity purification through its Nterminal polyhistidine tag using Ni-NTA agarose (Qiagen; 50 μL/100 μL IgG sepharose). Proteins were collected by centrifugation. This step was repeated once, and eluted proteins were combined and subsequently precipitated by adding four volumes of ice-cold acetone. After centrifugation, protein pellets were resuspended in sample buffer.

EXPERIMENTAL PROCEDURES

Yeast Strains

Yeast strains used in this study were S. cerevisiae CB199 auxotrophic for lysine and arginine (MATa, ura3-52, leu2-1, trp1-63, his3-200; arg4::natMX4, lys1::natMX3) and its variant expressing Pex14p fused at its C-terminus to a cleavage site (cs) for the tobacco etch virus (TEV) protease and the Protein A (PA) tag (MATa, ura3-52, leu2-1, trp1-63, his3-200; arg4::natMX4, lys1::natMX3, pex14::pex14-TEVcs-PA). CB199 was generated by chromosomal integration through homologous 2568



Article

Table 1. Molecular Components of Native Pex14p Membrane Complexesa

a

Proteins listed were classified as specifically enriched following both box plot analyses and a combinatorial statistical approach based on t-test and power analysis. Proteins in bold represent the Pex14p core complex while the other proteins are transient interactors. Proteins newly identified in this work as components of Pex14p complexes are marked with an asterisk. AP-AM, affinity purification after mixing; AP-PM, affinity purification prior to mixing; Subc. local., subcellular localization; P, peroxisomal; mult., multiple localizations; C, cytosolic; M, mitochondrial; N, nuclear; Var., variability of the quantification; RC, ratio count (i.e., number of peptide pairs); S, number of singlets (i.e., only the light form of the peptide was observed); N/A, not applicable; n.i., not identified; n.q., not quantifiable. A check mark represents meeting the criteria for specific interaction partner employing a t-test and power analysis (PA).

Nano-High-Pressure Liquid Chromatography−Electrospray Ionization−Tandem Mass Spectrometry (HPLC−ESI−MS/MS) Analysis

In AP-AM experiments, equal amounts of differentially labeled yeast cells (wet cell weight) were combined directly after harvesting, allowing for joint purification of the differentially labeled complexes. In AP-PM experiments, all purification steps were carried out separately for the differentially labeled yeast populations. Only after elution of the complexes, 12C6- and 13C6-labeled proteins (equal amounts based on the total protein concentrations determined for the homogenized membrane fractions) were combined prior to acetone precipitation, thereby diminishing exchange of binding partners. Both AP-AM and AP-PM experiments were performed in independent biological triplicates using 4 L of culture for each yeast strain.

Peptide mixtures were analyzed by nano HPLC−ESI−MS/MS using the UltiMate 3000 HPLC system (Dionex LC Packings, Idstein, Germany) directly coupled to an LTQ-Orbitrap XL instrument (Thermo Fisher Scientific, Bremen, Germany) as described previously17 with slight modifications. Briefly, peptides were separated using a 30 min linear gradient ranging from 4 to 40% ACN [in 0.1% (v/v) formic acid] followed by 40−80% ACN in 2 and 3 min at 80% ACN. The LTQ-Orbitrap XL, equipped with a nanoelectrospray ion source (Thermo Fisher Scientific) and distal coated SilicaTips (FS360-20-10-D, New Objective, Woburn), was operated using the following general mass spectrometric parameters: spray voltage, 1.6 kV; capillary voltage, 45 V; capillary temperature, 200 °C; tube lens voltage, 100 V. MS survey scans (m/z 300−1 500) with a resolution of 30 000 (at m/z 400) were acquired in the orbitrap. Simultaneously, the four most intense multiply charged ions were subjected to fragmentation by collisioninduced dissociation (CID) in the LTQ. The ion selection threshold was 500, and a dynamic exclusion of 60 s was applied.

