Quantitative proteomics analysis of cell cycle-regulated Golgi disassembly and reassembly

Abstract

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 isobaric tags for relative and absolute quantification approach with OFFGEL isoelectric focusing separation and liquid chromatography-matrix assisted laser desorption ionization-tandem mass spectrometry. In three independent experiments, a total of 1,193 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 with the interphase condition. Among these proteins, several COPI coatomer subunits (alpha, beta, gamma, and delta) are of particular interest. Altogether, this systematic quantitative proteomic study revealed candidate proteins of the molecular machinery that control the Golgi disassembly and reassembly processes in the cell cycle.

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 iTRAQ^TM labeling and the subsequent separation and protein identification of Golgi membrane treated with different cytosols. To quantitatively compare proteins associated with Golgi membrane (GM) 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 an OFFGEL apparatus to 8–12 fractions based on the pI of the peptides. Each fraction was then analyzed by reversed phase high pressure liquid chromatography-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). The gray portion in each bar represents the average pI of the peptides in that fraction and the dotted portion represents the S.D. the average pI of each fraction.

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.

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 versus 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 (lane 2), mitotic cytosol (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 three independent experiments were shown. The Golgi enzymes and SNARE proteins were not changed, whereas 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. RSA, rat serum albumin.