Chen, et al., 2009 1

Quantitative proteomics analysis of cell cycle regulated Golgi disassembly and reassembly Xuequn Chen1, Eric S. Simon2, Yi Xiang3, Maureen Kachman2, Philip C. Andrews2 and Yanzhuang Wang3 1. Department of Molecular & Integrative Physiology; 2. Department of Biological Chemistry; 3. Department of Molecular, Cellular and Developmental Biology; The University of Michigan, Ann Arbor, MI 48109 Running title: Comparative proteomics analysis of Golgi disassembly and reassembly Corresponding authors: Philip C. Andrews (andrewsp@umich.edu) and Yanzhuang Wang (yzwang@umich.edu) During mitosis, the stacked structure of the Golgi undergoes a continuous fragmentation process. The generated mitotic fragments are evenly distributed into the daughter cells and reassembled into new Golgi stacks. This disassembly and reassembly process is critical for Golgi biogenesis during cell division, but the underlying molecular mechanism is poorly understood. In this study, we have recapitulated this process using an in vitro assay and analyzed the proteins associated with interphase and mitotic Golgi membranes using a proteomic approach. Incubation of purified rat liver Golgi membranes with mitotic HeLa cell cytosol led to fragmentation of the membranes; subsequent treatment of these membranes with interphase cytosol allowed the reassembly of the Golgi fragments into new Golgi stacks. These membranes were then used for quantitative proteomics analyses by combining the iTRAQ approach with OFFGEL isoelectric focusing separation and LC-MALDI-MS/MS. In three independent experiments, a total of 1193 Golgi-associated proteins were identified and quantified. These included broad functional categories, such as Golgi structural proteins, Golgi resident enzymes, SNAREs, Rab GTPases, cargo and cytoskeletal proteins. More importantly, the combination of the quantitative approach with Western blotting allowed us to unveil 84 proteins with significant changes in abundance under the mitotic condition compared to the interphase condition. Among these proteins, several COPI coatomer subunits (alpha, beta, gamma and delta) are of particular interest. All together, this systematic quantitative proteomic study revealed candidate proteins of the molecular machinery that control the Golgi disassembly and reassembly processes in the cell cycle.

The Golgi complex is the central organelle in the secretory pathway, essential for post-translational modifications, sorting and trafficking of newly synthesized secretory and membrane proteins and lipids in all eukaryotic cells. The unique feature of the Golgi in almost all eukaryotic cells is the densely packed stacks of the flattened cisternal membranes. Processing enzymes in the Golgi complex, including those involved in modifying

bound oligosaccharides, are arranged across the stack in the cis to trans order in which they function. In animal cells, stacks are often interconnected to form a ribbon-like structure that is localized adjacent to the nucleus. Despite its complicated morphology and function, the Golgi apparatus is dynamic, capable of rapid disassembly and reassembly during mitosis or upon drug treatment. At the onset of mitosis, the characteristic

stacked organization of the Golgi apparatus undergoes extensive fragmentation. The mitotic Golgi fragments generated by this disassembly process are subsequently evenly distributed to the daughter cells, where they are reassembled into new Golgi stacks during cytokinesis (1,2). It is believed that the cell-cycle regulated Golgi disassembly and reassembly processes involve interactions between cytosolic and membrane proteins, and many studies have been performed in efforts to identify these proteins using biochemical and cell biology approaches. However, the underlying mechanism that controls the Golgi

disassembly and reassembly processes is still far from clear. For example, it is generally accepted that the microtubule cytoskeleton plays a role in organizing the Golgi structure (3). The Golgi interacts with the microtubules to maintain its perinuclear localization within the cytoplasm in interphase cells (4). During mitosis, rearrangement of the cytoskeleton facilitates the dispersal of the

http://www.jbc.org/cgi/doi/10.1074/jbc.M109.047084The latest version is at JBC Papers in Press. Published on January 7, 2010 as Manuscript M109.047084

Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 2

Golgi ribbon (1,2). However, the fragmentation of the Golgi during mitosis is far more extensive than cytoskeletal rearrangement. In addition, phosphorylation of Golgi structural proteins as well as vesicle formation is involved in mitotic Golgi fragmentation (5). Recently, several labs have attempted to comprehensively identify Golgi membrane proteins using organellar proteomics, a fundamental and fast-expanding research technology in proteomics and cell biology that combines biochemical fractionation and comprehensive protein identification (6-11). However, quantitative studies documenting the comprehensive protein changes in the Golgi membrane during the cell cycle have not been reported thus far. Analysis of the dynamics of proteins in the Golgi membrane during disassembly has the advantage of identifying not only those proteins that may be involved in this process, but also peripheral membrane proteins that exhibit changes, which are more likely to be specific regulators for the morphological change rather than contaminating membrane components. Biochemical reconstitution experiments have provided powerful tools with which we can dissect biological processes. One widely used method to study the Golgi disassembly and reassembly processes during the cell cycle involves purified Golgi membranes to which mitotic or interphase cytosol is added (12-14). After incubation, the membranes are separated from the cytosol by centrifugation through a sucrose cushion and then processed for biochemical and morphological analyses. This approach has contributed to the discovery and examination of many of the currently identified proteins that mediate Golgi membrane tethering (15,16), fusion (13,17-20) and cisternal stacking (21-23). When combined with modern quantitative proteomics approaches, this in vitro reconstitution system is expected to provide a

powerful tool to further dissect the molecular mechanism that regulates Golgi membrane dynamics during the cell cycle. In this study, we applied iTRAQ (isobaric tags for relative and absolute quantification) and LC-MALDI-MS/MS analysis to quantify protein changes on Golgi membrane stacks during the cell cycle using membranes provided by the in vitro reconstitution system. iTRAQ is a recently developed chemical labeling reagent (24) that has quickly gained popularity in proteomics within the past few years (25,26). It has the advantage of simultaneous

identification and quantitative comparison of several (up to 8) protein samples in the same experiment. This mass spectrometry-based technology allows us to quantitatively analyze proteins that are associated with the Golgi when the membranes are intact during interphase and when they are fragmented during mitosis. The ability to multiplex samples minimizes experimental variations and allows control samples to be run at the same time, resulting in the ability to detect relatively small changes in protein levels. This unbiased strategy also helps us to understand the global changes in the protein composition of the Golgi when its morphology is changed during cell division. This study revealed candidate proteins involved in the regulation of Golgi morphological changes during the cell cycle.

EXPERIMENTAL PROCEDURES Reagents All reagents were from Sigma or Roche Applied Sciences, unless otherwise stated. Sequencing grade modified trypsin was from Promega (Madison, WI, USA). iTRAQTM reagents were from Applied Biosystems (Foster City, CA). SCX MicroSpinTM columns were from The Nest Group, Inc. (Southborough, MA). Zorbax C18 reversed-phase cartridge and Zorbax 300 SB C18 reversed-phase analytical column were purchased from Agilent (Palo Alto, CA). All chemicals were of analytical grade and used as received. The following antibodies were used: monoclonal antibodies against Bet1, Gos28, GM130, and HSP90 from BD Transduction Laboratories; monoclonal antibodies against α-actin (Sigma), ARF1 (1D9, Abcam), β-COP (M3A5, T. Kreis), GRASP65 (G. Warren), PP2A C subunit (Upstate), and α-tubulin (K. Gull); polyclonal antibodies against ARF1 (D. Shields and D. Sheff), cdc2 (Upstate), β-COP (EAGE, T. Kreis), ERK2 (Upstate), GM130 (MLO7, M. Lowe), GRASP55 (J. Seemann), GRASP65 (27), HSP70 (Synaptic Systems) α-Mannosidase I (J. Seemann) and II (K. Moremen), golgin-84 (A. Satoh), NSF (A. Price), p115 (D. Shields), Rab1A (Santa Cruz), Rab6 (Santa Cruz), Rab11 (Santa Cruz), rat serum albumin (G. Warren), sec31 (F. Gorelick), syntaxin 5 (A. Price), SNAP29 (Synaptic Systems), and TGN38 (G. Warren). Antibodies to β’-, γ-, δ- and ε-COPs were kindly provided by D. Sheff. The monoclonal antibody for phosphorylated GRASP65 was raised in mouse with recombinant