Gel Electrophoresis and Tryptic Digestion of Proteins

Separation of affinity-purified proteins on a 4−12% NuPage Bis-Tris gradient gel (Invitrogen) was performed according to the manufacturer’s instructions. Following visualization of proteins with colloidal Coomassie Brilliant Blue G-250, gel lanes were cut into 20 slices, which were destained, washed, subjected to tryptic in-gel digestion, and prepared for LC−MS analysis as described.16 2569



Article

Mass Spectrometric Data Analysis

threshold defines the minimum enrichment factor (mEF) for denominating a protein “specific” and may differ between replicates. Proteins with singlet(s) were set as outliers, and box plots were plotted using the software R (version 2.8.0.). The results of these outlier analyses performed for each individual replicate defined the initial set of proteins representing potentially specific interaction partners of Pex14p. To obtain a more stringent data set, the following filtering criteria were consecutively applied to the primary set of outliers: (1) on the level of protein identification, sequence coverage of ≥5% and a protein posterior error probability (PEP) of ≤0.01 were required; (2) a protein was only considered a bona f ide Pex14p interaction partner when defined as an outlier in at least two out of three AP-AM and AP-PM experiments, respectively; and (3) for proteins quantified based on a single peptide pair or featuring singlets, the respective MS spectra were inspected manually. In addition, a combinatorial approach including a one-sided t-test and power analysis was employed to individually determine the mEF for proteins expressed in two or three replicates. The standard deviation of the power analysis was the 75% quantile of the observed protein standard deviations. To be classified as specific Pex14p interaction partner, proteins were required to exhibit a p-value of 0.05 in the AP-AM data set, thereby failing to meet the criteria for Pex14p-interacting proteins applying t-test and power analysis for statistical data evaluation. Further transient interaction

DISCUSSION

To gain new insight into the molecular composition of the membrane-embedded peroxisomal matrix protein import machinery, we thoroughly studied native membrane protein complexes of its central component Pex14p via SILAC-AP-MS. The application of two distinct experimental tracks (AP-AM and AP-PM) combined with statistical approaches allowed us to identify specific interaction partners of the importomer and, moreover, to define its stable and transient partners. On the basis of these findings, we established the so far most detailed Pex14p interactome (Figure 5) composed of 9 core and 12 transient components. The results obtained with both statistical approaches were consistent, which strengthens the reliability of 2575



Article

Figure 6. Hrr25p, Esl2p, and Phb1p partially colocalize with peroxisomes. (A) Steady-state expression levels of the genomically integrated TAPtagged proteins Hrr25p, Esl2p, Phb1p, Phb2p, and Pex14p (control) as well as the specificity of anti-Protein A (PA) antibodies were tested by immunoblotting of acetone-precipitated proteins of 30 mg of yeast cell lysates using polyclonal anti-PA antibodies. Mitochondrial porin (load control) was detected by antiporin antibodies. (B) Postnuclear supernatants of oleic acid-induced yeast cells expressing TAP-tagged Hrr25p, Esl2p, Phb1p, or Phb2p were loaded onto linear 2.25−24% Optiprep gradients containing 18% sucrose. The fractions were subjected to SDS-PAGE and immunoblot analysis using antibodies against PA, thiolase (peroxisomal marker), and porin (mitochondrial marker).

the receptor docking and RING finger complex as well as Pex8p generally exhibited very high abundance ratios in both SILAC-AP-MS tracks, classifying Pex14p as core component of the peroxisomal importomer. Pex5p, however, appeared to be highly enriched in AP-PM replicates only, indicating that it only interacts transiently with the Pex14p core complex. The transient association of Pex5p with the importomer is in general agreement with its biological role as receptor for peroxisomal targeting signal 1- (PTS1-) containing proteins. Recently, Meinecke et al. provided evidence that upon docking

the interactions we report here. Through this unbiased strategy, we obtained an extended view on the molecular assembly of the peroxisomal importomer, including new links to further peroxisomal membrane constituents as well as components of other cellular compartments. The Pex14p Core Complex Defines the Peroxisomal Importomer

Our quantitative MS-based interaction data confirmed all the peroxins already known as constituents of the Pex14p membrane protein complex in S. cerevisiae.4 Components of 2576



Article

newly identified interactions between Pex14p and Pex11p, Pex25p, as well as the ABC transporter in follow-up studies.

