Interactions of an Arabidopsis RanBPM homologue with LisH-CTLH domain proteins revealed high conservation of CTLH complexes in eukaryotes

Abstract

BackgroundRanBPM (Ran-binding protein in the microtubule-organizing centre) was originally reported as a centrosome-associated protein in human cells. However, RanBPM protein containing highly conserved SPRY, LisH, CTLH and CRA domains is currently considered as a scaffolding protein with multiple cellular functions. A plant homologue of RanBPM has not yet been characterized.ResultsBased on sequence similarity, we identified a homologue of the human RanBPM in Arabidopsis thaliana. AtRanBPM protein has highly conserved SPRY, LisH, CTLH and CRA domains. Cell fractionation showed that endogenous AtRanBPM or expressed GFP-AtRanBPM are mainly cytoplasmic proteins with only a minor portion detectable in microsomal fractions. AtRanBPM was identified predominantly in the form of soluble cytoplasmic complexes ~230 – 500 kDa in size. Immunopurification of AtRanBPM followed by mass spectrometric analysis identified proteins containing LisH and CRA domains; LisH, CRA, RING-U-box domains and a transducin/WD40 repeats in a complex with AtRanBPM. Homologues of identified proteins are known to be components of the C-terminal to the LisH motif (CTLH) complexes in humans and budding yeast. Microscopic analysis of GFP-AtRanBPM in vivo and immunofluorescence localization of endogenous AtRanBPM protein in cultured cells and seedlings of Arabidopsis showed mainly cytoplasmic and nuclear localization. Absence of colocalization with γ-tubulin was consistent with the biochemical data and suggests another than a centrosomal role of the AtRanBPM protein.ConclusionWe showed that as yet uncharacterized Arabidopsis RanBPM protein physically interacts with LisH-CTLH domain-containing proteins. The newly identified high molecular weight cytoplasmic protein complexes of AtRanBPM showed homology with CTLH types of complexes described in mammals and budding yeast. Although the exact functions of the CTLH complexes in scaffolding of protein degradation, in protein interactions and in signalling from the periphery to the cell centre are not yet fully understood, structural conservation of the complexes across eukaryotes suggests their important biological role.

Elution Profile Analysis of SDS-induced Subcomplexes by Quantitative Mass Spectrometry

“…However, the determination of the EPD-value showed that these proteins can be separated from dynein and lebercilin and therefore can be considered to be a different submodule (EPD CTLH Յ0.101, n ϭ 12, EPD DYN Ն0.111, except for the protein C20orf11 (GID8) EPD DYN ϭ 0.058 and the protein ARMC8 EPD DYN ϭ 0.080). Some proteins of this miscellaneous set have been reported to be part of a complex termed CTLH that is thought to be involved in microtubular dynamics (23).…”

Studies of recombinant TWA1 reveal constitutive dimerization

“…Gid1, Gid4 and Gid5 also function in vacuole-mediated degradation of fructose 1,6-bisphosphatase, a pathway equivalent to metazoan endo-lysosomal protein degradation [14]. RanBPM-binding proteins have been identified in the plant Arabidopsis thaliana , which include orthologues of TWA1/Gid8, MAEA/Gid9, Rmnd5/Gid2 as well as an orthologue of mammalian WDR26, which has domains in common with muskelin and Gid7 [15]. The Rmnd5/Gid2 orthologue of the plant Lotus japonicus has E3 ligase activity in vitro [16].…”

Integrative meta-analysis of protein interaction data identified multiple GID/MRCTLH protein complexes in plants

“…AtRanBPM (AT1G35470) is known to be part of a protein complex of about 500 kDa (Tomaštíková et al, 2012) and immunoprecipitation of AtRamPBM identified different proteins similar to other components of the MRCTLH complex (Francis et al, 2013), suggesting that MRCTLH complexes also exist in Arabidopsis.…”

“…These data opens the possibility that complexes A and B correspond, in fact, to a single, unique, very big complex, bigger than the yeast GID and the mammal MRCTLH complexes. However, it was determined that AtRanBPM (AT1G35470) is located predominantly in the form of soluble cytoplasmic complexes of ~230 -500 kDa in size (Tomaštíková et al, 2012), which may correspond to the size of complex A or the size of complex B, but not to the size of both together. In any case, at the present, we don't know the possible role of AT4G09300.…”

“…Accordingly, the Arabidopsis RanBPM homologue is part of soluble cytoplasmic complexes of about 400 kDa in size, complexes that also include proteins that are homologue to other components of the GID/MRCTLH complex in mammals and yeast (Tomaštíková et al, 2012). The role of the Arabidopsis CTLH complex is unknown.…”