Protein Interaction Network of Human Protein Kinase D2 Revealed by

Aug 25, 2016 - Developing a Multiplexed Quantitative Cross-Linking Mass Spectrometry Platform for Comparative Structural Analysis of Protein Complexes...
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Protein Interaction Network of Human Protein Kinase D2 Revealed by Chemical Cross-Linking/Mass Spectrometry Björn Haü pl,† Christian H. Ihling,† and Andrea Sinz*,† †

Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120 Halle (Saale), Germany S Supporting Information *

ABSTRACT: We investigated the interaction network of human PKD2 in the cytosol and in Golgi-enriched subcellular protein fractions by an affinity enrichment strategy combined with chemical cross-linking/mass spectrometry (MS). Analysis of the subproteomes revealed the presence of distinct proteins in the cytosolic and Golgi fractions. The covalent fixation of transient or weak interactors by chemical cross-linking allowed capturing interaction partners that might otherwise disappear during conventional pull-down experiments. In total, 31 interaction partners were identified for PKD2, including glycogen synthase kinase-3 beta (GSK3B), 14−3−3 protein gamma (YWHAG), and the alpha isoform of 55 kDa regulatory subunit B of protein phosphatase 2A (PPP2R2A). Remarkably, the entire seven-subunit Arp2/3 complex (ARPC1B, ARPC2, ARPC3, ARPC4, ARPC5, ACTR3, ACTR2) as well as ARPC1A and ARPC5L, which are putative substitutes of ARPC1B and ARPC5, were identified. We provide evidence of a direct protein−protein interaction between PKD2 and Arp2/3. Our findings will pave the way for further structural and functional studies of PKD2 complexes, especially the PKD2/Arp2/3 interaction, to elucidate the role of PKD2 for transport processes at the trans-Golgi network. Data are available via ProteomeXchange with identifiers PXD003909 (enrichment from cytosolic fractions), PXD003913 (enrichment from Golgi fractions), and PXD003917 (subcellular fractionation). KEYWORDS: affinity enrichment, Arp 2/3 complex, chemical cross-linking, interaction network, mass spectrometry, protein kinase D



INTRODUCTION Protein kinase D (PKD) belongs to the family of protein kinase C (PKC)-related calmodulin-dependent serine/threonine kinases and comprises the three isoforms PKD1, PKD2, and PKD3.1 PKD (Figure 1) plays an important role in multiple physiological functions, such as cell proliferation or cytoskeletal dynamics, and PKD isoforms are implicated in various pathological processes.2,3 A pivotal role is attributed to PKD regarding the major cellular processes of transport vesicle formation at the Golgi apparatus.4 For example, the inactivation of PKD2 leads to an extensive tubulation of trans-Golgi network (TGN) membranes instead of vesicle formation and cargo delivery.5 So far, some of the underlying mechanisms have been elucidated, mainly by using cell biological approaches. Activated PKD interacts with diacylglycerol (DAG) of the TGN membrane via its first cysteine-rich domain (C1a)6,7 and phosphorylates phosphatidylinositol-4 kinase IIIβ (PI4KIIIβ).8 This leads to the recruitment of further effectors for vesicle shedding, for example, the ceramide transfer protein (CERT)9 and the oxysterol-binding protein (OSBP)10 that are involved in membrane modification at the site of vesicle formation. Furthermore, an interaction between the second cysteine-rich domain (C1b) of the PKD2 isoform and the small G-protein ADP-ribosylation factor 1 (ARF1) has been proposed.11 This interaction has been described to be crucial for the recruitment of PKD2 to the TGN membrane with © 2016 American Chemical Society

ARF1 acting as scaffold protein. Recently, the PKD2/ARF1 interaction, in the context of matrix metalloproteinase secretion, has been extended to a multiprotein complex embracing ADP-ribosylation factor-like protein 1 (ARL1) and Arfaptin-2 as additional members.12 Although a number of PKD2 substrates have been characterized to date, there is still a demand for identifying additional PKD2 interaction partners as this will enhance our understanding of PKD2-dependent cellular processes at the molecular level, such as the formation of transport vesicles at the TGN. To investigate the interactions of PKD2 in the cytosol and at the trans-Golgi network, we performed chemical cross-linking and mass spectrometry (MS). During the last 15 years, chemical cross-linking/MS has matured into an alternative technique for analyzing protein 3Dstructures, for elucidating protein−protein interactions, and for mapping interaction networks.13−19 The most prominent advantage of cross-linking/MS is that experiments can be performed with purified proteins under native-like conditions. Weak or transient protein−protein interactions are preserved as covalent bonds are introduced between molecules. The ultimate goal is to conduct cross-linking experiments in the cellular environment20 and in living cells,21 which is still a Received: June 3, 2016 Published: August 25, 2016 3686

DOI: 10.1021/acs.jproteome.6b00513 J. Proteome Res. 2016, 15, 3686−3699

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Journal of Proteome Research

Figure 1. Illustration of PKD2 domain structure. Protein kinase D isoforms share a common domain structure, exemplarily shown for the PKD2 isoform. The cysteine-rich domain is responsible for binding diacylglycerol (DAG) at the TGN membrane. Also, the pleckstrin homology (PH) domain is involved in membrane interaction, while the catalytic domain exhibits kinase activity.

All reagents used were at the highest available purity. Human PKD2 was obtained from Carna Biosciences as N-terminally tagged GST fusion protein.

L-90K, Rotor SW28, Beckman Coulter). The supernatants representing the cytosolic fractions were collected, quick-frozen in liquid nitrogen, and stored at −20 °C. Membrane pellets were resuspended in 6 mL of fractionation buffer. Next, enrichment of Golgi components from the membrane fractions was carried out according to an established procedure30 with minor modifications. In brief, the resuspended membrane fractions were mixed with 6 mL of a solution containing 2.3 M sucrose, 20 mM HEPES, pH 7.5 and 250 μL of 100 mM EDTA, pH 7.4. The resulting solutions containing 1.4 M sucrose were transferred to an ultracentrifugation tube and overlaid with 14 mL of a solution containing 1.2 M sucrose, 20 mM HEPES, pH 7.5 and 8 mL of a solution containing 0.8 M sucrose, 20 mM HEPES, pH 7.5, creating a discontinuous sucrose density gradient. This gradient was subjected to ultracentrifugation at 100 000g for 2.5 h. The 0.8 M/1.2 M sucrose interface represents the Golgi fraction and was extracted in a minimal volume (∼2 mL) with a syringe. The Golgi fraction was diluted in 20 mM HEPES, pH 7.5 at 1:4 (v/ v) ratio and ultracentrifuged at 100 000g for 1 h. The sedimented material was resuspended in 1 mL of fractionation buffer containing 0.1% (w/v) n-dodecyl-β-D-maltoside (DDM), incubated for 1 h and centrifuged at 16 000g for 30 min. The supernatants were collected, quick-frozen in liquid nitrogen, and stored at −20 °C.