of cargo-loaded Pex5p to Pex14p, these two peroxins form a large and highly dynamic aqueous pore permitting translocation of folded and oligomeric cargo proteins of various sizes across the peroxisomal membrane.5 Also, the identification of the PTS1-proteins Mdh3p, Cat2p, and Idp3p as transient interaction partners of Pex14p is explained by the association of the cargo-loaded receptor with the importomer. Mono- and polyubiquitination are important steps in the recycling of the receptor Pex5p and its release from the peroxisomal membrane. While polyubiquitination labels Pex5p for proteasomal degradation, monoubiquitination tags the receptor for recycling (reviewed by Platta and Erdmann25). Accordingly, we found ubiquitin transiently linked to the Pex14p core complex. Its presence in the complex is consistent with its association with Pex5p.28

Dyn2p as Part of the Pex14p Core Complex

Dyn2p, the cytoplasmic light chain dynein of S. cerevisiae, has recently been shown to copurify with Pex14p when used as bait in an AP-MS experiment and to partially localize to peroxisomes in a Pex14p-dependent manner.6 In accordance with these observations, we identified Dyn2p as genuine component of the Pex14p core complex. Dyneins represent a family of microtubule-associated motor proteins consisting of several subunits with different size and function. Cytosolic dyneins have been implicated in a range of cellular processes including intracellular transport of various membranous organelles, mitosis, cell polarization, and directed cell movement.38,39 The cytosolic light chain subunits have been proposed to function in binding cargo for dynein-driven transport along microtubules. In addition, cytosolic light chain dyneins such as LC8 are known to be involved in numerous dynein-independent protein−protein interactions (Valle et al.,39 Mohan and Hosur,40 and references therein). This led to the suggestion that LC8 is a hub protein that is part of many protein interaction networks and plays essential roles for the maintenance and regulation of their organization.41 In S. cerevisiae, for example, Dyn2p was reported as nucleoporin that acts as “molecular glue” by promoting the dimerization and stabilization of the Nup82 complex, a module of the nuclear pore complex (NPC), which in turn stabilizes the incorporation of this module into the NPC. Mapping of potential binding sites for Dyn2p in Nup159, the interaction partner of Dyn2p in the Nup82 complex, revealed the presence of several recognition sites. They all contained the consensus sequence L/T/I/VQT.6 Interestingly, this consensus sequence is also present in Pex14p at amino acid sequence position 182−184 and in its interaction partners Pex5p (amino acid residues 102− 104 and 356−358), Pex17p (residues 108−110), Pex13p (residues 286−288), Pex11p (residues 150−152), and Cat2p (residues 90−92). Hence, in future investigations it will be of great interest (i) to reveal whether the interaction occurs via these sequences as well as (ii) to study the precise role of Dyn2p in its association with Pex14p. On the basis of published data, a function in peroxisome motility is unlikely. While peroxisomes of mammalian and Drosophila cells are evidently associated with microtubules and transported along these cytoskeletal tracks in a dynein-mediated manner,42−46 S. cerevisiae uses a different intracellular transport system. Assisted by Myo2p, a class V myosin motor protein, peroxisomes are moved along actin filaments.47

Further PMP Connections

Our proteomics data confirm an association between Pex14p and Pex15p, a protein proposed to act downstream of the importomer in the process of receptor export and recycling.29 This peroxin has already been reported to be associated with the importomer by affinity purification of epitope-tagged Pex15p, and its association with Pex14p has been observed using the split-ubiquitin system.30,31 On the basis of the low number of quantification events for Pex15p in our work, it presumably represents an interaction partner of rather low stoichiometry. Further constituents of the Pex14p interactome are the PMPs Pex11p, Pex25p, and Pxa1p. The latter is a subunit of a heterodimeric ATP-binding cassette (ABC) membrane transporter required for the translocation of long-chain fatty acids across the peroxisomal membrane32,33 and represents a newly identified component of the Pex14p complex. The second subunit of this transporter, Pxa2p, was detected in three out of six experiments and was specifically enriched in single replicates only (see Supplemental Tables S2B and S3B in the Supporting Information). Its presence in Pex14p complexes, however, could be shown by immunoblot analyses (data not shown), thereby substantiating our evidence for an association between the ABC transporter and the importomer. The peroxins Pex11p and Pex25p belong to the Pex11 family, which in S. cerevisiae further includes Pex27p, a constituent of very low abundance34,35 not identified in our study. The members of this protein family have recently been reported to execute individual functions in different steps of peroxisome biogenesis.36 While Pex11p is required for the proliferation of pre-existing organelles, Pex25p is involved in both initiation of de novo peroxisome biogenesis from the ER as well as membrane elongation of peroxisomes already existing in the cell. The association of Pex25p with the Pex14p complex and, thus, the importomer is shown here for the first time; the Pex14p-Pex11p connection, though, has recently been reported in a genome-wide in vivo screen for protein−protein interactions using a protein-fragment complementation assay.37 Interestingly, the high-throughput data obtained in the latter study also revealed interactions of Pex11p with Pex13p, Pex10p, and Pex12p, which is in line with our findings that Pex11p assembles with the core components of the peroxisomal import machinery. Interactions of Pex14p with Pex11p and Pex25p were further confirmed in reverse AP-MS experiments (data not shown). Thus, it will be interesting to define the functional importance and spatial arrangement of the