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 3

GST-GRASP65 (aa 202-446) treated with mitotic HeLa cell cytosol. Secondary antibodies for immunofluorescence and for Western blotting were from Molecular Probes and Jackson

Immunoresearch Laboratories, respectively. Cell-free Golgi disassembly and reassembly assay Golgi membranes were purified from rat liver as previously described (28); interphase (IC) and mitotic (MC) cytosols were prepared from HeLa S3 cells as described in the supplemental materials (14). Cytosols were changed into related buffers using Bio-Spin 6 columns and cleared by centrifugation before they were used to treat Golgi membranes. The Golgi disassembly assay was performed as described previously (5). In brief, purified Golgi membranes (200 µg) were mixed with 10 mg of mitotic cytosol (the MC:Golgi ratio was based on the quantitation results of these two components in rat liver cells), 1 mM GTP and an ATP-regenerating system (10 mM creatine phosphate, 0.1 mM ATP, 20 µg/ml creatine kinase, 20 µg/ml cytochalasin B) in MEB buffer (50 mM Tris-HCl, pH7.4, 0.2 M sucrose, 50 mM KCl, 20 mM β-glycerophosphate, 15 mM EGTA, 10 mM MgCl2, 2 mM ATP, 1 mM GTP, 1 mM glutathione, and protease inhibitors), with a final volume of 1 ml. After incubation for 60 min at 37°C, mitotic Golgi fragments (MGF) were isolated and soluble proteins were removed by centrifugation (55,000 rpm for 30 min in a TLA55 rotor) through a 0.4 M sucrose cushion in KHM buffer (20 mM Hepes-KOH, pH7.0, 0.2 M sucrose, 60 mM KCl, 5 mM Mg(OAc)2, 2 mM ATP, 1 mM GTP, 1 mM glutathione, protease inhibitors) onto a 6 µl 2 M sucrose cushion. The membranes were resuspended in KHM buffer, and aliquots were succeeded either to fixation and processing for electron microscopy (EM) (5,20,23), to reassembly reactions described below, or kept frozen at -80°C until further use for the proteomics analysis. Incubation of Golgi membrane with interphase cytosol in KHM buffer was used as control. For Golgi reassembly, 200 µg of MGFs were resuspended in 4 mg IC in KHM buffer in the presence of an ATP regeneration system in a final volume of 300 µl and incubated at 37°C for 60 min. Membranes were pelleted by centrifugation as described above, processed for EM, or kept at -80°C for the proteomic analysis or Western blotting. All results were confirmed by 3 or more independent experiments.

For EM analysis, the percentage of membranes in cisternae or in vesicles was determined by the intersection method (12,23). Cisternae were defined as long membrane profiles with a length greater than 4 times their width, the later being not more than 60 nm. Normal cisterna ranges 20-30 nm in width and longer than 200 nm. Stacks were defined as two or more cisternae that were separated by no more than 15 nm and overlapped in parallel by more than 50% of their length. In-solution digestion and iTRAQTM labeling For quantitative proteomics analysis, membranes were collected under four experimental conditions: 1) incubation with buffer alone; 2) with interphase cytosol alone; 3) with mitotic cytosol alone; and 4) sequential incubation with mitotic and interphase cytosols. After incubation, Golgi membrane was separated from cytosol by centrifugation through a sucrose cushion and the Golgi membrane pellets were stored at -80°C until use. For in-solution digestion, Golgi membrane pellets were solubilized on ice in 50 µl buffer containing 0.5 M TEAB (triethyl ammonium bicarbonate), 8 M urea and 0.4% SDS. Protein concentrations were determined with the Bio-Rad Bradford assay kit. Golgi membrane proteins (30 µg) from each of the previously mentioned four groups were reduced with 5 mM TCEP (tris(2-carboxyethyl)phosphine) for 1 hour at 37 °C. Cysteines were then blocked with 10 mM MMTS (methyl methanethiosulfonate) for 20 min at room temperature. The protein solution was diluted four times with 0.5 M TEAB containing 3 µg trypsin (1:10 w/w) and incubated at 37 °C overnight. To quantitatively compare Golgi membrane treated with mitotic cytosol to those treated with interphase cytosol, multiplexed isobaric tags (iTRAQTM reagents) were used to label tryptic peptides from different conditions: 115 for interphase cytosol treatment, 116 for mitotic cytosol treatment, and 117 for sequential mitotic and interphase cytosol treatments. In some experiments, a 114 tag was also used to label Golgi membranes treated with buffer alone. The labeling procedure was performed according to the protocol provided by the manufacturer. The reaction was stopped by diluting the mixture with 10 volumes of H2O. OFFGEL isoelectric focusing and LC-MALDI-MS/MS The combined peptide mixture was first fractionated based on isoelectric points (pIs) on a

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 4

3100 OFFGEL Fractionator (Agilent Technologies) using IPG strip pH 3–10 (GE healthcare) with a 12-well manifold according to the manufacturer’s instruction. The iTRAQ-labeled peptides were diluted in the peptide-focusing buffer to a final volume of 1.8 ml, 150 µl of which was loaded into each well. The samples were focused with a maximum current of 50 µA until 50 kV-h were achieved. Peptide fractions were harvested and desalted using Pierce C18 spin columns, the resulting elute was dried using a vacuum centrifuge and resuspended in 40 µl 0.1% trifluoroacetic acid (TFA). The re-suspended peptide sample from each OFFGEL fraction was concentrated on a reversed-phase cartridge (Zorbax C18; 5 mm by 0.3 mm i.d.; 5 µm particles; Agilent) and then separated by reversed-phase column (Zorbax 300 SB C18 column, 75 µm x 150 mm, 3.5 µm particles) on an Agilent 1100 HPLC system with the following binary gradient (flow rate: 300 nl/min): 0 min, 6.5% B; 9 min, 6.5% B; 12 min, 15% B; 92 min, 45% B; 97 min, 60% B; 102 min, 100% B; 104 min, 100% B; 105 min, 6.5% B; 115 min, 6.5% B. Solvent A is 0.1% TFA and solvent B is 90% acetonitrile and 0.1% TFA. The column effluent was mixed with MALDI matrix (2 mg/ml α-cyano-4-hydroxycinnamic acid) through a 25 nl mixing tee and spotted on 1536 well OptiTOFTM MALDI target plates which were later analyzed by tandem mass spectrometry. The MALDI matrix was delivered to the mixing tee by external infusion pump (Harvard Apparatus, Holliston, MA) at 800 nl/min. The MS and MS/MS spectra were acquired on an Applied Biosystems 4800 Proteomics Analyzer (TOF/TOF) (Applied Biosystems/MDX Sciex, Foster City, CA) in positive ion reflection mode with a 200 Hz Nd:YAG laser operating at 355 nm. The accelerating voltage was 20 kV with 400 ns delay. For MS/MS spectra, the collision energy was 2 keV and the collision gas was air. Each MALDI plate was calibrated on nine calibration wells using standards from Applied Biosystems with a 20 ppm mass accuracy in the MS mode. Both MS and MS/MS data were then acquired in the sample wells using the instrument default calibration. Typical MS spectra were obtained with the minimum possible laser energy in order to maintain the best resolution. Single-stage MS spectra for the entire samples were first collected and in each sample well MS/MS spectra were acquired from the 12 most intense peaks above the signal to noise ratio threshold of 30.

