BACKGROUND
ras-related nuclear proteins  are abundant small gtpases, associated with ran-specific nuclear gefs , cytoplasmic gaps , and with ranbp <dig>  that stimulate rangtp hydrolysis in the cytoplasm  <cit> .
the arabidopsis genome contains three genes encoding atran  <cit> , two genes encoding atrangap related proteins  <cit>  and three genes for ranbp <dig> isoforms – ranbp1a, ranbp1b and ranbp1c  <cit> .
plant ran binding proteins  display significant homology with yeast and mammalian ranbps, but there is little evidence for their biological function  <cit> .
one ranbp in animal cells, ranbpm , was identified in a yeast two-hybrid screen with ran as a bait.
ranbpm comprises four domains – spry, lish, ctlh and cra and is homologous to the human ranbp <dig> protein  <cit> .
although ranbpm and ranbp <dig> have been shown to bind the ran protein, they do not contain a consensus ran-binding sequence  <cit> .
ranbpm was defined as a member of the scorpin family of proteins  with a unique domain organization  <cit> .
as reviewed in suresh et al.
<cit> , numerous protein interactions described for the ranbpm protein suggest its multiple roles in the regulation of protein stability, cell cycle regulation and other as yet undefined cellular processes.
ranbpm was reported to be a part of the large ctlh  complexes  <cit> .
ctlh complexes composed of lish, ctlh and cra domain containing proteins, transducin/wd <dig> repeat proteins, and armadillo repeat proteins have been found in mammals and yeast  <cit> .
mammalian and yeast ctlh complexes are structurally conserved but their biological function is still not fully understood.
in yeast, the ctlh complex of gid/vid proteins plays a role in vacuole and proteasome-dependent fructose- <dig> -bisphosphatase degradation  <cit> .
similarly, it has been suggested that ctlh complexes partake in lysosome and proteasome-dependent proteolysis in mammalian cells  <cit> .
data on proteins with spry, lish, ctlh or cra domain-containing proteins in plants are limited.
in pinus radiata, the spry domain containing protein, sepr <dig>  is the homologue of a trithorax-group member and is involved in plant reproduction and development  <cit> .
in oryza sativa, the lish domain-containing protein, oslis-l <dig> is required for male gametophyte formation and the first internode elongation  <cit> .
here we provide data on an arabidopsis homologue of ranbpm that belongs to the uncharacterized family of plant spry, lish, ctlh and cra domain-containing proteins.
we used in silico analysis, biochemical, proteomic and microscopic analyses in vivo and in situ to characterize atranbpm.
we found that the atranbpm protein is present predominantly in the form of large cytoplasmic protein complexes that are structurally homologous to the ctlh type of complexes described in mammals and budding yeast.
RESULTS
the arabidopsis homologue of ranbpm is a spry-domain containing protein
by homology search of the arabidopsis thaliana genome, we found a spry-domain containing protein atranbpm , which is a homologue of the human ranbpm  protein.
the atranbpm cdna contains a single open reading frame and consists of  <dig> amino acids.
atranbpm is a member of the hom <dig> gene family that comprises  <dig> genes in  <dig> plant species, particularly from the ortho <dig> subfamily.
in arabidopsis there are three paralogues of atranbpm  and one gene originating from the segmental duplication of chromosome  <dig>  .
the products of these genes are described as spry domain-containing proteins but their biological roles in plants have not yet been identified.
the atranbpm protein is composed of four domains – spry, lish, ctlh and cra .
spry, lish, ctlh and cra domain-containing proteins are widely spread across eukaryotes.
the protein encoded by atranbpm is annotated as a protein of unknown function .
as shown in figure 1b, atranbpm has close homologues in other plant species such as ricinus communis , vitis vinifera , populus trichocarpa , sorghum bicolor , zea mays  and oryza sativa japonica group  .
the arabidopsis ranbpm shows a close identity and similarity to its spry, lish, ctlh and cra domains with mammalian ranbpm and ranbp <dig>  .
however, there is an insertion of about  <dig> amino acids between the ctlh and cra domains in human proteins .