Prohibitin, Hrr25p, and Esl2p are New Transient Pex14p Interaction Partners

According to our quantitative AP-MS data, both subunits of the prohibitin complex, Phb1p and Phb2p, appeared to associate with the Pex14p core complex in a dynamic fashion. Interestingly, copurification of prohibitin with Pex14p complexes was already suggested in earlier work.4 In this work, subcellular colocalization studies using Optiprep density gradient centrifugation unequivocally confirmed the presence of a portion of prohibitin in peroxisomal fractions. Prohibitins are known to localize predominantly to mitochondria where they form multimeric ring structures in the inner membrane.48,49 They have been implicated in a number of processes essential for mitochondrial structure and function.50 Interestingly, in mammalian cells, prohibitins have also been reported 2577



■

to be present in nonmitochondrial locations such as the nucleus and the plasma membrane (Osman et al.,51 Mishra et al.,52 and references therein). Our data provide new evidence for the association of prohibitin with peroxisomes and may point to a potential structural and/or functional role in peroxisome biogenesis. Hrr25p is a serine/threonine kinase of the casein kinase (CK) 1 family belonging to the highly conserved δ/ε group and has been identified in our study as a new transient component associated with the Pex14p core complex. In support of our quantitative MS-based interaction data, density gradient centrifugation of a PNS revealed that a fraction of Hrr25p was indeed associated with peroxisomes. Hrr25p is the only CK1 in budding yeast lacking a lipid membrane anchor and has been reported to exert a number of cellular functions that include vesicular trafficking,53,54 gene expression,55 ribosome biogenesis,56,57 DNA repair, chromosome segregation, and cell division.58,59 Furthermore, Hrr25p has recently been reported to be involved in regulatory processes of peroxisome biogenesis; it has been suggested to be a positive effector of glucose repression at the gene level.60 Data proving or disproving a possible implication in the formation or functionality of peroxisomes, however, were not provided. Of further note, AP-MS-based studies of different research groups recently provided evidence for an interaction between Hrr25p and Esl2p,61,62 a protein of unknown function that has also been identified as part of the Pex14p interactome established in this work. Through density gradient centrifugation experiments, we confirmed its partial association with peroxisomes. The transient interaction of the CK1 Hrr25p with the Pex14p complex raises the question whether reversible phosphorylation events play a regulatory role in the biogenesis of peroxisomes. Hence, it will be of future interest to obtain information about the phosphorylation status of the different components involved in peroxisomal protein import processes. Interestingly, a phosphorylation-dependent activation of Pex11p has recently been reported to play a role in the control of peroxisome dynamics; here, the cyclin-dependent kinase Pho85p has been suggested to mediate phosphorylation of Pex11p.63

■

Article

ASSOCIATED CONTENT

S Supporting Information *

Supplemental tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Address: Institute of Biology II, Functional Proteomics, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany. Phone: +49 761 203 2690. Fax: +49 761 203 2601. E-mail: [email protected]. Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Ed Hurt (Heidelberg), Nikolaus Pfanner (Freiburg), and Thomas Langer (Cologne) for kindly providing us with anti-Dyn2p and anti-Phb2p antibodies as well as Nadine Stoepel, Magdalena Pawlas, and Christian Bunse for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (Grant SFB 642), Excellence Initiative of the German Federal & State Governments (Grant EXC 294 BIOSS), the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen (MIWF; Protein Research Department), and the Bundesministerium für Bildung und Forschung. Cecile Brocard was supported by the EliseRichter Program of the Austrian Science Fund (FWF; Grant B39-V09).