Cell Culture and Harvest

Affinity Enrichment and Chemical Cross-Linking

HeLa cells (kindly provided by Dr. Cordelia Schiene-Fischer) were cultured in Dulbecco’s modified Eagle’s medium (Biochrom) containing 10% (v/v) of FCS (Biochrom) at 37 °C and an atmospheric CO2 concentration of 10% in standard cell culture flasks. The cells were subcultured to large cell culture dishes (150 mm diameter) and grown to a density of 80−90%. The culture medium was depleted and cells were harvested on ice in 5 mL of PBS (Biochrom) using a cell scraper. After two rounds of washing with 50 mL of PBS per tube of cell suspension (500g, 4 °C, 5 min), the combined cell pellet was equilibrated by washing with fractionation buffer (20 mM HEPES, 250 mM sucrose, pH 7.5). Subsequently, the cell pellet was resuspended at a ratio of 1:5 in ice-cold fractionation buffer containing protease inhibitors (Complete EDTA-free, Roche) and quick-frozen in liquid nitrogen for storage at −20 °C.

All affinity enrichment and cross-linking experiments were carried out between 0 to 4 °C, either in batch mode or using spin columns (Pierce Screw Cap, Thermo Fisher Scientific). First, 20 μg of glutathione sepharose beads (4B, GE Healthcare) was prepared by washing three times with binding buffer (50 mM HEPES, 150 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 10% (v/v) glycerol, pH 7.5). Subsequently, 20 (cytosolic fractions) or 10 μg (Golgi-enriched fractions) of GST-PKD2 in 200 μL of binding buffer was bound to the affinity matrix by incubation for 1 h. For the GST and matrix control samples, equimolar amounts of GST (Thermo Fisher Scientific) or binding buffer were applied. Beads with affinitycaptured GST-PKD2 and control samples were incubated with 800 μL of cytosolic fractions or 100 μL of Golgi-enriched fractions for 2 h. Chemical cross-linking was carried out with 5 mM bis(sulfosuccinimidyl) glutarate (BS2G-D0/D4) (Thermo Fisher Scientific) for 2 h on ice. Control samples without chemical cross-linker were treated with an equal volume of DMSO. The cross-linking reactions were quenched by the addition of ammonium bicarbonate to a final concentration of 20 mM. After the cross-linking reactions, the sepharose beads were washed three times with 400 μL of binding buffer containing 500 mM NaCl. The beads were resuspended in 40 μL of denaturation solution (8 M urea, 400 mM ammonium

challenging task due to high sample complexity and broad dynamic range of proteins in the cell. Most of the common structural proteomics approaches rely on the use of homobifunctional amine-reactive cross-linking reagents,22,23 where the protein of interest is usually purified by affinity enrichment.24 This process requires a reliable quantification of protein binding partners to distinguish enriched proteins from nonregulated ones. Also, nonspecific binding to the affinity matrix has to be ruled out.25 To this end, one of the most promising approaches is the use of label-free quantification (LFQ) of identified proteins based on the signal intensities of peptides assigned by MS/MS.26−29 In this study, we investigated the interaction networks of human PKD2 in the cytosol as well as in Golgi-enriched subcellular protein fractions in HeLa cells, employing an affinity enrichment chemical cross-linking/MS approach. Gaining insights into the interaction network of PKD2 will further enhance our knowledge of the intercellular pathways engaging PKD2, especially its role in transport processes at the TGN.



EXPERIMENTAL PROCEDURES

Proteins and Reagents

Subcellular Fractionation and Enrichment of Golgi Proteins

All fractionation steps were carried out between 0 and 4 °C. HeLa cell pellets resuspended in fractionation buffer were thawed on ice and homogenized by 20 strokes in a precooled Dounce homogenizer. The homogenates were subjected to centrifugation at 1000g for 10 min to sediment nonlysed cells and nuclei. The postnuclear supernatants (PNSs) were centrifuged at 100 000g for 1 h in an ultracentrifuge (Optima 3687

DOI: 10.1021/acs.jproteome.6b00513 J. Proteome Res. 2016, 15, 3686−3699

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Journal of Proteome Research

Carbamidomethylation of cysteine was set as fixed modification and oxidation of methionine was set as variable modification. Protein identifications were filtered against a target decoy database (reversed sequence order) allowing a maximum false discovery rate of 1%. Additionally, the “match between runs” feature was enabled and label-free quantification of proteins (LFQ) was conducted using the MaxLFQ option28 with minimum ratio count set to value 1. The “proteinGroups” output file from MaxQuant was used for downstream data analysis. The MS proteomics data of the affinity enrichment crosslinking experiments have been deposited to the ProteomeXchange Consortium33 via the PRIDE34 partner repository with identifiers PXD003909 (cytosolic protein fractions) and PXD003913 (Golgi-enriched protein fractions).

bicarbonate), incubated at 400 rpm, 37 °C for 15 min, and subjected to in-solution digestion with Glu-C and trypsin. In-Solution Digestion of Proteins

Samples were digested as previously described31 using trypsin (sequencing grade, Promega) or a trypsin/Glu-C (sequencing grade, Promega) mixture at an enzyme/substrate ratio of 1:50. Liquid Chromatography−Mass Spectrometry

Peptide solutions were analyzed by LC−MS/MS on an Ultimate 3000 RSLC nano-HPLC system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) via a Nanospray Flex nano-ESI ion source (Thermo Fisher Scientific). Samples were loaded onto a RP C8 precolumn (Acclaim PepMap, 300 μm × 5 mm, 5 μm, 100 Å, Thermo Fisher Scientific) at a flow rate of 30 μL/min. After washing on the precolumn with 0.1% (v/v) TFA for 15 min at 30 μL/min, peptides were eluted and separated on a C18 separation column (Acclaim PepMap, 75 μM × 250 mm, 2 μm, 100 Å, Thermo Fisher Scientific) that had been equilibrated with solvent A (0.1% (v/v) FA). Elution of peptides from the separation column was performed with a gradient from 0 to 35% solvent B (ACN, 0.08% (v/v) FA) within 300 min at 300 nL/min. Peptides were directly transferred to the nano-ESI ion source and analyzed in datadependent MS/MS acquisition mode: High-resolution precursor ion scans (m/z 300−1500, R = 120 000 at m/z 200, AGC target value = 4.0e5, max. injection time = 50 ms) in the Orbitrap analyzer were acquired every 5 s. Within these 5 s the most abundant signals were selected for MS/MS experiments using CID (35% normalized collision energy) (isolation window 2 Th). Fragment ion spectra were acquired in the linear ion trap (precursor ions charge states 2−7, signal threshold = 5000 counts, AGC target value = 1.0e4, max. injection time = 50 ms). Fragmented precursor ions were dynamically excluded from CID for 60 s (mass tolerance window 10 ppm). Data acquisition and inspection of raw data were conducted via the Xcalibur software (version 3.0.63, Thermo Fisher Scientific).