Database search and bioinformatics analysis Database searching was performed using ABI’s ProteinPilotTM v3.0 using the Paragon algorithm (29). This software interacts directly with the Oracle database in which the mass spectrometer stores its data, and submits monoisotopic peak lists in batch to a local instance of the search engine for protein identities. No additional peak list filtering was specified. Peak lists were generated by the mass spectrometer during data acquisition based on a specified signal to noise threshold (30 in this case). ProteinPilotTM also performed protein grouping to remove redundant hits and comparative quantifications using iTRAQ ratios. In an effort to comprehensively identify both rat proteins (originated from the Golgi membranes) and human cytosolic proteins (recruited from HeLa cell cytosol), a combined human and rat database was created by manually concatenating IPI rat (version 3.49, with 40,131 of Rattus norvegicus proteins) and IPI human databases (version 3.53, with 73,748 of Homo sapiens proteins). All data sets were then searched against this database in ProteinPilot. All reported proteins were identified with 90% or greater confidence as determined by ProteinPilotTM Unused scores (≥1.0) with the corresponding false positive discovery rate below 1%. The ParagonTM algorithm in ProteinPilot software was used as the default search program with iTRAQ-labeled peptide as sample type, trypsin as the digestion agent, methyl methanethiosulfonate for cysteine modification and 4800 TOF/TOF as the instrument. The Protein and Peptide Summary results obtained from ProteinPilot v3.0 software were exported to Microsoft Excel. The iTRAQ peak area data were normalized for loading error by biased corrections calculated using ProteinPilotTM. The default ion intensity threshold in ProteinPilotTM for calculating peptide ratios was 40 counts. Hierarchical clustering was performed based on Pearson correlation using Cluster 3.0 program and clusters were viewed with the JAVA TREEVIEW program. The theoretical pIs of the peptides in each OFFGEL fraction were calculated using "Compute pI/Mw" tool accessible on the ExPASY website. The identified peptides with a percentage confidence greater than 90% were included in the peptide list for the calculation.

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 5

RESULTS

Applying iTRAQ-based quantitative proteomics to cell-free Golgi disassembly and reassembly assay In this study, large amounts of stacked Golgi membranes were isolated from rat liver as previously described (28). These membranes were routinely purified 80– to 100-fold (97.4 ± 4.2 from 10 preparations) over the homogenate, examined by measuring β-l,4-galactosyltransferase (GalT) activity. When analyzed by electron microscopy (EM), the membranes exhibited as stacked structures (Fig. 1A), over 60% of the cisternal membrane profiles remained in stacks, with the rest in vesicles and tubular structures (Fig. 1A, E). As demonstrated by EM, preparations of Golgi stacks were contaminated with minimal amount of single membrane sheets, presumably plasma membrane or endoplasmic reticulum; no mitochondria or nuclei contamination has been observed in the Golgi preparations. To mimic the interphase and mitotic conditions in the Golgi membranes, we used an established cell-free system (5) to mimic the Golgi disassembly and reassembly process during the cell cycle. In this in vitro assay, the isolated Golgi membranes were treated with interphase or mitotic HeLa cell cytosol. Interphase cytosol was prepared from large-scale cultures of HeLa S3 cells cultured in suspension. Mitotic cytosol was prepared from spinner cells that had been synchronized by nocodazole treatment for 20-24 h, which arrested 96-98% of the cells in prometaphase (for procedure see Supplemental Materials). When Golgi stacks were incubated with mitotic cytosol for 60 min at 37°C in the presence of ATP and an ATP regenerating system, the morphology of the membranes underwent dramatic changes. They were transformed into vesicles and short tubules (Fig. 1C, E), similar to that reported for mitotic Golgi profiles in HeLa cells (30). Incubation with buffer alone, or with interphase cytosol (Fig. 1B), did not result in dramatic morphological change, as most of the membranes remained in cisternae (Fig. 1E) and most of which were found in stacks (Fig. 1F). To reconstitute the reassembly of fragmented Golgi membrane, mitotic Golgi fragments (Fig. 1C) were isolated by centrifugation through a sucrose cushion and re-incubated with interphase cytosol in the presence of ATP, GTP and an ATP regenerating system. Golgi stacks reassembled after a 60-min incubation at 37°C (see

Fig. 1D); most of the membranes were fused to form new cisternae Fig. 1E), which subsequently generated stacked structures (Fig. 1F). This system allowed us to prepare large amounts of Golgi membranes of interphase and mitotic conditions with high purity. Since treatment of rat Golgi membranes with human cell cytosol faithfully mimics the morphological changes of the Golgi in the cell cycle, this experiment showed that the underlying mechanism in rat and human is conserved. More importantly, it also suggested that in this system, rat and human proteins are interchangeable. Thus by quantifying the amount of rat (from the Golgi membranes) and human (from HeLa cell cytosol) proteins associated with the Golgi membranes from different reaction, we could identify candidate proteins involved in the morphological change of the Golgi. In addition, the reversibility of the disassembly and reassembly process also allowed us to apply proteomic techniques to identify candidate proteins whose association with the Golgi membranes is regulated during the cell cycle. Taking advantage of this established in vitro reconstitution approach, we analyzed the global changes of proteins that were associated with interphase or mitotic Golgi membranes in order to delineate the underlying mechanism for the dramatic morphological changes of the Golgi structure during the cell cycle. For this purpose, equal amounts of isolated Golgi membranes were treated separately in four different conditions as shown in Fig. 1, and re-isolated by centrifugation through a sucrose cushion to remove the cytosolic proteins. The membranes were then solubilized, digested with trypsin, and the resulting tryptic peptides were labeled with iTRAQ reagents (as described in the Experimental procedures) followed by OFFGEL isoelectric focusing and LC-MALDI-MS/MS analysis. This allowed us to comprehensively quantify the protein changes in the Golgi membranes under different conditions. In this study, a total of three independent experiments were performed and the flowchart of these experiments is illustrated in Fig. 2. In these experiments, Golgi membranes treated with buffer alone were also labeled with the iTRAQ reagent, 114, as negative controls. However, since the membranes in this reaction were not treated with cytosol, there were few cytosolic proteins associated with the membranes. On the contrast, a number of cytosolic proteins ubiquitously associated with Golgi