a phylogenetic analysis performed using mega <dig> software  revealed that arabidopsis ranbpm grouped with homologues from other plant species.
the metazoan ranbpm homologues grouped in a separated clade.
the saccharomyces homologue, gid1/vid <dig>  appears to be ancestral to the plant and metazoan clades .
spry, lish, ctlh and cra conserved domains are mainly involved in protein-protein interactions.
the spry domain  was originally identified as a structural motif in ryanodine receptors  <cit> .
proteins with spry domains are involved in intracellular calcium release, as is the case for ryanodine receptors, in rna metabolism and in regulatory and developmental processes  <cit> .
the lish domain  functions as a stable homodimerization and dimerization motif and its contribution to the dynamics of microtubules has been suggested  <cit> .
located adjacent to the c-terminus of the lish domain is a c-terminal to the lish motif  that has a putative α-helical structure with an as yet undescribed function.
the cra domain  was found to be a motif in the c-terminal part of ranbpm and ranbp <dig> and represents a protein-protein interaction domain often found in ran-binding proteins  <cit> .
putative interaction sites of atranbpm were analysed using elm  and sumosp  <dig>  databases  .
there are common motifs in the atranbpm amino acid sequence, such as phosphorylation consensus sites for phosphoinositide-3-oh kinase related kinases, a protein kinase a phosphorylation site, a mitogen-activated protein kinase phosphorylation site and docking motif, and a putative cyclin recognition site.
further, the atranbpm amino acid sequence possesses forkhead-associated phosphopeptide binding domains and a y-based sorting signal which might act in endosomal and secretory pathways.
protein expression levels, cellular distribution and molecular forms of atranbpm
the atranbpm coding sequence  was cloned into gateway-compatible plant binary vectors for n-terminal gfp and c-terminal gfp fusion.
arabidopsis cell suspension cultures were transformed according to mathur et al.
<cit> .
transformed arabidopsis plants were obtained through the floral-dip method  <cit> .
an antibody against a peptide from the c-terminal part of the atranbpm molecule was produced in rabbits and affinity purified using the immunogenic peptide.
when cell lines expressing gfp-atranbpm were analysed by western blotting of separated arabidopsis extracts, the antibody recognized a band of 52 kda, the predicted molecular mass  of atranbpm, and a band corresponding to the mw of gfp-atranbpm .
western blot further showed that the protein levels of atranbpm were much lower in arabidopsis seedlings than cell cultures.
in cultured arabidopsis cells, there was no significant difference in atranbpm protein levels between dividing cells and stationary growing non-dividing cells .
similarly, there was no difference in atranbpm protein levels between four, eight and 15 day old arabidopsis seedlings.
our data correspond to those on atranbpm expression found in the publically available affymetrix expression databases, the arabidopsis efp browser  <cit> , and the genevestigator database  <cit>  where atranbpm has a constant level of expression in analysed tissues as well as during seedling development.
the cellular distribution of atranbpm was analysed using differential centrifugation.
as shown in figure 3c, the majority of the protein was cytoplasmic and only small amounts sedimented either in low speed pellets or in the microsomal pellet .
the fact that atranbpm was also sedimented from a high-speed cytoplasmic s <dig> extract to pellet p <dig> indicated that the protein might be present in the form of higher mw complexes.
the separation of s <dig> high speed extract by native polyacrylamide gel electrophoresis  confirmed cell fractionation data and suggested that atranbpm is a component of protein complexes with a molecular mass of around ~ <dig> – 500 kda .
further, we immunopurified gfp-atranbpm using the gfp trap.
separation of purified gfp-atranbpm by native page followed by western blotting showed the presence of complexes of similar size as observed when high-speed supernatant s <dig> was analysed .
altogether these data showed that atranbpm is present predominantly in the form of large cytoplasmic complexes.
analysis of atranbpm protein complexes by mass spectrometry
to analyse putative interactors of atranbpm in the complexes, gfp-atranbpm and copurified proteins were separated by sds-page followed by silver staining, and specific bands were excised and analysed by maldi-ms .
gfp immunopurification performed in extracts from wild type cell was used as a negative control.