Data Analysis and Statistical Evaluation of LFQ Intensities

Analysis of data processed with MaxQuant was carried out with the Perseus software (version 1.5.1.6). LFQ intensities of identified proteins stored in the “proteinGroups” file were loaded as expression values for a pairwise comparison of each sample to the matrix control sample. Proteins identified only with modified peptides or in the decoy database and potential contaminants were discarded. Afterward, LFQ intensities were logarithmized and replicates were grouped for each sample. The sample groups were filtered for proteins with at least four (biological duplicates) or six (biological triplicates) valid values for LFQ intensities in at least one group. Missing values were imputed from the total matrix based on a normal distribution using default software parameters. Statistical analysis was conducted to compare LFQ intensities of each sample replicate group with the matrix control replicate group using a Student’s t test with a significance threshold of p value ≤0.01. The logarithmized ratios of the means of both groups were plotted against the negative logarithmized p values as “volcano” plots to visualize significant differences of LFQ intensities. Enrichment factors of proteins were calculated as the ratios of mean LFQ intensities in each sample and mean LFQ intensities in the matrix controls. Overlap of proteins with significant LFQ intensity differences were assigned and visualized with a Venn diagram generator (Oliveros, J. C. Venny. An interactive tool for comparing lists with Venn diagrams, version 2.0, 2007−2015, http://bioinfogp.cnb.csic.es/tools/venny/index.html). Enriched proteins were further filtered regarding their identification and quantification data. A minimum of two matched peptides in each replicate analysis and an overall LFQ intensity ratio ≥2 were set as criteria for a confident assignment.

Protein Identification and Label-Free Quantification

All experiments were conducted as biological replicates, that is independent cell culture and fractionation experiments, performing analyses as triplicate for all PKD2 and matrix control samples. Cross-linked matrix and GST control samples were performed as biological triplicates (Golgi-enriched fractions) or duplicates (cytosolic fractions). Also, two technical replicates, that is, affinity enrichment, chemical cross-linking, and MS analysis, were carried out. Raw files from LC−MS/MS measurements were analyzed using the MaxQuant software (version 1.5.2.8)27 by combining every file for each condition, that is, cytosolic fractions or Golgi-enriched fractions. Protein identification was carried out with the Andromeda search engine32 by matching MS and MS/MS data to the human reference proteome downloaded from the UniProt database (http://www.uniprot.org, FASTA file; download date: 201501-20; 89 734 entries) and an integrated database of common contaminants (245 entries). Database searches were conducted with standard settings for Orbitrap precursor ion MS analysis and measurement of fragment ions in the linear ion trap, allowing maximum mass differences of 20 ppm (first search)/ 4.5 ppm (main search) and 0.5 Da. Trypsin and Glu-C were set as specific proteolytic enzymes, allowing up to three missed cleavage sites for comparison with in-silico digestion products.

Proteome Analysis of Subcellular Fractionation

Samples from each fractionation step of three independent biological experiments were mixed with an equal volume of solubilization buffer (100 mM HEPES, 4% (w/v) SDS, 5% (v/ v) β-mercaptoethanol) and incubated at 95 °C for 5 min. The solubilized samples were centrifuged at 16 000g (4 °C, 30 min), and the supernatants were subjected to acetone precipitation. Precipitated proteins were sedimented by centrifugation at 16 000g (4 °C, 20 min) and supernatants were decanted. Proteins were resuspended in denaturation solution, followed by homogenization in an ultrasonication bath for 10 min. Protein concentrations were determined by Bradford protein assay (Bio-Rad). The samples were adjusted to a protein concentration of 1 μg/μL and subjected to in-solution digestion with trypsin. Mass spectrometric analyses of peptide mixtures were carried out as described above; MS/MS analysis was 3688

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Figure 2. Analytical strategy to identify protein kinase D2 (PKD2) interaction partners. (A) Homogenates of HeLa cells are subjected to subcellular fractionation to obtain cytosolic and Golgi-enriched protein fractions. (B) Affinity-captured GST-PKD2 is incubated with target protein fractions, followed by chemical cross-linking. After washing steps, covalently fixed protein complexes are eluted by in-solution digestion. XL = chemical crosslinking. (C) Peptide mixtures are separated by nano-HPLC and analyzed by nano-ESI-MS/MS on an Orbitrap Fusion instrument. Protein identification and label-free quantification (LFQ) is carried out with the MaxQuant software. The Perseus software is used for downstream data analysis and statistical evaluation of LFQ intensities. Identification of interaction sites between PKD2 and enriched binding partners is performed by the StavroX software.53 The StravoX logo is courtesy of Michael Götze.

Annotation of Proteins and Annotation Enrichment Analysis

performed using a data-dependent decision tree method (DDDT).35 Electron transfer dissociation (ETD) was performed for peptides with charge state 3 and m/z 300−650, charge state 4 and m/z 300−900, charge state 5 and m/z 300− 950, and precursor ions with charge state 6−8. CID was used for precursor ions with charge state 2, charge state 3 and m/z 650−1200, charge state 4 and m/z 900−1200, and charge state 5 and m/z 950−1200. Raw data were processed with the Proteome Discoverer software (version 1.4.1.14, Thermo Fisher Scientific). Protein identification was carried out using the Sequest HT search engine with spectrum matching weighted to either c- and z-ions (ETD) or b- and y-ions (CID). Database searches were performed against the reviewed human reference proteome of the Uniprot database (date: 2015-02-10, 20 222 entries) with trypsin as enzyme (up to three missed cleavages, precursor ion and fragment ion mass tolerances: 10 ppm and 0.6 Da, oxidation of methionine: dynamic modification, carbamidomethylation of cysteine: fixed modification). Search results were combined for validation with Percolator against a decoy database at false discovery rates of 1% (strict) and 5% (relaxed). The MS proteomics data of the subcellular fractionation experiments have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with identifier PXD003917.