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 6

membranes when treated with either interphase or mitotic cytosol. For this reason, Golgi membranes treated with interphase cytosol were used as controls in all experiments in order to better uncover mitosis specific Golgi protein changes. To quantitatively measure the amount of protein in the membranes, considerations were taken into the experimental design to reduce variations. First, we conducted duplicate biological experiments that separated the peptide mixture in the first dimension into 8 fractions based on their pIs using an OFFGEL isoelectric focusing system. This allowed us to assess the reproducibility of the experimental results. Next, to confirm the results from the duplicate experiments and also extend our findings, we conducted a third experiment identical to the duplicates, except that there were 12 fractions of peptides collected from the first dimensional OFFGEL separation. Using the third experiment as an example, we analyzed the characteristics of peptide separation by OFFGEL isoelectric focusing. It was found that the numbers of total and unique peptides identified from each OFFGEL fraction were similar except in the middle fractions, #6 and #7 (Fig. 3A). To evaluate the efficient separation of peptides by their pIs, the peptides identified in each OFFGEL fraction were first filtered by their confidence scores to exclude any hit below 90% confidence, after which the theoretical pIs were calculated using an online software tool Compute pI/MW. As a representative, the results from the third experiment are shown in Fig. 3B. As expected, the calculated pIs increased in a nearly linear fashion from the acidic end (fraction #1) to the basic end (fraction #12) with the middle 4 fractions (#5 to #8) at a slower slope. Proteomic survey of an enriched stacked Golgi membrane fraction When the peptides were analyzed by LC-MS/MS, three data sets from three independent biological experiments were collected. Over 14,000 MS/MS spectra were acquired and searched in a combined IPI rat and IPI human database using the ProteinPilotTM software. Protein identifications required an Unused score of 1.0 or greater corresponding to a 90% or more confidence and less than 1% false discovery rate. 60-70% of these spectra were identified with over 90% confidence. The numbers of proteins identified in each data set and common between different data sets are reported in Figure S1. The protein grouping

function in ProteinPilotTM and the combined database not only allowed us to comprehensively identify both rat and human proteins but also to distinguish between them when there are unique peptide sequences detected for rat or human. If the identified peptides for a given gene name matched sequences identical between the corresponding human and rat protein, this protein was then assigned as a “common” protein (Supplemental table 1). Using these criteria, a total of 1193 unique proteins were identified in three data sets (Supplemental table 2), 632 assigned as rat proteins, 334 as human proteins and 227 as “common” proteins. These proteins were then categorized based on their annotated subcellular localizations in the databases and literatures (Supplemental table 2) and an overview is shown in Fig. 4A. Half (50%, 606 proteins) of these proteins are known or candidate bona fide Golgi (30%) or cargo proteins (18%), or proteins in transit to other subcellular organelles such as the lysosome (2%). The second largest category includes cytosolic proteins (29%), followed by cytoskeletal proteins (8%). Since isolated Golgi membrane were incubated with excess cytosolic proteins as experimentally designed, it was expected that a significant number of cytosolic proteins would be present in the Golgi sample mainly through protein interactions with Golgi resident proteins as well as certain degree of non-specific adsorption. Ubiquitin conjugating proteins (Fig. 4B, 52 proteins, 4% of total) have been related to protein sorting and trafficking (31-33) and post-mitotic Golgi membrane reassembly (19,34). These cytosolic proteins may be involved in regulation of Golgi membrane dynamics during the cell cycle. The rest of the proteins are those with unknown identity or with uncharacterized subcellular localizations (3%), or those likely to be contaminants from the ER (2%), mitochondria (1%) and nucleus (7%). The 348 identified Golgi and associated proteins (Fig. 4A) can be further categorized according to their molecular functions, as shown in Fig. 4B and Supplemental table 2. These include Golgi enzymes (20%), golgins and Golgi structural proteins (5%), Golgi integral (14%) and peripheral membrane proteins (7%), kinases (8%), coat proteins for vesicle formation such as coatomers, ARF and adaptor proteins (10%), NSF, SNAREs and membrane fusion machineries (4%), Rabs and protein tethers (7%), ubiquitin conjugating proteins (16%) and protein chaperones (9%). Multiple

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 7

families of Golgi structural proteins are well represented (Fig. 4B) including the golgins/GRASP proteins as well as coat and adaptor proteins and membrane fusion proteins. These proteins have been shown to be involved in regulation of Golgi membrane dynamics during the cells cycle (1,2,5,35,36). Quantitative proteomics analysis of protein changes in mitotic Golgi disassembly and reassembly In all three data sets, the iTRAQ ratios of identified proteins were normalized using the automatic Bias Correction function in the ProteinPilotTM software to compensate for the minor differences during sample mixing. When necessary, the data between the different cytosol reactions were further adjusted according to the mean ratios of a group of Golgi enzymes which are expected to remain unchanged during the experimental procedures. The overall quality of the quantification and normalization is evaluated with the histogram of iTRAQ ratio distribution from identified proteins above the confidence threshold, 90%. Figure 5 shows the histograms of the protein ratios for MC vs. IC (116:115) (Fig. 5A) and MC→IC vs. IC treatment (117:115) (Fig. 5B). These histograms indicate normal distributions centered around 1.0 (mean ± S.D. are 0.97 ± 0.33 and 0.99 ± 0.37, respectively). The protein ratio histograms exhibit quite broad distributions (0.2 to 2.2 or more, which is relatively large deviations) indicating the presence of differentially regulated proteins. Supplemental table 3 shows protein changes from 84 unique proteins detected in three experiments that showed significant changes (MC vs. IC) with a p-value ≤ 0.05 for statistical significance and a threshold of 20% (mean ratios >1.20 or <0.80) for biological significance. This cutoff value is supported by our experience in a large number of projects where changes with 20% threshold and p-value ≤ 0.05 have been validated by Western blots (37). We next applied the hierarchical clustering method, a commonly used method for microarray analysis, to the proteomics results of the differentially regulated proteins (Supplemental table 3). The iTRAQ ratio of each protein was used to perform the cluster analysis using Pearson Correlation and the results were visualized using Java TreeView (Fig. 6A). It became obvious that different clusters of proteins are present. Some proteins were up-regulated with mitotic cytosol treatment (bright

yellow squares in the middle lane) whereas other proteins were down-regulated with mitotic cytosol treatment and the changes were then reversed under Golgi reassembly conditions (dark squares in the middle lanes and lighter squares in the third lanes). As an indication of the efficient clustering, the different subunits of the same protein complexes (e.g. chaperonin-containing T-complex subunits, Ccts and COPI coatomers, Cops) were clustered together. Closer analysis of selected clusters (Fig. 6B; Table 1) revealed proteins clustered together in several major patterns. One group of proteins including actin (Actg1), Rab11a and subunits of the chaperonin-containing T-complex (Tcp1 and Ccts) showed a significant increase in association with Golgi upon MC treatment then returned to the control level in the cases of actin and Rab11a or below the control level in the cases of T-complex subunits (Fig. 6B) after subsequent IC treatment. In contrast, a cluster containing COPI coatomer subunits (Cops), the heat shock protein hsc70 (Hspa8) and tubulin subunits (Tubs) showed a dramatic decrease upon mitotic cytosol treatment then returned back to control level (Fig. 6B). It has been shown that COPI vesicle formation is involved in mitotic Golgi fragmentation (5,20); whether other proteins mentioned above are part of this machinery requires further investigation. Several golgins (e.g. Golgas and Golphs) and GRASP (Gorasp) proteins such as GRASP65 have been shown to be involved in Golgi structure formation, phosphorylation of these proteins is required for Golgi membrane unstacking during mitosis (1,23). However, their total abundance did not appear to have any dramatic change (Table 2). In addition, the Golgi structural proteins, enzymes and SNARE proteins [e.g. Golgi SNAP receptor complex member 1 (Gosr1) and syntaxin 5a (Stx5a)], as expected, maintained the same level during the experimental procedures (Fig. 6D; Table 2). To validate the iTRAQ-based results, we conducted Western blot analysis on a large number of proteins, including many in the major clusters shown above (Fig. 7). We first compared the protein levels in the two cytosols. Most proteins, including ARF and coat proteins, Rabs and tethering complexes, some kinases, heat shock proteins, actin and tubulin, have comparable amount in both interphase and mitotic cytosols (Fig. 7A). These results indicate that the change of Golgi-associated proteins was not due to

