Oligomeric assembly and interactions within human RuvBL1 and RuvBL2 complexes
Résumé
The two closely related eukaryotic AAA+ proteins, RuvBL1 (TIP49) and RuvBL2 (TIP48), are essential components of large multi-protein complexes involved in diverse cellular processes. While the molecular mechanisms of RuvBL1 and RuvBL2 remain unknown, oligomerization is likely to be important for their function together or individually and different oligomeric forms might underpin different functions. Several experimental approaches were used to investigate the molecular architecture of the RuvBL1/2 complex and the role of the ATPase-insert domain (Domain II) for its assembly and stability. Analytical ultracentrifugation showed that RuvBL1 and RuvBL2 were mainly monomeric and each co-existed with small proportions of dimers, trimers and hexamers. Adenine nucleotides induced hexamerisation of RuvBL2 but not RuvBL1. In distinction, the RuvBL1/2 complexes contained single and double hexamers together with smaller forms. The role of the ATPase-insert domain (Domain II) in complex assembly was examined by size exclusion chromatography using deletion mutants of RuvBL1 and RuvBL2. Significantly, catalytically competent dodecameric RuvBL1/2 complexes lacking Domain II in one or both proteins could be assembled but the loss of Domain II in RuvBL1 destabilised the dodecamer. The composition of the RuvBL1/2 complex was analysed by mass spectrometry. Several species of mixed RuvBL1/2 hexamers with different stoichiometries were seen in the spectra of the RuvBL1/2 complex. A number of our results indicate that the architecture of the human RuvBL1/2 complex does not fit the recent structural model of the yeast Rvb1/2 complex.
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