Proteins identified from each subcellular fractionation step were subjected to GO (gene ontology)36 annotation according to their cellular location. For this, Uniprot identifiers of proteins, with a minimum of two peptides matched with at least medium confidence, were searched with the AmiGO Slimmer application (version 1.8, http://amigo1.geneontology.org/cgibin/amigo/slimmer)37 for matches to a user-defined GO slim term subset including the following entries: GO:0005634 nucleus, GO:0005739 mitochondrion, GO:0005783 endoplasmic reticulum, GO:0005794 Golgi apparatus, GO:0005829 cytosol, GO:0005840 ribosome, GO:0005856 cytoskeleton, and GO:0005886 plasma membrane. The Uniprot database was set as source for GO term assignment. Visualization of Protein−Protein Interactions

Protein interactions were visualized with the Cytoscape software (version 3.3.0).38 Each identified interaction partner was set as a distinct node and set into relationship to PKD2 by assignment of connecting edges. Additionally, interactions between identified proteins were mapped by integrating data from the BioGRID interaction database (version 3.4, http:// thebiogrid.org).39 For this purpose, gene names from the final results table of identified PKD2 interaction partners were compared to Osprey custom network files downloaded from BioGRID containing previously published interaction data of each protein. 3689

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Figure 3. Proteins identified in subcellular fractions and abundance of Golgi marker proteins. Data originate from three independent biological replicate experiments. (A) Proteins identified from each subcellular fractionation step were annotated with GO terms representing major cellular components. The assignment rate of each GO term under investigation was plotted for each subcellular fraction. (B) The representative Golgi marker proteins beta-1,4-galactosyltransferase 1 (B4GALT1) and giantin (GOLGB1) were quantified by counting peptide spectral matches (MS/MS count) across all subcellular fractions. Asterisks indicate a significant enrichment in the Golgi-enriched fraction compared with the homogenate. *ttest p value ≤0.02. ** t-test p value ≤0.01. GOLGB1 was not identified in cytosolic fractions (n/a). 3690

DOI: 10.1021/acs.jproteome.6b00513 J. Proteome Res. 2016, 15, 3686−3699

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Figure 4. Protein analysis in cytosolic fractions. (A) Statistical analysis of label-free quantification (LFQ) data. For an assignment of significantly enriched proteins, LFQ intensities of proteins identified in affinity enrichment/cross-linking (PKD2-XL) and the conventional pull-down experiment (PKD2) were evaluated using t-test statistics. The logarithmized expression ratios were plotted against the negative logarithmized t-test p value. Significantly enriched and depleted proteins (p ≤ 0.01) are shown as red and black dots versus the background of nonregulated proteins (black circles). Enriched proteins matching the filtering criteria of the final results table were annotated with their gene names (red letters). Black letters indicate overlapping proteins. Analysis embraces data from three biological replicate experiments with two technical replicates each. (B) Overlap analysis of significantly enriched proteins. Enriched proteins identified in PKD2-XL samples were evaluated for overlapping protein identifications in the control samples (GST-XL, GST, Matrix-XL) and the pull-down experiment without chemical cross-linking (PKD2). 3691

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Enrichment of PKD2 Protein Interaction Partners from Cytosolic Fractions

RESULTS

Affinity Enrichment of PKD2 Interaction Partners Combined with Chemical Cross-Linking/MS

Cross-linking of PKD2 was performed according to Figure 2 using cytosolic and Golgi-enriched fractions, and the affinity enrichment experiments were monitored by SDS-PAGE analysis (Figure S1). Coomassie Brillant Blue staining of separated proteins revealed signal shifts to the higher molecular weight region in the cross-linked PKD2 samples compared with the PKD2 controls, indicating on-bead formation of PKD2 protein complexes. After cross-linking, the cytosolic protein fractions containing PKD2−protein complexes were subjected to in-solution digestion and LC−MS/MS analysis. All sample replicates, including matrix and GST controls, were simultaneously processed with MaxQuant to ensure a sample-wide consistency of the normalization and LFQ algorithms. The matrix control comprises all proteins binding to the affinity support itself, which is occasionally referred to as the “bead proteome”.25 In addition, we included a matrix control sample with chemical cross-linking into our workflow to identify proteins that are nonspecifically cross-linked to the affinity matrix, potentially via attachment to the amine group of immobilized glutathione. Also, GST control samples, either with or without subjecting them to chemical cross-linking, were considered for identifying potential nonspecific interactors of the GST-PKD2 fusion protein. Differences of LFQ intensities of GST-PKD2 with the matrix control samples (Figure 4A) as well as with the remaining control samples (Figure S2-A) were visualized as “volcano” plots. In total, after the removal of potential contaminants and false-positives, 2413 protein groups were identified exhibiting quantitative information. (For detailed data and representative fragment ion spectra of single-peptide identifications, see Table S2 and Figure S4.) Of those, 24 proteins were found to be enriched in the crosslinked GST-PKD2 sample, while 14 proteins were enriched in the GST control, 10 in the cross-linked GST control, 7 in the GST-PKD2 sample, and 3 in the cross-linked matrix control. In particular, the number of enriched proteins was nearly 4-fold increased upon chemical cross-linking of GST-PKD2 samples compared with the conventional pull-down experiment. Proteins found to be enriched in either of the control samples had to be considered as potential nonspecific interactors and were accordingly rejected as specific PKD2 interaction partners. For the respective assignment, enriched proteins from paired evaluations were compared by overlap analysis (Figure 4B). Notably, no overlap of enriched proteins was detected for the cross-linked GST-PKD2 sample, and only one overlap (nuclear pore complex protein ELYS, AHCTF1) was found for the GSTPKD2 pull-down sample without cross-linking (Figure S2B). Overall, this indicates a low interference of PKD2 binding to cytosolic proteins between the affinity-captured bait protein and GST alone or the affinity matrix. The comparison of the GST-PKD2 samples with or without cross-linking (Figure 4B) revealed an overlap of four enriched proteins: Protein kinase D2, alpha-tubulin 2, ADP/ATP translocase 3, and human DnaJ protein 2. The vast majority of 20 proteins were exclusively identified upon chemical cross-linking of PKD2, while only two proteins were found in the conventional pull-down experiment. This impressively underlines the strength of our approach in capturing specific PKD2 binding partners by a covalent fixation, thus efficiently removing nonspecific binders and recovering interaction partners despite extensive washing steps. Proteins assigned as PKD2 binding partners after overlap analysis and

