It has been demonstrated that most proteins required for mitosis are contained in protein complexes. In order to understand how mitotic proteins function, we have to define the mitotic protein complexes first.

In this Work Package, we will adapt the iTAP technology for purifying a subset of mitotic protein complexes from human cells. In the stable cell lines or cell pools expressing tagged mouse transgenes, the endogenous human genes will be knocked down by RNAi, whereas the mouse transgenes will not be affected by the specific siRNAs, resulting in effective gene replacement in human cells. The tandem affinity purification tag will allow purification of native protein complexes. These complexes will be further characterized using mass spectrometry technology.

This study is expected to provide the first systematic view of protein interaction networks of genes necessary for mitosis in mammalian cells. The data generated also constitute the basis for analysis of cell-cycle dependent phosphorylation state of these mitotic protein complexes by mass spectrometry.

1) To generate stable human cell lines or cell pools bearing tagged mouse transgenes

2) To define the protein complexes using tandem affinity purification and mass spectrometry

Tony Hyman (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany)

Jan-Michael Peters (Research Institute of Molecular Pathology, Vienna, Austria)

Richard Durbin (Wellcome Trust Sanger Institute, Hinxton, UK)

1) Generation of stable human cell lines bearing tagged mouse genes

Results from MitoCheck labs and others have shown that most mouse genes can functionally replace their human orthologs. On the other hand, sufficient sequence diversity at the mRNA level allows specific and efficient knocking down of the endogenous human genes by RNAi without affecting expression of the mouse genes. This finding led to our decision of using mouse BACs instead of human ones to tag the genes of interest and to establish stable human cell pools expressing tagged mouse transgenes.

TAP tagging has been used successfully in yeast cells. In Drosophila, knocking down the endogenous gene transiently by RNAi has been shown to dramatically increase the yields of precipitated complex by TAP purification. This so-called iTAP technique however does not translate well in human cells. We have tested various tags and have developed a LAP-tag based tandem affinity purification procedure that yields satisfactory results in human cells. In the course of the MitoCheck project, we will generate more than 100 stable human cell pools bearing tagged mouse transgenes of interest.

2) Defining mitotic protein complexes

We are taking two approaches to isolate mitotic protein complexes of interest. First, we use conventional immunoprecipitation techniques for those mitotic proteins with available antibodies. Second, we will pull down proteins that form complexes with the tagged mitotic proteins (bait) by means of tandem affinity purification. Both methods are well established. We shall be able to provide information on the interacting partners of about 150 bait proteins at the end of the MitoCheck project.

- Wapl controls the dynamic association of cohesin with chromatin.
Küng S, Hegemann B, Peters BH, Lipp JJ, Schleiffer A, Mechtler K, Peters JM. Cell. 2006 Dec 1;127(5):955-67. (PMID: 17113138)

- Sororin Is Required for Stable Binding of Cohesin to Chromatin and for Sister Chromatid Cohesion in Interphase.
Schmitz J, Watrin E, Lenart P, Mechtler K, Peters JM. Curr Biol. 2007 Apr 3;17(7):630-6 (PMID: 17349791)

- HPLC techniques for proteomics analysis--a short overview of latest developments.
Mitulovic G, Mechtler K. Brief Funct Genomic Proteomic. 2006 Dec; 5(4):249-60. (PMID: 17124183)

- Cleaning of raw peptide MS/MS spectra: improved protein identification following deconvolution of multiply charged peaks, isotope clusters, and removal of background noise.
Mujezinovic N, Raidl G, Hutchins JRA, Peters JM, Mechtler K, Eisenhaber F. (PMID: 16955515)