neither atranbpm protein nor bands corresponding to the mw of proteins copurified with gfp-atranbpm were observed in the negative controls and only background contamination such as hsps and cytoskeletal proteins were detected by maldi-ms .
we found that proteins reproducibly copurifying with atranbpm belong to ctlh-domain containing proteins including lish , cra and u-box , and to the transducin/wd- <dig> domain-containing proteins at5g <dig>  and at5g <dig> .
alternatively to maldi-ms, we used lc-maldi-ms/ms analysis of eluates from the gfp trap to confirm the interaction of gfp-atranbpm with the proteins mentioned above.
proteins  copurified with atranbpm protein were identified by both maldi-ms and lc-maldi-ms/ms analysis.
the values before and after the slash in the columns “no.
peptides” and “sequence coverage” refer to maldi-ms and lc-maldi-ms/ms data, respectively.
homologues of human ctlh-complex members reported by kobayashi et al.,  <cit>  , proteins in the complex described by umeda et al.
<cit>  , yeast homologues in the gid complex published by regelmann et al.
<cit>  .
database search indicated that the proteins copurified with atranbpm were homologous to protein components of the human ctlh complex  <cit> .
the ctlh complex is annotated in corum  as a complex with a putative function in regulating cell migration, nucleokinesis, chromosome segregation and microtubule dynamics.
components of the ctlh complex are conserved amongst animal species and their presence is predicted for other eukaryotes such as plants and fungi.
a relatively lower degree of homology with individual mammalian and plant members of ctlh complexes  is due to the fact that part of the molecule separating highly conserved domains contains insertions and deletions of various lengths, differing among plant, mammalian and/or yeast homologues.
the lc-maldi-ms/ms analysis also identified among proteins copurified with atranbpm armadillo-repeat-containing protein  .
however, the protein was not proven to be a homologue of the human armadillo repeat armc <dig>  a member of the ctlh complex  <cit> .
further, yippee family proteins  and yippee-like protein  were copurified with atranbpm and identified by lc-maldi-ms/ms .
yippee-like proteins belong to the ypel family of proteins whose members have been described as interactors of the human ranbpm  <cit> .
proteins at5g <dig>  at5g <dig>  at3g <dig>  and at3g <dig>  copurified with atranbpm were predicted to interact within a functional network in the aranet database  .
together these data suggest that plant atranbpm protein, like its yeast and mammalian homologues, is a component of a cytoplasmic multiprotein complex where it interacts with lish-ctlh domain-containing proteins and armadillo-repeat-containing proteins.
in vivo localization of gfp-atranbpm and atranbpm immunofluorescence labelling in arabidopsis cultured cells and seedlings
in cultured cells of arabidopsis, the gfp-atranbpm signal was localized in the cytoplasm and in the perinuclear area of interphase cells, with a weaker signal detected in nuclei .
we observed no gain-of-function phenotype of arabidopsis seedlings expressing gfp-atranbpm from gateway vectors.
similarly to cultured cells, the gfp-atranbpm signal was localized in the cytoplasm, in nuclei and accumulated in the vicinity of nuclei in dividing zone of roots .
in differentiated root cells, gfp-atranbpm was cytoplasmic and in the perinuclear area .
c-terminal gfp fusions showed a similar localization pattern when expressed transiently in cultured cells of arabidopsis .
immunofluorescence analyses showed that the atranbpm protein was distributed patchily in the cytoplasm and nuclei; in non-dividing cells it accumulated in the perinuclear region and in dividing cells, the signal was slightly enriched in the area of the mitotic spindle and the phragmoplast .
further we compared localization of the endogenous protein and gfp-atranbpm by double labelling with anti-atranbpm and anti-gfp antibodies.
we confirmed a similar localization pattern for atranbpm and expressed atranbpm with a cytoplasmic signal slightly enriched in the perinuclear area and a weaker nuclear signal .
to determine whether the atranbpm protein plays a role in microtubule organization like the truncated version of human ranbpm  <cit> , we studied the colocalization of atranbpm with the centrosomal protein, γ-tubulin.
in acentrosomal plant cells, γ-tubulin is present with dispersed sites of microtubule nucleation in the cytoplasm, with nuclei, and on membranes and microtubules  <cit> .