The aim of this study was to identify novel binding partners of human PKD2 from cytosolic and Golgi-enriched protein fractions to elucidate the role of PKD2 for transport processes at the TGN (Figure 2). Our approach relied on an affinitybased enrichment of PKD2 interaction partners, followed by their covalent fixation with chemical cross-linking, and allowed capturing interactions of PKD2 with both cytosolic and Golgi protein effectors. This yielded insights into the role of PKD2 for membrane recruitment and cytoskeletal dynamics as well as into PKD’s function at the Golgi compartment itself. Chemical cross-linking has proven highly advantageous as it allows capturing transient and weak interactions that are often elusive in pull-down experiments. These PKD2 interaction partners that were found to be enriched by chemical cross-linking were identified by MS using an LFQ approach with the MaxQuant software. This strategy eliminated nonspecifically enriched proteins by quantification and normalization of background proteins that inevitably bind to the affinity matrix.29 Qualitative Assessment of Subcellular Fractionation and Golgi Protein Enrichment

To evaluate the efficiency of the subcellular fractionation step and the enrichment of Golgi proteins, samples of the homogenate, PNS, cytosol, membrane, and Golgi-enriched fractions were subjected to an in-depth proteome analysis. After in-solution digestion, samples were analyzed using a datadependent decision-tree MS/MS method (DDDT)35 employing CID or ETD fragmentation of tryptic peptides. Proteins were identified and assigned to their location in the major cellular compartments. For each fractionation step, the following mean numbers of proteins were identified in three biological replicates: 2682 ± 148 (homogenate), 2631 ± 40 (PNS), 2155 ± 96 (cytosolic fraction), 2581 ± 162 (membrane fraction), and 2195 ± 376 (Golgi-enriched fraction). (For detailed data, see Table S1.) The fraction-dependent protein assignments for each GO term are shown in Figure 3A. Proteome analysis confirmed that Golgi-assigned proteins increased nearly 2-fold during fractionation from ∼5% in the homogenate fraction to ∼9% in the Golgi-enriched fraction. A similar trend was observed for proteins assigned to the endoplasmic reticulum and the plasma membrane. On the contrary, protein assignments exhibited nearly constant percentages for ribosome, cytoskeleton, and mitochondrion across all fractions under investigation. Furthermore, comparatively high percentages of protein assignments to the nucleus and the cytosol were detected for each fraction, yet a decreased assignment of proteins to the nucleus and the cytosol was found during the course of fractionation, along with an increase in membranous protein content. Prior to cross-linking, the maximum numbers of Golgi proteins were identified in the Golgi fractions, and, similarly, the maximum numbers of cytosolic proteins were identified in the cytosolic fractions. We further estimated the success of the Golgi enrichment step based on the abundance of specific marker proteins. Here spectral counting revealed significantly increased numbers of peptide spectral matches (PSMs) for the representative Golgi markers beta-1,4-galactosyltransferase 1 (B4GALT1) and giantin (GOLGB1) in Golgi-enriched fractions (Figure 3B). 3692

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Journal of Proteome Research Table 1. Identification and Statistics of Proteins Enriched in Cytosolic Fractionsa Uniprot entry Q9HB19 O15143 P63151 O15144 P49841 O43684 O15511 P61158 P67870 P61160 P28482 P61981 Q13526 Q92747

protein name

gene name

enrichment factor

t-test p value

Ø peptides

Ø sequence coverage (%)

Pleckstrin homology domain-containing family A member 2 Actin-related protein 2/3 complex subunit 1B serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B alpha isoform Actin-related protein 2/3 complex subunit 2 glycogen synthase kinase-3 beta mitotic checkpoint protein BUB3 Actin-related protein 2/3 complex subunit 5 actin-related protein 3 casein kinase II subunit beta actin-related protein 2 mitogen-activated protein kinase 1 14−3−3 protein gamma peptidyl-prolyl cis−trans isomerase NIMA-interacting 1 actin-related protein 2/3 complex subunit 1A

PLEKHA2 ARPC1B PPP2R2A

30.4 22.0 15.5

2.0 × 10−4 1.8 × 10−3 2.2 × 10−4

9 10 14

32 33 35

ARPC2 GSK3B BUB3 ARPC5 ACTR3 CSNK2B ACTR2 MAPK1 YWHAG PIN1 ARPC1A

13.3 12.3 8.2 8.0 7.2 6.8 5.8 5.6 3.6 2.9 2.6

3.9 4.7 7.7 1.2 4.1 5.6 2.3 2.3 8.5 5.7 8.9

10−4 10−5 10−4 10−4 10−4 10−3 10−3 10−3 10−3 10−3 10−3

8 5 8 4 14 4 11 4 14 2 3

31 21 31 27 48 19 30 15 48 23 16

× × × × × × × × × × ×

a

Protein complexes were subjected to in-solution digestion before nano-HPLC/nano-ESI-MS/MS was conducted. Proteins were identified and quantified with MaxQuant. LFQ intensities from three biological replicates and two technical replicates were statistically evaluated using Perseus with a threshold for significant enrichment of t-test p value ≤0.01. Enrichment factors were calculated as ratios of mean LFQ intensities between the PKD2-cross-linked sample and matrix control. Mean values of identification data were averaged across all replicate samples. Data were filtered for identified proteins with an enrichment factor ≥2 and at least two peptides matched in each replicate analysis.

filtering of identification and quantitation data are listed in Table 1. Strikingly, five subunits (ARPC1B, ARPC2, ARPC5, ACTR3, ACTR2) of the seven-subunit Arp2/3 complex and ARPC1A, a putative substitute of ARPC1B, were identified to be enriched in PKD2 samples upon chemical cross-linking. Notably, the ARPC4 subunit was also found to be significantly enriched (factor 4.7, t-test p value =1.40 × 10−5), although it did not pass data filtering criteria for minimal peptide count in one replicate experiment and was therefore not considered in the final results table. Using a more relaxed significance threshold for statistical analysis (t-test p value ≤0.05), the enrichment of subunits ARPC3 and ARPC5L, a putative substitute of ARPC5, was increased. Conclusively, we were able to enrich the entire seven-subunit Arp2/3 complex as well as two associated proteins by a covalent fixation using our chemical cross-linking strategy.

affinity enrichment cross-linking approach largely outnumbered the conventional pull-down experiment. Conclusively, 40 proteins were enriched exclusively after the cross-linking step compared with only 22 proteins exclusively found in the pulldown experiment without cross-linking, while 46 enriched proteins were shared between both conditions. Proteins significantly enriched in the Golgi fraction and matching the applied data filtering criteria are listed in Table 2. Strikingly, the ACTR2 subunit of the Arp2/3 complex was detected as specific PKD interaction partner in cytosolic as well as in Golgi fractions. Also, the 55 kDa regulatory subunit B of serine/ threonine-protein phosphatase 2A and the glycogen synthase kinase-3 protein family (GSK3A/B) were found to be enriched in both fractions.



