| Abstract: | Ran-binding protein in microtubule organising centre (RanBPM) was originally isolated as a protein that binds to the small GTPase Ran. Recently our group and other groups reported that RanBPM was associated with several proteins and composed a large protein complex. Here, we used tandem MS with an antibody against RanBPM to purify this complex from a soluble extract of HEK293 cells: we identified Muskelin, p48EMLP, p44CTLH, and the novel armadillo-repeat proteins ARMC8alpha and ARMC8beta as components. In RanBPM, Muskelin, p48EMLP, and p44CTLH we found LisH/CTLH motifs, which are present in proteins involved in microtubule dynamics, cell migration, nucleokinesis, and chromosome segregation. We renamed the 20S large protein complex the CTLH complex. The N-terminal 364 amino acids of ARMC8alpha and ARMC8beta were completely conserved, suggesting that these proteins are probably alternatively spliced products from the same gene. We confirmed the in vivo association of each component by co-immunoprecipitation assays with Cos-7 cells in which these components were exogenously overexpressed. A pull-down assay with bacterially-expressed Twa1 revealed binding of each in vitro-translated component to Twa1. Finally, we confirmed the cellular localization of these proteins. Taken together, our results reveal that RanBPM, ARMC8alpha, ARMC8beta, Muskelin, p48EMLP, and p44CTLH form complexes in cells. |