■

ABBREVIATIONS ABC, ATP-binding cassette; AP-MS, affinity purification mass spectrometry; AP-AM, affinity purification after mixing; APPM, affinity purification prior to mixing; CID, collision-induced dissociation; CK, casein kinase; mEF, minimum enrichment factor; NPC, nuclear pore complex; PA, Protein A; PEP, posterior error probability; PMP, peroxisomal membrane protein; PNS, postnuclear supernatant; PTS, peroxisomal targeting signal; SGD, Saccharomyces Genome Database; SILAC, stable isotope labeling with amino acids in cell culture; TAP, tandem affinity purification; TEV, tobacco etch virus

CONCLUDING REMARKS

Membrane-embedded macromolecular complexes execute important functions in a multitude of biological processes such as protein transport, signal transduction, and the biogenesis of organelles. Because of major technical challenges, however, studies of large membrane protein complexes are still greatly underrepresented in interaction proteomics endeavors. The hydrophobicity and insertion of multiprotein complexes into lipid bilayers require purification strategies specifically tailored to effectively extract them from membranes while maintaining their native composition. We advocate the use of quantitative AP-MS approaches to obtain an unbiased and thorough view on such molecular machines. Importantly, our strategy enabled us to investigate the molecular composition of the peroxisomal importomer without the necessity of organelle isolation or further affinity-based purification steps and stringent washing conditions to minimize unspecific binding partners. This opens up the possibility for delineating new binding partners that had not been known to be associated with protein complexes and/or organelles before and for providing insight into their dynamic nature.

■

REFERENCES

(1) Wanders, R. J.; Waterham, H. R. Biochemistry of mammalian peroxisomes revisited. Annu. Rev. Biochem. 2006, 75, 295−332. (2) Girzalsky, W.; Platta, H. W.; Erdmann, R. Protein transport across the peroxisomal membrane. Biol. Chem. 2009, 390, 745−751. (3) Rayapuram, N.; Subramani, S. The importomer-A peroxisomal membrane complex involved in protein translocation into the peroxisome matrix. Biochim. Biophys. Acta 2006, 1763, 1613−1619. (4) Agne, B.; Meindl, N. M.; Niederhoff, K.; Einwachter, H.; Rehling, P.; Sickmann, A.; Meyer, H. E.; Girzalsky, W.; Kunau, W. H. Pex8p: an intraperoxisomal organizer of the peroxisomal import machinery. Mol. Cell 2003, 11, 635−646. (5) Meinecke, M.; Cizmowski, C.; Schliebs, W.; Kruger, V.; Beck, S.; Wagner, R.; Erdmann, R. The peroxisomal importomer constitutes a large and highly dynamic pore. Nat. Cell Biol. 2010, 12, 273−277. (6) Stelter, P.; Kunze, R.; Flemming, D.; Hopfner, D.; Diepholz, M.; Philippsen, P.; Bottcher, B.; Hurt, E. Molecular basis for the functional interaction of dynein light chain with the nuclear-pore complex. Nat. Cell Biol. 2007, 9, 788−796.