DISCUSSION We identified 31 specific PKD2 protein interaction partners from cytosolic and Golgi fractions using an affinity enrichment strategy combined with cross-linking/MS. A preceding proteomic evaluation of the proteins contained in the subcellular fractions had revealed an enrichment of distinct proteins. This analysis also disclosed the limitations of cellular fractionation attributed to the inevitably imperfect nature of separation, multiple localizations of identified proteins, and the high sensitivity of modern MS technology.40,41 Fractionation provided the starting material for identifying specific PKD2 interactors from the Golgi compartment and the cytosol. Remarkably, the identification of specific PKD2 protein binding partners validates the efficiency of quantification and statistical evaluation used herein. Furthermore, the expected enrichment of PKD2 in samples comprising the GST-PKD2 fusion protein confirmed the validity of our analytical strategy. In general, the PKD2 interaction partners identified in this study exhibit comparably low abundances, which on the one hand implicates low experimental interferences with high-abundant proteins and on the other hand demonstrates the power of the chemical cross-linking approach for protein retrieval. The covalent fixation of PKD2 binding proteins points to direct interactions

Enrichment of PKD2 Protein Interaction Partners from Golgi Fractions

In addition to identifying PKD2 protein binding partners from the cytosol, we extended our analysis to Golgi-enriched protein fractions, resulting in the identification of 1425 protein groups with quantitative information. (For detailed data and representative fragment ion spectra of single-peptide identifications, see Table S2 and Figure S5.) Proteins enriched after chemical cross-linking were detected in each pairwise comparison with the matrix control (Figure 5A, Figure S3A). In total, 103 proteins were found to be enriched in the crosslinked GST-PKD2 sample, while four proteins were enriched in the GST control, 43 in the cross-linked GST control, 76 in the GST-PKD2 sample, and 46 in the cross-linked matrix control sample. In contrast with the cytosolic protein fraction, an overlap of several enriched proteins was detected between GST-PKD2 and control samples (Figure 5B, Figure S3B). These overlapping proteins have to be considered as potential nonspecific interactors and were accordingly removed from the GST-PKD2 data sets. Nevertheless, the majority of enriched proteins were identified from GST-PKD2 samples. Again, the 3693

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Figure 5. Protein analysis in Golgi-enriched fractions. (A) Statistical analysis of label-free quantification (LFQ) data. For an assignment of significantly enriched proteins, LFQ intensities of proteins identified in affinity enrichment/cross-linking (PKD2-XL) and the conventional pulldown experiment (PKD2) were evaluated using t-test statistics. The logarithmized expression ratios were plotted against the negative logarithmized t-test p value. Significantly enriched and depleted proteins (p ≤ 0.01) are shown as red and black dots versus the background of nonregulated proteins (black circles). Enriched proteins matching the filtering criteria of the final results table were annotated with their gene names (red letters). Black letters indicate overlapping proteins. Analysis embraces data from three biological replicate experiments with two technical replicates each. (B) Overlap analysis of significantly enriched proteins. Enriched proteins identified in PKD2-XL samples were evaluated for overlapping protein identifications in the control samples (GST-XL, GST, Matrix-XL) and the pull-down experiment without chemical cross-linking (PKD2). 3694

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Journal of Proteome Research Table 2. Identification and Statistics of Proteins Enriched in Golgi Fractionsa Uniprot entry Q3ZCM7 P49840 Q9NY65 P54709 P15291 Q96EY1 O75131 O94973 P46782 Q9UBS4 O75427 P50995 P26639 P63151 Q9HCN8 Q99832 P61160

protein name tubulin beta-8 chain glycogen synthase kinase-3 alpha tubulin alpha-8 chain sodium/potassium-transporting ATPase subunit beta-3 beta-1,4-galactosyltransferase 1 DnaJ homologue subfamily A member 3, mitochondrial copine-3 AP-2 complex subunit alpha-2 40S ribosomal protein S5 DnaJ homologue subfamily B member 11 leucine-rich repeat and calponin homology domain-containing protein 4 annexin A11 threonine−tRNA ligase, cytoplasmic serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B alpha isoform Stromal cell-derived factor 2-like protein 1 T-complex protein 1 subunit eta actin-related protein 2

gene name

enrichment factor

Ø peptides

Ø sequence coverage (%)

TUBB8 GSK3A TUBA8 ATP1B3 B4GALT1 DNAJA3 CPNE3 AP2A2 RPS5 DNAJB11 LRCH4

18.8 17.9 17.1 8.9 7.4 5.7 5.4 4.4 3.9 3.8 3.1

10−6 10−5 10−5 10−3 10−4 10−4 10−4 10−3 10−3 10−3 10−3

9 4 13 4 4 3 6 5 5 4 3

22 16 39 22 17 8 14 7 29 13 6

ANXA11 TARS PPP2R2A

2.8 2.5 2.4

3.08 × 10−3 5.81 × 10−4 4.48 × 10−4

3 3 3

7 7 10

SDF2L1 CCT7 ACTR2

2.3 2.2 2.1

1.11 × 10−5 7.97 × 10−3 5.31 × 10−3

4 5 4

32 17 15

t-test p value 3.04 1.31 7.02 9.77 1.57 8.28 5.45 1.77 1.74 5.43 6.59

× × × × × × × × × × ×

a

Protein complexes were subjected to in-solution digestion before nano-HPLC/nano-ESI-MS/MS was conducted. Proteins were identified and quantified with the MaxQuant software. LFQ intensities from three biological replicates and two technical replicates were statistically evaluated using Perseus with a threshold for significant enrichment of t-test p value ≤0.01. Enrichment factors were calculated as ratios of mean LFQ intensities between the PKD2-XL sample and the matrix control. Mean values of identification data were averaged across all replicate samples. Data were filtered for identified proteins with an enrichment factor ≥2 and at least two peptides matched in each replicate analysis.