we found that the arabidopsis homologue atranbpm did not colocalize with γ-tubulin-positive sites either present with the nuclear envelope or on microtubular arrays .
as a portion of ranbpm was reported to be associated with the ran protein in mammalian cells  <cit>  we performed double immunofluorescence labelling of atranbpm and ran protein.
in cultured cells of arabidopsis, ran was localized in the cytoplasm and in nuclei but only a small fraction of atranbpm colocalized with nuclear ran.
discussion
ranbpm protein was first characterized in human cells as a centrosomal protein involved in microtubule nucleation which colocalized with γ-tubulin at centrosomes and at ectopic nucleation sites  <cit> .
the data on ranbpm initiated an investigation of the role of the rangtpase pathway and its role in chromatin-mediated microtubule nucleation and spindle assembly  <cit> .
however, later it was found that antibody against the 55 kda form of ranbpm that was characterized by nakamura et al.
<cit>  did not recognize the full length 90 kda ranbpm protein.
the truncated form of ranbpm  was shown to be an incorrectly translated product of the ranbpm gene and moreover, only the truncated version but not the whole ranbpm molecule was active in microtubule nucleation  <cit> .
the complete ranbpm molecule thus does not show the same properties as the truncated version and it was depicted as a scaffold protein that links and modulates interactions between cell surface receptors and their intracellular signalling pathways  <cit> .
the molecular mass of plant atranbpm  corresponds to that of the truncated ranbpm rather than to the whole human ranbpm molecule.
however, the reason for the apparent discrepancy in size of the full length human and plant ranbpm molecules is an insertion of  <dig> amino acids between ctlh and cra domains in the plant protein and the presence of a long stretch of proline and glutamine residues on the n-terminal part of human ranbpm molecule.
we found that atranbpm maintained the properties of the full length ranbpm of mammals:  while the truncated version of human ranbpm is present in centrosomal and ectopic microtubule nucleation sites  <cit> , atranbpm protein in acentrosomal plant cells did not colocalize with microtubule nucleation sites positive for γ-tubulin;  proteins of the γ-tubulin nucleating machinery were not identified by maldi-ms or lc-maldi-ms/ms among proteins interacting with atranbpm, nor were they found in coexpression databases and a biological role of atranbpm other than in processes relating to centrosomes and microtubules was thus suggested;  the presence of highly conserved spry, lish, ctlh and cra domains in atranbpm indicated its function in mediating multiple protein interactions that were described for the whole molecule of human ranbpm  <cit> .
atranbpm protein was not specifically enriched with microtubules in dividing cells or localized in putative microtubule nucleation sites.
subcellular localization of atranbpm corresponded to published data on the subcellular localization of the whole ranbpm molecule in mammalian cells  <cit> .
since a weak nuclear signal, observed for human ranbpm was suggested to be a consequence of over-expression of its tagged version  <cit> , we analysed by immunofluorescence the nuclear localization for endogenous atranbpm protein.
a smaller portion of atranbpm was present in nuclei and the possibility that the nuclear signal observed for gfp-atranbpm or atranbpm-gfp resulted from over expression of gfp fusions was thus excluded.
we observed only partial colocalization of ran and atranbpm in nuclei.
it would be interesting to address the question whether transport of the atranbpm complex between nucleus, perinuclear area and cytoplasm might be regulated by a weak interaction between atranbpm and ran as it was suggested for mammalian ranbpm  <cit> .
heterogeneity in the localization of mammalian ranbpm might depend on interacting proteins  <cit> .
conserved domains of ranbpm protein are responsible for interactions and complex formations with a physiologically divergent group of proteins.
as reviewed by suresh et al.
<cit> , ranbpm is a modulator/protein stabilizer, a regulator of transcriptional activity, and has cell cycle and neurological functions.
ranbpm interacts with a wide range of receptors  <cit> , acts as a scaffolding protein  <cit> , and is involved in signal transduction pathways  <cit> , and the apoptotic pathways  <cit> .
plant homologues of ranbpm belong to genes with unknown functions.
we found that the product of atranbpm is predominantly a cytoplasmic protein that is a part of protein complexes with a molecular mass of approximately  <dig> – 500 kda.
a large protein complex of ranbpm was described by nishitani et al.