2578



Article

(7) Gingras, A. C.; Gstaiger, M.; Raught, B.; Aebersold, R. Analysis of protein complexes using mass spectrometry. Nat. Rev. Mol. Cell. Biol. 2007, 8, 645−654. (8) Oeljeklaus, S.; Meyer, H. E.; Warscheid, B. New dimensions in the study of protein complexes using quantitative mass spectrometry. FEBS Lett. 2009, 583, 1674−1683. (9) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 2002, 1, 376−386. (10) Blagoev, B.; Kratchmarova, I.; Ong, S. E.; Nielsen, M.; Foster, L. J.; Mann, M. A proteomics strategy to elucidate functional proteinprotein interactions applied to EGF signaling. Nat. Biotechnol. 2003, 21, 315−318. (11) Tackett, A. J.; DeGrasse, J. A.; Sekedat, M. D.; Oeffinger, M.; Rout, M. P.; Chait, B. T. I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions. J. Proteome Res. 2005, 4, 1752−1756. (12) Wang, X.; Huang, L. Identifying dynamic interactors of protein complexes by quantitative mass spectrometry. Mol. Cell. Proteomics 2008, 7, 46−57. (13) Mousson, F.; Kolkman, A.; Pijnappel, W. W.; Timmers, H. T.; Heck, A. J. Quantitative proteomics reveals regulation of dynamic components within TATA-binding protein (TBP) transcription complexes. Mol. Cell. Proteomics 2008, 7, 845−852. (14) Goldstein, A. L.; McCusker, J. H. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 1999, 15, 1541−1553. (15) Knop, M.; Siegers, K.; Pereira, G.; Zachariae, W.; Winsor, B.; Nasmyth, K.; Schiebel, E. Epitope tagging of yeast genes using a PCRbased strategy: more tags and improved practical routines. Yeast 1999, 15, 963−972. (16) Wiese, S.; Gronemeyer, T.; Ofman, R.; Kunze, M.; Grou, C. P.; Almeida, J. A.; Eisenacher, M.; Stephan, C.; Hayen, H.; Schollenberger, L.; Korosec, T.; Waterham, H. R.; Schliebs, W.; Erdmann, R.; Berger, J.; Meyer, H. E.; Just, W.; Azevedo, J. E.; Wanders, R. J.; Warscheid, B. Proteomics characterization of mouse kidney peroxisomes by tandem mass spectrometry and protein correlation profiling. Mol. Cell. Proteomics 2007, 6, 2045−2057. (17) Kaller, M.; Liffers, S. T.; Oeljeklaus, S.; Kuhlmann, K.; Röh, S.; Hoffmann, R.; Warscheid, B.; Hermeking, H. Genome-wide characterization of miR-34a induced changes in protein and mRNA expression by a combined pulsed SILAC and microarray analysis. Mol. Cell. Proteomics 2011, 10, M111.010462. (18) Cox, J.; Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 2008, 26, 1367−1372. (19) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20, 3551− 3567. (20) Tukey, J. W. Data-based graphics: visual display in the decades to come. Stat. Sci. 1990, 5, 327−339. (21) Motulsky, H. Intuitive Biostatistics; Oxford University Press: New York, 1995. (22) Shannon, P.; Markiel, A.; Ozier, O.; Baliga, N. S.; Wang, J. T.; Ramage, D.; Amin, N.; Schwikowski, B.; Ideker, T. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003, 13, 2498−2504. (23) Erdmann, R.; Veenhuis, M.; Mertens, D.; Kunau, W. H. Isolation of peroxisome-deficient mutants of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 5419−5423. (24) Dodt, G.; Gould, S. J. Multiple PEX genes are required for proper subcellular distribution and stability of Pex5p, the PTS1 receptor: Evidence that PTS1 protein import is mediated by a cycling receptor. J. Cell Biol. 1996, 135, 1763−1774. (25) Platta, H. W.; Erdmann, R. Peroxisomal dynamics. Trends Cell. Biol. 2007, 17, 474−484.