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 8

the availabilities of the proteins in interphase or mitotic cytosol. We then compared the proteins associated with either interphase or mitotic Golgi membranes (Fig. 7B). As expected, Golgi resident proteins such as TGN38 and enzymes such as α-Mannosidase (Man) II had no change across all conditions. Consistent with our iTRAQ results, the total amount of proteins such as GM130, golgin 84 and GRASP65 did not change, although GRASP55 (indicated by the band shift) and GRASP65 (recognized by the phosphospecific antibody, pGRASP65) were highly phosphorylated (Fig. 7B). Previous studies demonstrated that these proteins are modified during mitosis, but they do not detach from the membranes, nor are they degraded during mitosis (22,23). SNARE proteins and cargos (e.g. rat serum albumin, RSA) were among the unchanged proteins as indicated by the iTRAQ results (Fig. 6D). The unique patterns of changes of COPI coatomer subunits were confirmed in the Western blot results (Fig.7B) with antibodies against five out of all six subunits identified by iTRAQ. COPI vesicle budding has been shown to be part of the process to sequester the Golgi membranes into vesicles during mitosis (5). The reduction of coat proteins on the membranes is likely due to uncoating after the generation of the vesicles. The different patterns of the heat shock proteins and the cytoskeleton proteins actin and tubulin were also confirmed (Fig. 7B). All together, the comprehensive Western analysis results are consistent with the iTRAQ-based quantitative proteomics results and provided strong support to our findings in this study.

DISCUSSION Organellar proteomics combines subcellular fractionation with mass spectrometry and provides a powerful approach to identifying the protein compositions of each subcellular compartment. Because of the central role of the Golgi apparatus in cell biology and physiology, it has been a very active research subject of modern organellar proteomics. However, due to some limitations in gel-based protein separation techniques and relatively lower sensitivity of mass spectrometers in the past, early studies of Golgi proteomes reported a relatively small number of identified proteins. In an early two-dimensional gel study (10), 73 proteins were identified from isolated rat liver Golgi stacks. In another study, the protein compositions of Golgi fractions isolated from basal and maximal

functional states of the mammary glands were compared using 2D gel electrophoresis, and 45 proteins were identified (8). A subsequent mass spectrometry analysis using 1D SDS-PAGE led to the identification of 81 proteins from purified hepatic Golgi apparatus (6). Recent progress in proteomic technology has enabled more comprehensive high-throughput profiling strategies of enriched Golgi fractions, resulting in hundreds of protein identifications. In a shotgun proteomics study, 421 proteins were characterized in Golgi fractions isolated from rat liver (7). In a more recent study using redundant peptide counting approach, a quantitative map of the rough ER, smooth ER and Golgi apparatus isolated from rat liver homogenates was conducted (38). In this study, 193 proteins unique to Golgi were reported together with another 405 proteins found in both the ER and the Golgi. For the 193 proteins found only in Golgi/COPI vesicles, terminal sugar transferases, CASP, golgin, SNAREs, and a subset of Rabs and COGs (the conserved oligomeric Golgi complex proteins) were prominent (38). The overall goal of the current study was to identify proteins associated with Golgi membranes in different phases of the cell cycle and therefore to unveil the molecular mechanisms underlying the morphological change of Golgi membranes. For this purpose, proteins in isolated Golgi membranes treated with mitotic cytosol were comprehensively compared to those treated with interphase cytosol using the iTRAQ labeling technique combined with LC-MALDI-MS/MS analysis. This allowed us to identify and quantitatively analyze a total of 1193 of Golgi membrane and associated proteins. The identified known and potential Golgi intrinsic and associated proteins include the following major functional categories: forty two Golgi structural proteins, such as GRASP55, GRASP65 and GM130, that are involved in Golgi structure formation; fifty Golgi resident enzymes involved in various steps of glycoprotein modifications such as mannosidases I and II; seven SNARE proteins for membrane fusion; twenty five Rab GTPases for vesicle tethering and thirty six COPI and COPII vesicle coat protein subunits. More importantly, the combination of the quantitative approach with Western blotting allowed us to unveil eighty-four proteins with significant changes in abundance under mitotic condition compared to interphase condition (Fig. 6, 7 and Supplemental table 3). The specificities of these protein changes to Golgi

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 9

membrane disassembly process in the cell cycle were further supported by the following evidence: 1) many of these protein changes were detected in more than one experiment; 2) multiple or all subunits of several protein complexes were found changed in the same direction and to a similar degree; 3) the protein changes under mitotic condition were reversed upon a subsequent interphase cytosol treatment which is consistent with the reversible morphological changes under these conditions. Among these proteins, several COPI coatomer subunits (e.g. alpha, beta, gamma and delta) are of particular interest. COPI vesicle formation in interphase cells is believed to mediate intra-Golgi transport and retrograde transport for the Golgi to the endoplasmic reticulum-Golgi intermediate compartment (39). In interphase cells, COPI coat proteins are enriched on Golgi membranes. COPI vesicle formation is also thought to be part of the mitotic fragmentation process to sequester the Golgi membranes into vesicles (5). As the COPI coat is not a stable structure, it falls off the vesicles for preparation of subsequent membrane fusion. As shown in Fig. 7B, the membrane associated COPI coat proteins were reduced after mitotic cytosol treatment and returned back to control level after subsequent interphase cytosol treatment. The signaling protein Rab11a is a small GTPase associated with the trans-Golgi network (TGN) and endosomes and is thought to be involved in endosome to TGN recycling (40-42). Recent studies showed that Rab11 is required for the formation of the cleavage furrow, or contractile ring, for separation of the two daughter cells in cytokinesis (43-46). Although it has been thought that the membrane is likely from the endosomes, as Rab11 is located on both endosome and TGN membranes, it is unclear whether the Golgi can also provide the required membranes for cytokinesis. Our results showed that the association of Rab11a is increased during mitosis (Figure 6B), demonstrating that Golgi membrane could function as the donor membrane for the formation of the cleavage furrow. Another interesting group of proteins are the cytoskeletal proteins. For example, the amount of membrane associated actin gamma 1 (Actg1) was dramatically increased upon mitotic cytosol treatment compared with control interphase cytosol

treatment, and this amount was reduced when the mitotic Golgi fragments were further treated with the interphase cytosol to allow Golgi assembly to occur (Fig. 6B, 7B). In contrast, the microtubule components, such as tubulin isoforms alpha 1a (Tuba1a), 1b (Tuba1b), 3a (tuba3a) and beta 5 (Tubb5), were decreased when the Golgi membranes were fragmented upon mitotic cytosol treatment and were restored when further treated with interphase cytosol (Fig. 6C, 7B). The formation and positioning of the Golgi ribbon-like structure is thought to be dependent on microtubule organization and the centrosome (47-50). The centrosome nucleates polarized microtubule assembly and creates a radial array of microtubules with minus ends embedded in the centrosome and plus ends extended in all directions towards the cell periphery. Golgi membrane-associated minus end-directed microtubule motors, mainly dynein, move the Golgi stacks inward and cause concentration of the Golgi stacks around the centrosome. ER derived vesicles track along microtubules to allow collection of the cargo proteins in the Golgi apparatus positioned at the cell center, and then move back towards the cell periphery along microtubules to various destinations including the plasma membrane. Thus the ribbon formation and localization of the Golgi may facilitate protein transport in mammalian cells. Our results demonstrated that the interaction between the Golgi membranes and the microtubule cytoskeleton is reduced during mitosis, suggesting that dissociation of Golgi membranes from microtubule cytoskeleton may be part of the mechanism underlying Golgi fragmentation at the onset of mitosis. On the other hand, the role for actin microfilaments in Golgi organization is so far unclear, although it has been indicated that actin plays a role in ER to Golgi transport (51). Our results suggest that the actin cytoskeleton is selectively associated with mitotic Golgi vesicles instead of intact Golgi cisternal membranes, a feature important for vesicle transport. These possibilities need further investigations. Taken together, this quantitative proteomics study has provided a near complete accounting of proteins in the Golgi apparatus of rat liver under both interphase and mitotic conditions. The data provide an important foundation for the resolution of long-standing problems regarding the secretory pathway and biogenesis of this membrane organelle.

