Differential proteomic screen to evidence proteins ubiquitinated upon mitotic exit in cell-free extract of Xenopus laevis embryos.
Post-translational modification of proteins via ubiquitination plays a crucial role in numerous vital functions of the cell. Polyubiquitination is one of the key regulatory processes involved in regulation of mitotic progression. Here we describe a differential proteomic screen dedicated to identification of novel proteins ubiquitinated upon mitotic exit in cell-free extract of Xenopus laevis embryo. Mutated recombinant His6-tagged ubiquitin (Ubi (K48R)) was added to mitotic extract from which we purified conjugated proteins, as well as associated proteins in nondenaturing conditions by cobalt affinity chromatography. Proteins eluted from Ubi (K48R) supplemented and control extracts were compared by LC-MS/MS analysis after monodimensional SDS-PAGE. A total of 144 proteins potentially ubiquitinated or associated with them were identified. Forty-one percent of these proteins were shown to be involved in ubiquitination and/or proteasomal degradation pathway confirming the specificity of the screen. Twelve proteins, among them ubiquitin itself, were shown to carry a "GG" or "LRGG" remnant tag indicating their direct ubiquitination. Interestingly, sequence analysis of ubiquitinated substrates carrying these tags indicated that in Xenopus cell-free embryo extract supplemented with Ubi (K48R) the majority of polyubiquitination occurred through lysine-11 specific ubiquitin chain polymerization. The potential interest in this atypical form of ubiquitination as well as usefulness of our method in analyzing atypical polyubiquitin species is discussed.
Bazile
Franck
F
CNRS UMR 6061, Institute of Genetics & Development, University of Rennes 1, Mitosis & Meiosis Group, IFR 140 GFAS, 35 043 Rennes Cedex, France.
Gagné
Jean-Philippe
JP
Mercier
Geneviève
G
Lo
Ken Sin
KS
Pascal
Aude
A
Vasilescu
Julian
J
Figeys
Daniel
D
Poirier
Guy G
GG
Kubiak
Jacek Z
JZ
Chesnel
Franck
F
D000818:Animals; D002474:Cell-Free System; D004625:Embryo, Nonmammalian / Q000201:enzymology; D005260:Female; D006639:Histidine / Q000378:metabolism; D008938:Mitosis / Q000502:physiology*; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination; D029867:Xenopus Proteins / Q000378:metabolism*; D014982:Xenopus laevis
D020543:Proteome; D011994:Recombinant Proteins; D025801:Ubiquitin; D029867:Xenopus Proteins; D006639:Histidine