(26) Erdmann, R.; Blobel, G. Giant peroxisomes in oleic acid-induced Saccharomyces cerevisiae lacking the peroxisomal membrane protein Pmp27p. J. Cell Biol. 1995, 128, 509−523. (27) Marshall, P. A.; Krimkevich, Y. I.; Lark, R. H.; Dyer, J. M.; Veenhuis, M.; Goodman, J. M. Pmp27 promotes peroxisomal proliferation. J. Cell Biol. 1995, 129, 345−355. (28) Platta, H. W.; Girzalsky, W.; Erdmann, R. Ubiquitination of the peroxisomal import receptor Pex5p. Biochem. J. 2004, 384, 37−45. (29) Platta, H. W.; Grunau, S.; Rosenkranz, K.; Girzalsky, W.; Erdmann, R. Functional role of the AAA peroxins in dislocation of the cycling PTS1 receptor back to the cytosol. Nat. Cell Biol. 2005, 7, 817−822. (30) Rosenkranz, K.; Birschmann, I.; Grunau, S.; Girzalsky, W.; Kunau, W. H.; Erdmann, R. Functional association of the AAA complex and the peroxisomal importomer. FEBS J. 2006, 273, 3804− 3815. (31) Eckert, J. H.; Johnsson, N. Pex10p links the ubiquitin conjugating enzyme Pex4p to the protein import machinery of the peroxisome. J. Cell Sci. 2003, 116, 3623−3634. (32) Shani, N.; Valle, D. A Saccharomyces cerevisiae homolog of the human adrenoleukodystrophy transporter is a heterodimer of two half ATP-binding cassette transporters. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 11901−11906. (33) Hettema, E. H.; van Roermund, C. W.; Distel, B.; van den Berg, M.; Vilela, C.; Rodrigues-Pousada, C.; Wanders, R. J.; Tabak, H. F. The ABC transporter proteins Pat1 and Pat2 are required for import of long-chain fatty acids into peroxisomes of Saccharomyces cerevisiae. EMBO J. 1996, 15, 3813−3822. (34) Ghaemmaghami, S.; Huh, W. K.; Bower, K.; Howson, R. W.; Belle, A.; Dephoure, N.; O’Shea, E. K.; Weissman, J. S. Global analysis of protein expression in yeast. Nature 2003, 425, 737−741. (35) Rottensteiner, H.; Stein, K.; Sonnenhol, E.; Erdmann, R. Conserved function of pex11p and the novel pex25p and pex27p in peroxisome biogenesis. Mol. Biol. Cell 2003, 14, 4316−4328. (36) Huber, A.; Koch, J.; Kragler, F.; Brocard, C.; Hartig, A. A subtle interplay between three pex11 proteins shapes de novo formation and fission of peroxisomes. Traffic 2012, 13, 157−167. (37) Tarassov, K.; Messier, V.; Landry, C. R.; Radinovic, S.; Serna Molina, M. M.; Shames, I.; Malitskaya, Y.; Vogel, J.; Bussey, H.; Michnick, S. W. An in vivo map of the yeast protein interactome. Science 2008, 320, 1465−1470. (38) Hook, P.; Vallee, R. B. The dynein family at a glance. J. Cell Sci. 2006, 119, 4369−4371. (39) Vallee, R. B.; Williams, J. C.; Varma, D.; Barnhart, L. E. Dynein: An ancient motor protein involved in multiple modes of transport. J. Neurobiol. 2004, 58, 189−200. (40) Mohan, P. M.; Hosur, R. V. Structure-function-folding relationships and native energy landscape of dynein light chain protein: nuclear magnetic resonance insights. J. Biosci. 2009, 34, 465− 479. (41) Barbar, E. Dynein light chain LC8 is a dimerization hub essential in diverse protein networks. Biochemistry 2008, 47, 503−508. (42) Schrader, M.; Burkhardt, J. K.; Baumgart, E.; Lüers, G.; Spring, H.; Völkl, A.; Fahimi, H. D. Interaction of microtubules with peroxisomes. Tubular and spherical peroxisomes in HepG2 cells and their alterations induced by microtubule-active drugs. Eur. J. Cell Biol. 1996, 69, 24−35. (43) Rapp, S.; Saffrich, R.; Anton, M.; Jakle, U.; Ansorge, W.; Gorgas, K.; Just, W. W. Microtubule-based peroxisome movement. J. Cell Sci. 1996, 109, 837−849. (44) Wiemer, E. A.; Wenzel, T.; Deerinck, T. J.; Ellisman, M. H.; Subramani, S. Visualization of the peroxisomal compartment in living mammalian cells: dynamic behavior and association with microtubules. J. Cell Biol. 1997, 136, 71−80. (45) Schrader, M.; King, S. J.; Stroh, T. A.; Schroer, T. A. Real time imaging reveals a peroxisomal reticulum in living cells. J. Cell Sci. 2000, 113, 3663−3671. 2579