<cit>  and ideguchi et al.
<cit> .
later the complex was designated as a ctlh complex  <cit> .
five from eleven ctlh domain-containing proteins that were identified in databases were described as being a part of the ctlh complex and an interaction within the complex via lish-ctlh domains was suggested  <cit> .
in saccharomyces cerevisiae all four ctlh domain-containing proteins present in the genome are part of the ctlh-like complex gid.
the gid complex is suggested to be involved in proteasome-dependent glucose-induced degradation of fructose- <dig> -bisphosphatase  <cit> .
there are twenty one ctlh-domain containing proteins encoded in the arabidopsis genome and of these, six proteins were identified in our experiments to form ctlh-like complexes with atranbpm protein.
though we identified the same spectrum of atranbpm interacting proteins in several independent experiments it cannot be ruled out that the members of ctlh complexes exist and remain to be identified.
we found that the antibody raised against a peptide from the atranbpm sequence did not work in immunopurification experiments.
the immunopurifications performed with a panel of antibodies against the atranbpm protein might help to disclose the presence of other putative members of plant ctlh complexes.
alternatively, immunopurification of the ctlh complex or pull down experiments via proteins that copurified with atranbpm might extend our knowledge of the composition and protein interactions of newly identified ctlh complexes.
all the members of the ctlh complexes that we identified belong to yet uncharacterized plant lish-ctlh domain-containing proteins.
protein at1g <dig> is a homologue of the human twa <dig> protein found in yeast by two-hybrid analysis to interact with ranbpm  <cit> .
lish, cra, and ring/u-box domain-containing proteins of unknown function , copurified with atranbpm, are homologous to human maea and rmd <dig>  respectively, that are members of the ctlh complex annotated by kobayashi et al.
<cit> .
similarly, lish and ctlh domain-containing proteins such as ranbp <dig> or the wd repeat domain  <dig> , were suggested to be putative members of the ctlh complex  <cit> .
we found that uncharacterized arabidopsis transducin/wd- <dig> domain-containing proteins at5g <dig> and at5g <dig> which copurified with atranbpm, are homologues of human wdr <dig> protein and thus belong to components of the plant ctlh complex.
corresponding to our microscopic data on cytoplasmic and nuclear localizations of atranbpm, the members of the human ctlh complex, proteins ranbpm, rmd <dig>  wdr <dig>  twa <dig>  and maea, were shown to have predominantly cytoplasmic and a weaker nuclear localization  <cit> .
a component of the human ctlh complex, the armadillo repeat-containing protein armc <dig>  when exogenously expressed, upregulates the proteolytic degradation of ectopically expressed α-catenin, and thus was suggested to play an important role in the ubiquitin-independent and proteasome-dependent degradation of α-catenin  <cit> .
α-catenin is known to function as a link protein between cadherins and actin-containing filaments of the cytoskeleton  <cit> ; thus a complex might regulate molecular adhesion.
we identified armadillo-repeat-containing protein  among proteins copurified with atranbpm but its homology with armadillo repeat armc <dig>  a member of the human ctlh complex, was not proven.
however, we found that expression of copurified protein at4g <dig>  a homologue of rmd <dig> protein, correlated strongly with expression of the armadillo repeat-containing protein at3g <dig>  one of the homologues of armc <dig>  and with expression of the plant adhesion molecule rabgap/tbc domain-containing protein at3g <dig> .
it is tempting to speculate whether similar function in connecting external signals in adhesion with the cell centre analogous to the cadherin and catenin pathway in animals might exist for plant ctlh complexes.
however, further experimental data are needed to prove this hypothetical function of the ctlh complexes in plants.
besides copurification with ctlh complex proteins, we found that plant atranbpm interacts with yippee proteins  and yippee-like protein .
it was shown in human cells that ranbpm binds members of ypel  family of proteins involved in a cell division-related functions  <cit> .
our data showed that binding with yippee proteins might present one of the atranbpm multiple protein interactions in plant cells too.