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 10

REFERENCES 1. Wang, Y. (2008) Golgi apparatus inheritance. in The Golgi apparatus. State of the art 110 years after

Camillo Golgi’s discovery (Mironov, A., Pavelka, M., and Luini, A. eds.), Springer-Verlag Gmbh., Wien-New York. pp 580-607

2. Shorter, J., and Warren, G. (2002) Annu Rev Cell Dev Biol 18, 379-420 3. Liu, Z., Vong, Q. P., and Zheng, Y. (2007) Dev Cell 12, 839-840 4. Brownhill, K., Wood, L., and Allan, V. (2009) Semin Cell Dev Biol 5. Tang, D., Mar, K., Warren, G., and Wang, Y. (2008) J Biol Chem 283, 6085-6094 6. Bell, A. W., Ward, M. A., Blackstock, W. P., Freeman, H. N., Choudhary, J. S., Lewis, A. P., Chotai,

D., Fazel, A., Gushue, J. N., Paiement, J., Palcy, S., Chevet, E., Lafreniere-Roula, M., Solari, R., Thomas, D. Y., Rowley, A., and Bergeron, J. J. (2001) J Biol Chem 276, 5152-5165

7. Wu, C. C., MacCoss, M. J., Mardones, G., Finnigan, C., Mogelsvang, S., Yates, J. R., 3rd, and Howell, K. E. (2004) Mol Biol Cell 15, 2907-2919

8. Wu, C. C., Taylor, R. S., Lane, D. R., Ladinsky, M. S., Weisz, J. A., and Howell, K. E. (2000) Traffic 1, 963-975

9. Wu, C. C., Yates, J. R., 3rd, Neville, M. C., and Howell, K. E. (2000) Traffic 1, 769-782 10. Taylor, R. S., Wu, C. C., Hays, L. G., Eng, J. K., Yates, J. R., 3rd, and Howell, K. E. (2000)

Electrophoresis 21, 3441-3459 11. Mogelsvang, S., and Howell, K. E. (2006) Curr Opin Cell Biol 18, 438-443 12. Misteli, T., and Warren, G. (1994) J Cell Biol 125, 269-282 13. Rabouille, C., Kondo, H., Newman, R., Hui, N., Freemont, P., and Warren, G. (1998) Cell 92, 603-610 14. Rabouille, C., Misteli, T., Watson, R., and Warren, G. (1995) J Cell Biol 129, 605-618 15. Shorter, J., and Warren, G. (1999) J Cell Biol 146, 57-70 16. Satoh, A., Wang, Y., Malsam, J., Beard, M. B., and Warren, G. (2003) Traffic 4, 153-161 17. Kondo, H., Rabouille, C., Newman, R., Levine, T. P., Pappin, D., Freemont, P., and Warren, G. (1997)

Nature 388, 75-78 18. Rabouille, C., Levine, T. P., Peters, J. M., and Warren, G. (1995) Cell 82, 905-914 19. Wang, Y., Satoh, A., Warren, G., and Meyer, H. H. (2004) J Cell Biol 164, 973-978 20. Xiang, Y., Seemann, J., Bisel, B., Punthambaker, S., and Wang, Y. (2007) J Biol Chem 282, 21829-

21837 21. Barr, F. A., Puype, M., Vandekerckhove, J., and Warren, G. (1997) Cell 91, 253-262 22. Shorter, J., Watson, R., Giannakou, M. E., Clarke, M., Warren, G., and Barr, F. A. (1999) Embo J 18,

4949-4960 23. Wang, Y., Seemann, J., Pypaert, M., Shorter, J., and Warren, G. (2003) Embo J 22, 3279-3290 24. Ross, P. L., Huang, Y. N., Marchese, J. N., Williamson, B., Parker, K., Hattan, S., Khainovski, N.,

Pillai, S., Dey, S., Daniels, S., Purkayastha, S., Juhasz, P., Martin, S., Bartlet-Jones, M., He, F., Jacobson, A., and Pappin, D. J. (2004) Mol Cell Proteomics 3, 1154-1169

25. Zieske, L. R. (2006) J Exp Bot 57, 1501-1508 26. Aggarwal, K., Choe, L. H., and Lee, K. H. (2006) Brief Funct Genomic Proteomic 5, 112-120 27. Wang, Y., Satoh, A., and Warren, G. (2005) J Biol Chem 280, 4921-4928 28. Wang, Y., Taguchi, T., and Warren, G. (2005) Purification of Rat Liver Golgi Stacks. in Cell Biology: A

Laboratory Handbook, 3rd Edition (Celis, J. ed.), Elsevier Science (USA), San Diego. pp 33-39 29. Shilov, I. V., Seymour, S. L., Patel, A. A., Loboda, A., Tang, W. H., Keating, S. P., Hunter, C. L.,

Nuwaysir, L. M., and Schaeffer, D. A. (2007) Mol Cell Proteomics 6, 1638-1655 30. Lucocq, J. M., Berger, E. G., and Warren, G. (1989) J Cell Biol 109, 463-474 31. Angers, A., Ramjaun, A. R., and McPherson, P. S. (2003) J Biol Chem 32. Bonifacino, J. S., and Traub, L. M. (2003) Annu Rev Biochem 72, 395-447 33. Scott, P. M., Bilodeau, P. S., Zhdankina, O., Winistorfer, S. C., Hauglund, M. J., Allaman, M. M.,

Kearney, W. R., Robertson, A. D., Boman, A. L., and Piper, R. C. (2004) Nat Cell Biol 6, 252-259 34. Meyer, H. H., Wang, Y., and Warren, G. (2002) Embo J 21, 5645-5652 35. Barr, F. A., and Short, B. (2003) Curr Opin Cell Biol 15, 405-413 36. Seemann, J., Jokitalo, E., Pypaert, M., and Warren, G. (2000) Nature 407, 1022-1026

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 11

37. Keshamouni, V. G., Michailidis, G., Grasso, C. S., Anthwal, S., Strahler, J. R., Walker, A., Arenberg, D. A., Reddy, R. C., Akulapalli, S., Thannickal, V. J., Standiford, T. J., Andrews, P. C., and Omenn, G. S. (2006) J Proteome Res 5, 1143-1154

38. Gilchrist, A., Au, C. E., Hiding, J., Bell, A. W., Fernandez-Rodriguez, J., Lesimple, S., Nagaya, H., Roy, L., Gosline, S. J., Hallett, M., Paiement, J., Kearney, R. E., Nilsson, T., and Bergeron, J. J. (2006) Cell 127, 1265-1281

39. Duden, R. (2003) Mol Membr Biol 20, 197-207 40. Zerial, M., and McBride, H. (2001) Nat Rev Mol Cell Biol 2, 107-117 41. Pfeffer, S. (2005) Biochem Soc Trans 33, 627-630 42. Pfeffer, S. R., Soldati, T., Geissler, H., Rancano, C., and Dirac-Svejstrup, B. (1995) Cold Spring Harb