Table 3. Identified Protein PKD2 Interaction Partnersa source fraction

PKD2 interaction

C C C/G C C C/G

direct secondary secondary secondary secondary secondary

G

secondary

40S ribosomal protein S5 annexin A11 AP-2 complex subunit alpha-2 beta-1,4-galactosyltransferase 1 copine-3 DnaJ homologue subfamily A member 3, mitochondrial

G G G G G G

novel novel novel novel novel novel

DnaJ homologue subfamily B member 11

G

novel

glycogen synthase kinase-3 alpha leucine-rich repeat and calponin homology domain-containing protein 4 mitotic checkpoint protein BUB3 peptidyl-prolyl cis−trans isomerase NIMA-k

G G

novel novel

C C

novel novel

Pleckstrin homology domain-containing family A member 2 sodium/potassium-transporting ATPase subunit beta-3

C G

novel novel

stromal cell-derived factor 2-like protein 1

G

novel

T-complex protein 1 subunit eta threonine−tRNA ligase, cytoplasmic

G G

novel novel

interacting protein glycogen synthase kinase-3 beta 14−3−3 protein gamma actin-related protein 2/3 complex casein kinase II subunit beta mitogen-activated protein kinase 1 serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B alpha isoform tubulin

evidence/context

ref

PKD regulates GSK3B during cell morphogenesis PKD generates a 14−3−3 binding motif on PI4KIIIβ PKD phosphorylates the Arp2/3 complex NPF cortactin PKD and CK2 are recruited by the COP9 signalosome PKCμ/PKD activates MAPK1 signaling PP2A counteracts PKD-mediated phosphorylation of PI4KIIIβ microtubules control PKD activity to maintain Golgi integrity PKC isoenzymes phosphorylate S6K annexins act as scaffolds for PKC isoenzymes AP-2 complex regulates postendocytotic trafficking traffic of B4GALT1 at the Golgi compartment copines participate in membrane trafficking PKD is activated in mitochondria in response to oxidative stress formation of a multichaperone complex binding nascent proteins GSK3(B) is involved in post-Golgi membrane trafficking phosphorylation of LRCH4 during mitosis

44 52 45 54 55 52

PKD isoenzymes associate with the mitotic apparatus PIN1 controls the regulation of conventional PKC isoenzymes PLEKHA2 links PI3K signaling to cytoskeletal interactions Golgi-located PKD regulates trafficking of cation ATPase ATP7B formation of a multichaperone complex binding nascent proteins CCT activity is implicated to cytoskeletal organization tRNA synthetase activity during protein synthesis and noncanonical functions

66 67

56 57 58 59 60 61 62 63 64 65

68 69 63 70 71

a

PKD2 interaction partners identified from cytosolic (C) and Golgi (G) fractions using the affinity enrichment/cross-linking/MS approach were assigned as direct, secondary, or novel interactors of PKD2 based on literature data and database mining. 3695

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Figure 6. PKD2 interaction network. All interacting proteins identified in data sets from cytosolic (red lines) and Golgi (green lines) fractions and PKD2 were set as gene-name-annotated nodes and related to each other by connecting edges. Interactions showing additional evidence from literature mining are shown as double lines; novel interaction partners of PKD2 are presented as single lines. Interactions between PKD2 interactors (black dashed lines) were assigned based on data deposited in the BioGRID interaction database, and node clusters were visualized by color mapping. The interaction between PKD2 and ARPC5 was recently identified in an affinity MS study (black solid line) (BioGRID).49

glycogen synthase kinase-3 beta (GSK3B) has been revealed in the context of endothelial cell morphogenesis.44 Furthermore, PKD has been reported to exert a regulatory function in the Arp2/3 complex-induced actin polymerization pathway via phosphorylation of cortactin.45 The underlying processes are important for controlling actin remodeling, regarding polarized cell motility via formation of lamellipodia,46 and might also be relevant for vesicle formation.47 More precisely, cortactin has been shown to colocalize with subunits of the Arp2/3 complex and to accelerate Arp2/3 complex-driven polymerization of actin, while phosphorylation of cortactin by PKD at Ser-298 generates a 14−3−3 binding motif. However, we did not find cortactin to be enriched in our cross-linking samples, but we confidently identified cortactin in all data sets. Thus, the identification of Arp2/3 complex subunits probably implies a direct interaction between PKD2 and the Arp2/3 complex. Regulatory functions of nucleation promoting factors (NPFs), such as cortactin, toward Arp2/3 as well as the necessity of phosphorylation to induce Arp2/3 activity have recently been discussed.48 In this respect, a direct interaction of PKD2 with Arp2/3 might point toward a complex PKD2-dependent regulatory assembly including NPF recruitment as well as a direct induction of nucleation activity by phosphorylation. To the best of our knowledge, there is no information available to date about a direct functional relationship between PKD2 and the entire Arp2/3 complex, and our study provides direct evidence of this interaction for the first time. Interestingly, PKD2 recently has been identified as an interaction partner in an affinity capture/MS study with the ARPC5 subunit of the Arp2/3 complex as bait protein.49 Also, we were able to identify 14−3−3 protein gamma as cytosolic interaction partner of PKD2, pointing to the formation of a 14−3−3 regulatory interaction, possibly in the context of cortactin phosphorylation. Because 14−3−3 proteins are involved in a wide variety of cellular processes,50 a direct interaction between PKD2 and 14−3−3 gamma protein seems plausible, irrespective of cortactin signaling. Regarding carrier formation at the Golgi apparatus, a multiprotein complex, including a 14−3−3 gamma dimer, has been described to be stabilized by PKD activity.51 This stabilizing effect is attributed