CONCLUSIONS
ranbpm protein was shown to have numerous apparently functionally unrelated protein interactors, including membrane receptors, cytoplasmic and nuclear proteins  <cit> .
this suggests its role in a variety of cellular processes.
our biochemical and proteomic studies showed that atranbpm is a component of plant homologues of ctlh complexes.
the fact that ctlh complexes are present in mammals, yeast and in plants suggests their structural conservation in evolution.
ctlh complexes thus might represent a module with important but not fully understood biological functions.

 ras-related nuclear proteins are abundant small gtpases, associated with ran-specific nuclear gefs, cytoplasmic gaps, and with ranbp that stimulate rangtp hydrolysis in the cytoplasm. the arabidopsis genome contains three genes encoding atran, two genes encoding atrangap related proteins and three genes for ranbp isoforms – ranbp1a, ranbp1b and ranbp1c. plant ran binding proteins display significant homology with yeast and mammalian ranbps, but there is little evidence for their biological function. one ranbp in animal cells, ranbpm, was identified in a yeast two-hybrid screen with ran as a bait. ranbpm comprises four domains – spry, lish, ctlh and cra and is homologous to the human ranbp protein. although ranbpm and ranbp have been shown to bind the ran protein, they do not contain a consensus ran-binding sequence. ranbpm was defined as a member of the scorpin family of proteins with a unique domain organization. as reviewed in suresh et al., numerous protein interactions described for the ranbpm protein suggest its multiple roles in the regulation of protein stability, cell cycle regulation and other as yet undefined cellular processes. ranbpm was reported to be a part of the large ctlh complexes. ctlh complexes composed of lish, ctlh and cra domain containing proteins, transducin/wd repeat proteins, and armadillo repeat proteins have been found in mammals and yeast. mammalian and yeast ctlh complexes are structurally conserved but their biological function is still not fully understood. in yeast, the ctlh complex of gid/vid proteins plays a role in vacuole and proteasome-dependent fructose- -bisphosphatase degradation. similarly, it has been suggested that ctlh complexes partake in lysosome and proteasome-dependent proteolysis in mammalian cells. data on proteins with spry, lish, ctlh or cra domain-containing proteins in plants are limited. in pinus radiata, the spry domain containing protein, sepr is the homologue of a trithorax-group member and is involved in plant reproduction and development. in oryza sativa, the lish domain-containing protein, oslis-l is required for male gametophyte formation and the first internode elongation. here we provide data on an arabidopsis homologue of ranbpm that belongs to the uncharacterized family of plant spry, lish, ctlh and cra domain-containing proteins. we used in silico analysis, biochemical, proteomic and microscopic analyses in vivo and in situ to characterize atranbpm. we found that the atranbpm protein is present predominantly in the form of large cytoplasmic protein complexes that are structurally homologous to the ctlh type of complexes described in mammals and budding yeast.  the arabidopsis homologue of ranbpm is a spry-domain containing protein by homology search of the arabidopsis thaliana genome, we found a spry-domain containing protein atranbpm, which is a homologue of the human ranbpm protein. the atranbpm cdna contains a single open reading frame and consists of amino acids. atranbpm is a member of the hom gene family that comprises genes in plant species, particularly from the ortho subfamily. in arabidopsis there are three paralogues of atranbpm and one gene originating from the segmental duplication of chromosome. the products of these genes are described as spry domain-containing proteins but their biological roles in plants have not yet been identified. the atranbpm protein is composed of four domains – spry, lish, ctlh and cra. spry, lish, ctlh and cra domain-containing proteins are widely spread across eukaryotes. the protein encoded by atranbpm is annotated as a protein of unknown function. as shown in figure 1b, atranbpm has close homologues in other plant species such as ricinus communis, vitis vinifera, populus trichocarpa, sorghum bicolor, zea mays and oryza sativa japonica group. the arabidopsis ranbpm shows a close identity and similarity to its spry, lish, ctlh and cra domains with mammalian ranbpm and ranbp. however, there is an insertion of about amino acids between the ctlh and cra domains in human proteins. a phylogenetic analysis performed using mega software revealed that arabidopsis ranbpm grouped with homologues from other plant