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Open Access Research article

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

Eva Tomaštíková1, Věra Cenklová2, Lucie Kohoutová3, Beáta Petrovská1, Lenka Váchová2, Petr Halada3, Gabriela Kočárová3 and Pavla Binarová3*

Author Affiliations

1 Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany AS CR, v.v.i., Sokolovská 6, Olomouc, 772 00, Czech Republic

2 Institute of Experimental Botany, AS CR, v.v.i., Sokolovská 6, 772 00, Olomouc, Czech Republic

3 Institute of Microbiology, AS CR, v.v.i., Vídeňská 1083, 142 20, Prague 4, Czech Republic

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BMC Plant Biology 2012, 12:83  doi:10.1186/1471-2229-12-83

Published: 7 June 2012

Abstract

Background

RanBPM (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.

Results

Based 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.

Conclusion

We 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.

Keywords:
Arabidopsis homologue of RanBPM; CTLH-complex; LisH-CTLH domain proteins