on the molecular level, aside from potential interconnected proteins. In accordance with the multiplicity of PKD signaling, the pool of identified interactors exhibits a certain functional heterogeneity. As such, the observed interactions represent snapshots of protein complexes assembled in various cellular processes engaging PKD2. Along with that, and also as a consequence of the above-mentioned limitations of subcellular fractionation, the interactors identified cover multiple cellular destinations, which is not surprising due to the stimulusdependent broad range of PKD localization (cytosol, plasma membrane, Golgi apparatus, nucleus, mitochondria).42 We were able to show a distinct enrichment of proteins related to the Golgi apparatus in the DDM-solubilized Golgi fractions, yet GO database analysis of assigned interaction partners using the EMBL-EBI QuickGO browser (http://www.ebi.ac.uk/ QuickGO) 43 revealed only one Golgi-related protein (B4GALT1). Two further proteins, exhibiting a certain evidence of Golgi localization, were assigned in the cytosolic fractions (ARPC2, MAPK1). The majority of identified interactors originate from distinct cytoplasmic compartments, nevertheless showing evidence of membrane and cytoskeletal interactions (Table 3) and thus potentially being involved in PKD2-mediated Golgi interaction. Apparently, the protein pool enriched in the Golgi fractions exhibits a certain bias toward tubulin (TUBB8, TUBA8), chaperones (DNAJA3, DNAJB11, CCT7), and ribosomal proteins (RPS5) that are occasionally mentioned in the context of nonspecific binding to affinity supports.25 However, because our approach succeeded in sorting out overlapping nonspecifically enriched proteins between PKD2 samples and control experiments, those proteins have to be classified as bona fide PKD2 interactors. Because an exhaustive characterization of all identified interaction partners is beyond the scope of this work, we provide a summary of identified PKD2 interaction partners in Table 3, focusing on their known or putative relationship with PKD. For a number of protein interaction partners identified herein, a functional relationship to PKD has been described. For instance, a direct interaction (i.e., phosphorylation) with 3696

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Journal of Proteome Research to the phosphorylation of PI4KIIIβ by PKD, enabling 14−3−3 protein binding to stimulate lipid kinase activity.52 On the contrary, the activating effect of PKD toward PI4KIIIβ is discussed to be counteracted by protein phosphatase 2A (PP2A). Strikingly, we identified the alpha isoform of the PP2A 55 kDa regulatory subunit B to be enriched upon chemical cross-linking in both cytosolic and Golgi protein fractions. Thus, our data give a hint on direct interactions between PKD2 with 14−3−3 protein gamma and PP2A. However, because PI4KIIIβ was not identified in our experiments, we are currently unable to distinguish whether the enrichment of 14−3−3 gamma and PP2A originates from a direct functional relationship between PI4KIIIβ and PKD2 or another interaction of PKD2 within the 14−3−3 signaling network. Furthermore, it might also be possible that the interaction between PKD2 and PP2A occurs in a 14−3−3-independent manner. Because chemical cross-linking allows a covalent fixation of transient or weak interactors, which might otherwise disappear during multiple washing steps, our strategy is advantageous compared with traditional pull-down experiments and most likely points toward direct interactions of significantly enriched proteins. Overall, our approach revealed a number of proteins, for which evidence regarding PKD interaction, either direct (GSK3B) or indirect (YWHAG, Arp2/3 complex, CK2, MAPK1, PP2A, tubulin), has already been described. Also, we discovered novel interactors that have so far not been described to be involved in PKD2 signaling (Table 3). A network map of PKD2 interactions arising fom our data as well as from previous reports is shown in Figure 6. The results obtained herein on the protein network of PKD2 will serve as the basis for further structural and functional studies between PKD2 and its interaction partners. For instance, interaction interfaces will be elucidated by cross-linking/MS and data analysis with the StavroX software (Figure 2).53 Special focus will be laid on the PKD2/Arp2/3 interaction that is crucial for controlling nucleation of actin polymerization and branching of filaments. We envision that further cross-linking experiments will reveal molecular details of this interaction.



AUTHOR INFORMATION

Corresponding Author

*Tel: +49-345-5525170. Fax: +49-345-5527026. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Raw data are available via ProteomeXchange with identifiers PXD003909 (enrichment from cytosolic fractions), PXD003913 (enrichment from Golgi fractions) and PXD003917 (subcellular fractionation).



ACKNOWLEDGMENTS B.H. is funded by the DFG (project Si 867/16-1). A.S. gratefully acknowledges financial support for this project by the DFG and the region of Saxony Anhalt. Alexander Becher (group of Prof. Thomas Seufferlein, University Medical Center Ulm) is acknowledged for introducing B.H. to the preparation of Golgi fractions. We are grateful to Dr. Cordelia SchieneFischer and Prof. Gunter Fischer for providing their cell culture facilities.





ABBREVIATIONS ARF1, ADP-ribosylation factor 1; ARL1, ADP-ribosylation factor-like protein 1; BS2G, bis(sulfosuccinimidyl) glutarate; CERT, ceramide transfer protein; CID, collision-induced dissociation; DAG, diacylglycerol; DDDT, data-dependent decision tree; DDM, n-dodecyl-β-D-maltoside; DMEM, Dulbecco’s modified Eagle’s medium; ETD, electron transfer dissociation; GO, gene ontology; HPLC, high-performance liquid chromatography; LFQ, label-free quantification; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NPF, nucleation promoting factor; OSBP, oxysterol-binding protein; PI4KIIIβ, phosphatidylinositol-4 kinase IIIβ; PKD, protein kinase D; PNS, postnuclear supernatant; TCEP, tris(2carboxyethyl)phosphine; TGN, trans-Golgi network; XL, cross-linking

CONCLUSIONS An affinity enrichment strategy combined with chemical crosslinking/MS allowed mapping the protein interaction networks of human PKD2 in the cytosol and the Golgi apparatus. Our approach disclosed a number of proteins for which functional relationships to PKD have already been described. Markedly, novel PKD2 interactors, such as the entire Arp 2/3 complex, have been discovered in this study. Further structural and functional studies are required to elucidate the interactions discovered herein in more detail to enhance our understanding of PKD2 signaling processes.



plots) of LFQ intensities and overlap analysis for Golgienriched fractions. (PDF) Figure S4. Representative fragment ion spectra of singlepeptide protein identifications from enrichment experiments with cytosolic fractions (extracted from MaxQuant Viewer tab). (PDF) Figure S5. Representative fragment ion spectra of singlepeptide protein identifications from enrichment experiments with Golgi-enriched fractions (extracted from MaxQuant Viewer tab). (PDF) Table S1. Protein identification data for subcellular fractionation experiments (extracted from Proteome Discoverer results files). (XLS) Table S2. Protein identification data for enrichment experiments (extracted from MaxQuant “proteinGroups” output file). (XLS)

ASSOCIATED CONTENT



S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.6b00513. Figure S1. SDS-PAGE analysis of enrichment experiments. Figure S2. Statistical evaluation (“volcano” plots) of LFQ intensities and overlap analysis for cytosolic fractions. Figure S3. Statistical evaluation (“volcano”

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Journal of Proteome Research

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DOI: 10.1021/acs.jproteome.6b00513 J. Proteome Res. 2016, 15, 3686−3699