Symp Quant Biol 60, 221-227 43. Matheson, J., Yu, X., Fielding, A. B., and Gould, G. W. (2005) Biochem Soc Trans 33, 1290-1294 44. Agop-Nersesian, C., Naissant, B., Ben Rached, F., Rauch, M., Kretzschmar, A., Thiberge, S., Menard,

R., Ferguson, D. J., Meissner, M., and Langsley, G. (2009) PLoS Pathog 5, e1000270 45. Giansanti, M. G., Belloni, G., and Gatti, M. (2007) Mol Biol Cell 18, 5034-5047 46. Schonteich, E., Pilli, M., Simon, G. C., Matern, H. T., Junutula, J. R., Sentz, D., Holmes, R. K., and

Prekeris, R. (2007) Eur J Cell Biol 86, 417-431 47. Rios, R. M., Sanchis, A., Tassin, A. M., Fedriani, C., and Bornens, M. (2004) Cell 118, 323-335 48. Linstedt, A. D. (2004) Cell 118, 271-272 49. Barr, F. A., and Egerer, J. (2005) J Cell Biol 168, 993-998 50. Infante, C., Ramos-Morales, F., Fedriani, C., Bornens, M., and Rios, R. M. (1999) J Cell Biol 145, 83-

98 51. Valderrama, F., Duran, J. M., Babia, T., Barth, H., Renau-Piqueras, J., and Egea, G. (2001) Traffic 2,

717-726

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 12

Table 1: Major Golgi associated protein changes when treated with mitotic cytosol

Accession

Gene Symbol

Protein name

Exp. #

Unique peptides

(Rat)

Unique Peptides (human)

Common peptides

Unused score

%Cov

116: 115

PVal 116: 115

EF 116: 115

117: 115

PVal 117: 115

EF 117: 115

IPI00896224.1 Actg1 Actin, cytoplasmic 2 1 0 0 15 53.5 56.3 2.00 0.00 1.43 0.96 0.88 1.92 2 0 0 16 45.9 46.4 1.51 0.02 1.37 1.24 0.47 3.71 3 0 0 16 49.8 62.4 2.03 0.00 1.45 1.23 0.05 1.19 IPI00792677.1 Tuba1b Tubulin alpha-1B 2 0 0 14 38.2 45.4 0.42 0.00 1.67 0.89 0.37 1.36 chain 3 0 0 18 54.6 65.6 0.37 0.01 1.93 0.75 0.06 1.36

IPI00007752.1 Tubb2c Tubulin beta-2C chain 2 0 0 14 4.0 41.3 0.41 0.04 1.88 0.82 0.32 1.70

3 0 1 18 57.8 58.6 0.44 0.05 2.22 0.76 0.13 1.60 IPI00197579.2 Tubb5 Tubulin beta-5 chain 2 0 0 17 47.8 51.3 0.63 0.03 1.42 1.00 1.00 7.83 3 0 0 15 8.1 43.2 0.54 0.01 1.38 0.82 0.02 1.11 IPI00643158.1 Tubb6 Tubulin beta-6 chain 3 0 0 21 5.7 27.6 0.40 0.03 1.77 0.94 0.30 6.68 IPI00373289.1 Arcn1 Coatomer subunit 1 0 0 4 8.2 16.0 0.66 0.00 1.12 0.94 0.57 1.38 delta 3 0 0 5 12.8 17.0 0.74 0.04 1.32 0.83 0.10 1.27 IPI00646493.1 Copa Coatomer subunit 1 0 1 4 12.0 5.2 0.65 0.01 1.28 0.97 0.77 1.25 alpha 2 0 0 4 8.2 3.2 0.46 0.04 2.04 0.94 0.89 3.35 3 0 18 0 35.4 17.5 0.58 0.00 1.37 0.88 0.11 1.23

IPI00295851.4 Copb1 Coatomer subunit beta 1 0 0 3 4.0 3.3 0.53 0.03 1.84 1.19 0.09 1.35

2 0 0 1 2.0 2.0 0.26 0.69 3 0 1 12 24.1 20.0 0.44 0.00 1.34 0.86 0.09 1.27 IPI00220219.6 Copb2 Coatomer subunit 3 0 8 0 15.7 11.5 0.53 0.01 1.47 0.82 0.29 1.84 Beta’ IPI00783982.1 Copg Coatomer subunit 1 0 1 3 6.0 5.4 0.54 0.03 1.50 1.10 0.85 1.76 gamma 3 0 10 0 23.3 16.9 0.46 0.00 1.30 0.86 0.06 1.28 IPI00208205.1 Hspa8 1 0 0 17 41.2 36.8 0.79 0.01 1.14 1.27 0.54 2.33

Heat shock cognate 71 kDa protein 2 0 0 18 56.2 45.8 0.57 0.01 1.46 0.89 0.71 1.95

3 0 0 20 67.0 52.5 0.62 0.00 1.25 0.96 0.54 1.09 The iTRAQ were used to label tryptic peptides: 115 for interphase cytosol treatment, 116 for mitotic cytosol treatment, and 117 for sequential mitotic and interphase cytosol treatments. Three independent biological experiments were performed and indicated in "Exp. #" column. In ProteinPilot software, the true ratio for the protein is expected to be found between Mean * EF (error factor) and Mean ÷ EF with 95% confidence. The Unused ProtScore is a measurement of all the peptide evidence for a protein that is not better explained by a higher ranking protein. The ProtScore = -log (1-%confidence), therefore ProScore 1.0 corresponds to 90% confidence and 2.0 corresponds to 99% confidence. Based on the protein grouping results in ProteinPilot, the numbers of peptides unique

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 13

to rat or human protein sequences or common between rat and human protein sequences are listed for each protein in the columns “Unique peptides (rat)”, “Unique peptides (human)”, and “Common peptides”.

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 14

Table 2. Abundance of Golgin and related proteins had no significant change when treated with mitotic cytosol

Accession Gene

Symbol Protein name Exp. #

Unique peptide Score %Cov

116:115

117: 115

PVal 116:115

EF 116:115

PVal 117:115

EF 117:115

(rat)a IPI00209187.1 Golga2 GM130 1 8 14.0 9.0 0.99 0.96 0.91 1.28 0.89 2.20 2 13 22.2 18.4 0.94 0.90 0.79 1.65 0.61 1.57 3 14 37.5 25.4 1.01 0.92 0.92 1.20 0.25 1.17 IPI00194277.3 Golga5 1 10 18.1 16.7 0.91 0.72 0.46 1.37 0.08 1.47

Golgin subfamily A member 5 2 10 17.5 19.5 0.80 0.76 0.15 1.40 0.21 1.60

3 16 26.7 23.5 1.10 1.02 0.38 1.26 0.68 1.10 IPI00560077.1 Golph3 Golgi phosphoprotein 3 1 3 6.1 13.4 0.86 0.81 0.34 1.68 0.13 1.42 3 4 9.8 13.8 0.83 0.93 0.34 1.63 0.50 1.32 IPI00368095.3 Golim4 1 12 26.0 22.3 0.96 0.81 0.50 1.14 0.06 1.25

similar to Golgi phosphoprotein 4 2 14 26.3 23.4 1.14 1.19 0.62 1.74 0.51 1.76

3 16 34.0 27.6 1.05 1.03 0.38 1.12 0.45 1.07 IPI00207306.3 Gorasp1 GRASP65 3 5 8.0 13.1 0.83 0.63 0.85 16.43 0.22 2.56 IPI00362488.1 Gorasp2 GRASP55 1 4 9.2 12.3 0.82 0.80 0.29 3.34 0.04 1.21 2 2 3.7 6.8 0.87 0.94 0.75 73.11 0.64 3.71 3 5 10.1 13.2 1.37 1.06 0.34 2.26 0.69 1.44 IPI00324618.3 Uso1b 1 2 6.0 4.8 0.78 0.59 0.08 2.58 0.33 4.91