Article

(46) Kural, C.; Kim, H.; Syed, S.; Goshima, G.; Gelfand, V. I.; Selvin, P. R. Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement? Science 2005, 308, 1469−1472. (47) Hoepfner, D.; van den Berg, M.; Philippsen, P.; Tabak, H. F.; Hettema, E. H. A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae. J. Cell Biol. 2001, 155, 979−990. (48) Ikonen, E.; Fiedler, K.; Parton, R. G.; Simons, K. Prohibitin, an antiproliferative protein, is localized to mitochondria. FEBS Lett. 1995, 358, 273−277. (49) Tatsuta, T.; Model, K.; Langer, T. Formation of membranebound ring complexes by prohibitins in mitochondria. Mol. Biol. Cell 2005, 16, 248−259. (50) Merkwirth, C.; Langer, T. Prohibitin function within mitochondria: essential roles for cell proliferation and cristae morphogenesis. Biochim. Biophys. Acta 2009, 1793, 27−32. (51) Osman, C.; Merkwirth, C.; Langer, T. Prohibitins and the functional compartmentalization of mitochondrial membranes. J. Cell Sci. 2009, 122, 3823−3830. (52) Mishra, S.; Ande, S. R.; Nyomba, B. L. The role of prohibitin in cell signaling. FEBS J. 2010, 277, 3937−3946. (53) Murakami, A.; Kimura, K.; Nakano, A. The inactive form of a yeast casein kinase I suppresses the secretory defect of the sec12 mutant. Implication of negative regulation by the Hrr25 kinase in the vesicle budding from the endoplasmic reticulum. J. Biol. Chem. 1999, 274, 3804−3810. (54) Lord, C.; Bhandari, D.; Menon, S.; Ghassemian, M.; Nycz, D.; Hay, J.; Ghosh, P.; Ferro-Novick, S. Sequential interactions with Sec23 control the direction of vesicle traffic. Nature 2011, 473, 181−186. (55) Ho, Y.; Mason, S.; Kobayashi, R.; Hoekstra, M.; Andrews, B. Role of the casein kinase I isoform, Hrr25, and the cell cycle-regulatory transcription factor, SBF, in the transcriptional response to DNA damage in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 581−586. (56) Schäfer, T.; Maco, B.; Petfalski, E.; Tollervey, D.; Böttcher, B.; Aebi, U.; Hurt, E. Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit. Nature 2006, 441, 651−655. (57) Ray, P.; Basu, U.; Ray, A.; Majumdar, R.; Deng, H.; Maitra, U. The Saccharomyces cerevisiae 60 S ribosome biogenesis factor Tif6p is regulated by Hrr25p-mediated phosphorylation. J. Biol. Chem. 2008, 283, 9681−9691. (58) Hoekstra, M. F.; Liskay, R. M.; Ou, A. C.; DeMaggio, A. J.; Burbee, D. G.; Heffron, F. HRR25, a putative protein kinase from budding yeast: association with repair of damaged DNA. Science 1991, 253, 1031−1034. (59) Katis, V. L.; Lipp, J. J.; Imre, R.; Bogdanova, A.; Okaz, E.; Habermann, B.; Mechtler, K.; Nasmyth, K.; Zachariae, W. Rec8 phosphorylation by casein kinase 1 and Cdc7-Dbf4 kinase regulates cohesin cleavage by separase during meiosis. Dev. Cell 2010, 18, 397− 409. (60) Saleem, R. A.; Knoblach, B.; Mast, F. D.; Smith, J. J.; Boyle, J.; Dobson, C. M.; Long-O’Donnell, R.; Rachubinski, R. A.; Aitchison, J. D. Genome-wide analysis of signaling networks regulating fatty acidinduced gene expression and organelle biogenesis. J. Cell Biol. 2008, 181, 281−292. (61) Gavin, A. C.; Aloy, P.; Grandi, P.; Krause, R.; Boesche, M.; Marzioch, M.; Rau, C.; Jensen, L. J.; Bastuck, S.; Dumpelfeld, B.; Edelmann, A.; Heurtier, M. A.; Hoffman, V.; Hoefert, C.; Klein, K.; Hudak, M.; Michon, A. M.; Schelder, M.; Schirle, M.; Remor, M.; Rudi, T.; Hooper, S.; Bauer, A.; Bouwmeester, T.; Casari, G.; Drewes, G.; Neubauer, G.; Rick, J. M.; Kuster, B.; Bork, P.; Russell, R. B.; Superti-Furga, G. Proteome survey reveals modularity of the yeast cell machinery. Nature 2006, 440, 631−636. (62) Petronczki, M.; Matos, J.; Mori, S.; Gregan, J.; Bogdanova, A.; Schwickart, M.; Mechtler, K.; Shirahige, K.; Zachariae, W.; Nasmyth, K. Monopolar attachment of sister kinetochores at meiosis I requires casein kinase 1. Cell 2006, 126, 1049−1064.

(63) Knoblach, B.; Rachubinski, R. A. Phosphorylation-dependent activation of peroxisome proliferator protein PEX11 controls peroxisome abundance. J. Biol. Chem. 2010, 285, 6670−6680.

2580


Identification of Core Components and Transient ... - ACS Publications

Recommend Documents