species. the metazoan ranbpm homologues grouped in a separated clade. the saccharomyces homologue, gid1/vid appears to be ancestral to the plant and metazoan clades. spry, lish, ctlh and cra conserved domains are mainly involved in protein-protein interactions. the spry domain was originally identified as a structural motif in ryanodine receptors. proteins with spry domains are involved in intracellular calcium release, as is the case for ryanodine receptors, in rna metabolism and in regulatory and developmental processes. the lish domain functions as a stable homodimerization and dimerization motif and its contribution to the dynamics of microtubules has been suggested. located adjacent to the c-terminus of the lish domain is a c-terminal to the lish motif that has a putative α-helical structure with an as yet undescribed function. the cra domain was found to be a motif in the c-terminal part of ranbpm and ranbp and represents a protein-protein interaction domain often found in ran-binding proteins. putative interaction sites of atranbpm were analysed using elm and sumosp databases. there are common motifs in the atranbpm amino acid sequence, such as phosphorylation consensus sites for phosphoinositide-3-oh kinase related kinases, a protein kinase a phosphorylation site, a mitogen-activated protein kinase phosphorylation site and docking motif, and a putative cyclin recognition site. further, the atranbpm amino acid sequence possesses forkhead-associated phosphopeptide binding domains and a y-based sorting signal which might act in endosomal and secretory pathways. protein expression levels, cellular distribution and molecular forms of atranbpm the atranbpm coding sequence was cloned into gateway-compatible plant binary vectors for n-terminal gfp and c-terminal gfp fusion. arabidopsis cell suspension cultures were transformed according to mathur et al.. transformed arabidopsis plants were obtained through the floral-dip method. an antibody against a peptide from the c-terminal part of the atranbpm molecule was produced in rabbits and affinity purified using the immunogenic peptide. when cell lines expressing gfp-atranbpm were analysed by western blotting of separated arabidopsis extracts, the antibody recognized a band of 52 kda, the predicted molecular mass of atranbpm, and a band corresponding to the mw of gfp-atranbpm. western blot further showed that the protein levels of atranbpm were much lower in arabidopsis seedlings than cell cultures. in cultured arabidopsis cells, there was no significant difference in atranbpm protein levels between dividing cells and stationary growing non-dividing cells. similarly, there was no difference in atranbpm protein levels between four, eight and 15 day old arabidopsis seedlings. our data correspond to those on atranbpm expression found in the publically available affymetrix expression databases, the arabidopsis efp browser, and the genevestigator database where atranbpm has a constant level of expression in analysed tissues as well as during seedling development. the cellular distribution of atranbpm was analysed using differential centrifugation. as shown in figure 3c, the majority of the protein was cytoplasmic and only small amounts sedimented either in low speed pellets or in the microsomal pellet. the fact that atranbpm was also sedimented from a high-speed cytoplasmic s extract to pellet p indicated that the protein might be present in the form of higher mw complexes. the separation of s high speed extract by native polyacrylamide gel electrophoresis confirmed cell fractionation data and suggested that atranbpm is a component of protein complexes with a molecular mass of around ~ – 500 kda. further, we immunopurified gfp-atranbpm using the gfp trap. separation of purified gfp-atranbpm by native page followed by western blotting showed the presence of complexes of similar size as observed when high-speed supernatant s was analysed. altogether these data showed that atranbpm is present predominantly in the form of large cytoplasmic complexes. analysis of atranbpm protein complexes by mass spectrometry to analyse putative interactors of atranbpm in the complexes, gfp-atranbpm and copurified proteins were separated by sds-page followed by silver staining, and specific bands were excised and analysed by maldi-ms. gfp immunopurification performed in extracts from wild type cell was used as a negative control. neither atranbpm protein nor bands corresponding to the mw of proteins copurified with gfp-atranbpm were observed in the negative controls and only background contamination such as hsps and cytoskeletal proteins were detected by maldi-ms. we found that proteins reproducibly copurifying with atranbpm belong to ctlh-domain containing proteins