General vesicular transport factor p115 2 4 7.4 7.4 0.35 0.43 0.04 2.56 0.06 3.99

3 4 14.7 9.9 0.28 0.49 0.01 2.23 0.00 1.45 IPI00210219.1 Gosr1 1 2 3.6 7.0 0.90 0.62 0.32 1.42 0.06 1.73

Golgi SNAP receptor complex member 1 2 2 4.6 7.7 1.12 1.54 0.56 1.74 0.34 3.33

3 6 12.6 26.4 0.94 0.87 0.72 1.55 0.17 1.27 IPI00785588.1 Stx5a Isoform 1 of syntaxin-5 2 5 7.7 19.1 1.19 0.85 0.53 2.71 0.59 2.95 3 4 7.5 11.0 0.85 0.87 0.54 2.10 0.39 1.55

a. Since there were only peptides unique to rat sequences detected, all these proteins are assigned to rat origin. Therefore, only the numbers of peptides unique to rat protein sequences are listed in the column “Unique peptides (rat)”. b. Uso1 showed decrease in both experiments. This was also confirmed by Western blotting (Figure 7B).

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 15

Footnotes Acknowledgements We gratefully acknowledge Drs. F. Gorelick, K. Gull, T. Kreis, M. Lowe, K. Moremen, A. Price, A. Satoh, J. Seemann, D. Sheff, D. Shields and G. Warren for generously providing antibodies. We thank J. Williams and D Tang for suggestions and reagents. We thank Sarah Volk for her assistance in OFFGEL electrophoresis. This work was supported by National Institute of Health grant P41 RR018627 to P. Andrews, and was partially supported by the Pardee Cancer Research Foundation, the National Institute of Health (GM087364) and American Cancer Society (RGS-09-278-01-CSM) to Y. Wang. Abbreviations ARF1, ADP-ribosylation factor-1; EM, electron microscopy; IC, interphase cytosol; MC, mitotic cytosol; MGF, mitotic Golgi fragments; iTRAQ, isobaric tags for relative and absolute quantification; NSF, N-ethylmaleimide-sensitive fusion protein; PP2A, protein serine/threonine phosphatase type 2A; RSA, rat serum albumin; SNAP, soluble NSF attachment protein; SNARE, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors; TGN, trans-Golgi network. FIGURE LEDENDS Figure 1. Morphology of Golgi membranes after incubation with interphase or mitotic cytosol. EM photographs showing purified Golgi membrane stacks (arrows) (A) incubated with interphase cytosol (B), mitotic cytosol (C), or with mitotic cytosol followed by interphase cytosol (D). Extensive fragmentation after mitotic cytosol treatment (C) yielded large vesicular profiles, small vesicular profiles (arrowheads), and remnants of stacked and single cisternae. E-F. Quantitation of EM micrographs from A-D by the intersection method to estimate the percentage of membranes in cisternae (E) and the percentage of cisternae in stacks (F). Results represent the mean of three independent experiments ± S.D. Figure. 2. Flow chart of iTRAQTM labeling and the subsequent separation and protein identification of Golgi membrane treated with different cytosols. To quantitatively compare proteins associated with Golgi membrane during the cell cycle, 30 μg of Golgi membrane proteins treated with different cytosols were solubilized, reduced, alkylated and digested with trypsin. Tryptic peptides were labeled with iTRAQ reagents and then mixed between different reactions. The mixture of iTRAQ labeled peptides was first separated on OFFGEL apparatus to 8-12 fractions based on the pI of the peptides. Each fraction was then analyzed by reversed phase HPLC-MALDI-MS/MS.

Figure 3. The number and average pI of identified peptides in each OFFGEL fraction. Each of the twelve bars represents one fraction collected from the IPG strip (13 cm length, pH 3-10). The fraction number increases with the pH value on the strip. The numbers of total and unique peptides in each of the twelve fractions identified with confidence 90% are plotted (A). The average pI of all the peptides in a single OFFGEL fraction is calculated using the Compute MW/pI program at the ExPASY website and plotted (B). Gray portion in each bar represents the average pI of the peptides in that fraction and the dotted portion represents the standard deviation (S.D.) of each fraction’s average pI. Figure 4. Subcellular and functional classification of the identified proteins. (A) Subcellular localization of proteins identified from the purified Golgi membrane. (B) Functional categories of identified known and potential Golgi associated proteins. Figure 5. Histograms of tryptic peptide distributions based on their iTRAQ ratios. Proteins identified from three separate experiments were pooled together and their distribution on iTRAQ ratios 116:115 (A) and 117:115 (B) were plotted in histograms between iTRAQ ratio 0 and 2.5 with the bin size at 0.05. Samples labeling: 115, interphase cytosol; 116, mitotic cytosol; 117, interphase mitotic cytosol.

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 16

Figure 6. Hierarchical clustering of Golgi membrane and associated proteins that showed significant changes upon treatment with mitotic cytosol. Hierarchical clustering using Pearson correlation was performed on the proteins in Supplemental table 3 to generate the heat map. As an overview, part of the clustering result is shown in (A). Two representative clusters showed unique patterns were selected and the averaged fold changes (MC and MC IC vs. IC) from three separate experiments were plotted for each protein (B and C). In contrast to the proteins showed significant changes, many Golgi structural proteins showed little change. Several such proteins were selected as representatives for a very distinct cluster, and their average ratios under the three indicated conditions were also plotted (D). Figure 7. Western blot analysis of major Golgi associated proteins. A. Protein levels in the cytosols. Equal amounts of interphase (lanes 1-2) and mitotic (lanes 3-4) HeLa cell cytosols were analyzed by SDS-PAGE and Western blotting for the indicated proteins. The protein level for almost all the tested proteins was comparable in interphase and mitotic cytosols. B. Analysis of Golgi membrane associated proteins. Purified rat liver Golgi membranes were incubated with either buffer (lane 1), interphase cytosol (IC, lane 2), mitotic cytosol (MC, lane 3), or with subsequent treatments with interphase and mitotic cytosol (MC IC, lane 4) followed by isolation of the total membranes by centrifugation. Equal fractions (by volume) of each reaction were analyzed by Western blotting for indicated proteins and representative images from 3 independent experiments were shown. The Golgi enzymes and SNARE proteins were not changed; while some of the Golgi structural proteins (e.g. GRASP65, indicated by the phospho-specific antibody, pGRASP65; and GRASP55, indicated by the band shift) were phosphorylated. The membrane association of COP proteins and some other cytosolic proteins changed after subsequent treatments. NSF, N-ethylmaleimide-sensitive fusion protein; SNAP, soluble NSF attachment protein; RSA, rat serum albumin.

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 17

Figure 1

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 18

Figure 2

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 19

Figure 3

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 20

Figure 4

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 21

Figure 5

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 22

Figure 6

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Chen, et al., 2009 23

Figure 7

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Yanzhuang WangXuequn Chen, Eric S. Simon, Yi Xiang, Maureen Kachman, Philip C. Andrews and

reassemblyQuantitative proteomics analysis of cell cycle regulated Golgi disassembly and

published online January 7, 2010J. Biol. Chem. 

  10.1074/jbc.M109.047084Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

  http://www.jbc.org/content/suppl/2010/01/07/M109.047084.DC1

by guest on August 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from