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Two-dimensional Blue Native/SDS Gel Electrophoresis of Multi-Protein Complexes from Whole Cellular Lysates

2003; Elsevier BV; Volume: 3; Issue: 2 Linguagem: Inglês

10.1074/mcp.t300010-mcp200

1535-9484

Margarita M. Camacho-Carvajal, Bernd Wollscheid, Ruedi Aebersold, Viktor Steimle, Wolfgang W. Schamel,

Endoplasmic Reticulum Stress and Disease

Identification and characterization of multi-protein complexes is an important step toward an integrative view of protein-protein interaction networks that determine protein function and cell behavior. The limiting factor for identifying protein complexes is the method for their separation. Blue native PAGE (BN-PAGE) permits a high-resolution separation of multi-protein complexes under native conditions. To date, BN-PAGE has only been applicable to purified material. Here, we show that dialysis permits the analysis of multi-protein complexes of whole cellular lysates by BN-PAGE. We visualized different multi-protein complexes by immunoblotting including forms of the eukaryotic proteasome. Complex dynamics after γ interferon stimulation of cells was studied, and an antibody shift assay was used to detect protein-protein interactions in BN-PAGE. Furthermore, we identified defined protein complexes of various proteins including the tumor suppressor p53 and c-Myc. Finally, we identified multi-protein complexes via mass spectrometry, showing that the method has a wide potential for functional proteomics. Identification and characterization of multi-protein complexes is an important step toward an integrative view of protein-protein interaction networks that determine protein function and cell behavior. The limiting factor for identifying protein complexes is the method for their separation. Blue native PAGE (BN-PAGE) permits a high-resolution separation of multi-protein complexes under native conditions. To date, BN-PAGE has only been applicable to purified material. Here, we show that dialysis permits the analysis of multi-protein complexes of whole cellular lysates by BN-PAGE. We visualized different multi-protein complexes by immunoblotting including forms of the eukaryotic proteasome. Complex dynamics after γ interferon stimulation of cells was studied, and an antibody shift assay was used to detect protein-protein interactions in BN-PAGE. Furthermore, we identified defined protein complexes of various proteins including the tumor suppressor p53 and c-Myc. Finally, we identified multi-protein complexes via mass spectrometry, showing that the method has a wide potential for functional proteomics. In the post-genomic era, there is an increasing need to develop methods that allow the analysis of multi-protein complexes (MPCs). 1The abbreviations used are: MPC, multi-protein complex; BN, blue native; BiP, heavy chain binding protein; Hsp, heat shock protein; IFN-γ, γ interferon; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem MS; WCL, whole-cell lysate; 2D, two-dimensional. 1The abbreviations used are: MPC, multi-protein complex; BN, blue native; BiP, heavy chain binding protein; Hsp, heat shock protein; IFN-γ, γ interferon; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem MS; WCL, whole-cell lysate; 2D, two-dimensional. This would provide an integrative view of the protein-protein interaction networks that define protein function and cell behavior. Methods to analyze the interactions of proteins with each other and to study MPCs in terms of their composition, dynamics, post-translational modifications, size, and abundance of their different subunits are highly desirable. So far, protein interactions in yeast have been determined at large scale using either a two-step affinity purification (1Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Functional organization of the yeast proteome by systematic analysis of protein complexes..Nature. 2002; 415: 141-147Google Scholar), immunoprecipitations (2Ho Y. Gruhler A. Heilbut A. Bader G.D. Moore L. Adams S.L. Millar A. Taylor P. Bennett K. Boutilier K. Yang L. Wolting C. Donaldson I. Schandorff S. Shewnarane J. Vo M. Taggart J. Goudreault M. Muskat B. Alfarano C. Dewar D. Lin Z. Michalickova K. Willems A.R. Sassi H. Nielsen P.A. Rasmussen K.J. Andersen J.R. Johansen L.E. Hansen L.H. Jespersen H. Podtelejnikov A. Nielsen E. Crawford J. Poulsen V. Sorensen B.D. Matthiesen J. Hendrickson R.C. Gleeson F. Pawson T. Moran M.F. Durocher D. Mann M. Hogue C.W. Figeys D. Tyers M. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry..Nature. 2002; 415: 180-183Google Scholar) of each individual protein, or alternatively using comprehensive two-hybrid screens (3Ito T. Tashiro K. Muta S. Ozawa R. Chiba T. Nishizawa M. Yamamoto K. Kuhara S. Sakaki Y. Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins..Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1143-1147Google Scholar, 4Uetz P. Giot L. Cagney G. Mansfield T.A. Judson R.S. Knight J.R. Lockshon D. Narayan V. Srinivasan M. Pochart P. Qureshi-Emili A. Li Y. Godwin B. Conover D. Kalbfleisch T. Vijayadamodar G. Yang M. Johnston M. Fields S. Rothberg J.M. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae..Nature. 2000; 403: 623-627Google Scholar). These approaches allow the detection of individual protein-protein interactions (interactomics), and combination of these datasets have led to the development of a network of protein-protein interactions (5Schwikowski B. Uetz P. Fields S. A network of protein-protein interactions in yeast..Nat. Biotechnol. 2000; 18: 1257-1261Google Scholar, 6von Mering C. Krause R. Snel B. Cornell M. Oliver S.G. Fields S. Bork P. Comparative assessment of large-scale data sets of protein-protein interactions..Nature. 2002; 417: 399-403Google Scholar). The drawback is that these approaches do not study protein complexes directly (complexomics). The properties of MPCs such as their relative abundance and exact composition are in many cases still unknown.Identification and analysis of MPCs requires their separation under native conditions. Native isoelectric focusing is one way to separate protein complexes based on their isoelectric point. The method is limited by the fact that many proteins tend to be insoluble close to their isoelectric point, hydrophobic proteins tend to precipitate at the basic pole, and the matrixes that are currently available (acrylamide or agarose) have a discrete pore size that limits the size of the complexes that can enter the gel ( 5 MDa) is currently unknown. We do not know whether this is a defined complex or whether large p53 aggregates form during the gel run. We observed three complexes for the proto-oncogene c-Myc (Fig. 3). Astonishingly, a large part of the c-Myc protein is present in the form of a discrete complex of at least 1 MDa. The small complex at the diagonal could correspond to c-Myc monomer and/or c-Myc-Max heterodimer. Because many interactions of Myc with other proteins have been described (28Sakamuro D. Prendergast G.C. New Myc-interacting proteins: A second Myc network emerges..Oncogene. 1999; 18: 2942-2954Google Scholar), the approach presented here should be informative to determine the predominant interaction partners of Myc within the cell. Transcription factor 2b and heat shock protein 40 (Hsp40) were found to be monomers or present in very small complexes that migrated within the diagonal (Fig. 3). The chaperone Hsp60 is known to form heptameric ring-like structures of 420 kDa (29Hutchinson E.G. Tichelaar W. Hofhaus G. Weiss H. Leonard K.R. Identification and electron microscopic analysis of a chaperonin oligomer from Neurospora crassa mitochondria..EMBO J. 1989; 8: 1485-1490Google Scholar) that were also detected by our novel method (Fig. 3). Hsp70 and Hsc70 bind to various substrates as well as to cofactors. They do not form identical complexes (Fig. 3), indicating that they might bind to different substrates/cofactors. The heavy chain binding protein (BiP) is a chaperone of the endoplasmic reticulum lumen complexed to distinct substrates (30Gething M.J. Role and regulation of the ER chaperone BiP..Sem. Cell. Dev. Biol. 1999; 10: 465-472Google Scholar). Indeed, most, if not all, BiP was found in different MPCs of similar sizes. The BAP membrane proteins form large MPCs in the mitochondria (BAP32/37) (31Nijtmans L.G.J. de Jong L. Artal Sanz M. Coates P.J. Berden J.A. Willem Back J. Muijsers A.O. van der Spek H. Grivell L.A. Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins..EMBO J. 2000; 19: 2444-2451Google Scholar) and the endoplasmic reticulum (BAP29/31) (32Schamel W.W. Kuppig S. Becker B. Gimborn K. Hauri H.P. Reth M. A high-molecular-weight complex of BAP29/BAP31 is involved in the retention of membrane-bound IgD in the endoplasmic reticulum..Proc. Nat. Acad. Sci. U. S. A. 2003; 100: 9861-9866Google Scholar), respectively. The presence of these complexes was confirmed by our approach (Fig. 3).Fig. 3Identification of MPCs by immunoblotting. HEK293 or J558Lδδm/mb1 (for the BAP proteins) WCLs were separated by 2D BN/SDS-PAGE (5.5–14 and 10%, respectively) and immunoblotted using antibodies against the indicated proteins.View Large Image Figure ViewerDownload (PPT)Identification of MPC Components by MS—It would be highly desirable to use WCL BN/SDS-PAGE to identify and characterize MPCs using a systematic proteomics approach. Silver staining of a 2D BN/SDS gel showed that that quite a number of spots migrate below the diagonal (white dotted lines), indicating their MPC nature (Fig. 4 A). The MPCs disappeared when the WCL was treated with SDS and boiled before loading on the BN gel (Fig. 4B).Fig. 4Visualization of MPCs on a 2D WCL BN/SDS gel and identification of MPC components by MS.A, WCL of HEK293 cells was prepared using Triton X-100 and separated by 2D BN/SDS-PAGE (5.5–17 and 10%, respectively). Corresponding spots