including lish, cra and u-box, and to the transducin/wd- domain-containing proteins at5g and at5g. alternatively to maldi-ms, we used lc-maldi-ms/ms analysis of eluates from the gfp trap to confirm the interaction of gfp-atranbpm with the proteins mentioned above. proteins copurified with atranbpm protein were identified by both maldi-ms and lc-maldi-ms/ms analysis. the values before and after the slash in the columns “no. peptides” and “sequence coverage” refer to maldi-ms and lc-maldi-ms/ms data, respectively. homologues of human ctlh-complex members reported by kobayashi et al.,, proteins in the complex described by umeda et al., yeast homologues in the gid complex published by regelmann et al.. database search indicated that the proteins copurified with atranbpm were homologous to protein components of the human ctlh complex. the ctlh complex is annotated in corum as a complex with a putative function in regulating cell migration, nucleokinesis, chromosome segregation and microtubule dynamics. components of the ctlh complex are conserved amongst animal species and their presence is predicted for other eukaryotes such as plants and fungi. a relatively lower degree of homology with individual mammalian and plant members of ctlh complexes is due to the fact that part of the molecule separating highly conserved domains contains insertions and deletions of various lengths, differing among plant, mammalian and/or yeast homologues. the lc-maldi-ms/ms analysis also identified among proteins copurified with atranbpm armadillo-repeat-containing protein. however, the protein was not proven to be a homologue of the human armadillo repeat armc a member of the ctlh complex. further, yippee family proteins and yippee-like protein were copurified with atranbpm and identified by lc-maldi-ms/ms. yippee-like proteins belong to the ypel family of proteins whose members have been described as interactors of the human ranbpm. proteins at5g at5g at3g and at3g copurified with atranbpm were predicted to interact within a functional network in the aranet database. together these data suggest that plant atranbpm protein, like its yeast and mammalian homologues, is a component of a cytoplasmic multiprotein complex where it interacts with lish-ctlh domain-containing proteins and armadillo-repeat-containing proteins. in vivo localization of gfp-atranbpm and atranbpm immunofluorescence labelling in arabidopsis cultured cells and seedlings in cultured cells of arabidopsis, the gfp-atranbpm signal was localized in the cytoplasm and in the perinuclear area of interphase cells, with a weaker signal detected in nuclei. we observed no gain-of-function phenotype of arabidopsis seedlings expressing gfp-atranbpm from gateway vectors. similarly to cultured cells, the gfp-atranbpm signal was localized in the cytoplasm, in nuclei and accumulated in the vicinity of nuclei in dividing zone of roots. in differentiated root cells, gfp-atranbpm was cytoplasmic and in the perinuclear area. c-terminal gfp fusions showed a similar localization pattern when expressed transiently in cultured cells of arabidopsis. immunofluorescence analyses showed that the atranbpm protein was distributed patchily in the cytoplasm and nuclei; in non-dividing cells it accumulated in the perinuclear region and in dividing cells, the signal was slightly enriched in the area of the mitotic spindle and the phragmoplast. further we compared localization of the endogenous protein and gfp-atranbpm by double labelling with anti-atranbpm and anti-gfp antibodies. we confirmed a similar localization pattern for atranbpm and expressed atranbpm with a cytoplasmic signal slightly enriched in the perinuclear area and a weaker nuclear signal. to determine whether the atranbpm protein plays a role in microtubule organization like the truncated version of human ranbpm, we studied the colocalization of atranbpm with the centrosomal protein, γ-tubulin. in acentrosomal plant cells, γ-tubulin is present with dispersed sites of microtubule nucleation in the cytoplasm, with nuclei, and on membranes and microtubules. we found that the arabidopsis homologue atranbpm did not colocalize with γ-tubulin-positive sites either present with the nuclear envelope or on microtubular arrays. as a portion of ranbpm was reported to be associated with the ran protein in mammalian cells we performed double immunofluorescence labelling of atranbpm and ran protein. in cultured cells of arabidopsis, ran was localized in the cytoplasm and in nuclei but only a small fraction of atranbpm colocalized with nuclear ran. discussion ranbpm protein was first characterized in human cells as a centrosomal protein involved in microtubule nucleation which colocalized with γ-tubulin at centrosomes and at ectopic nucleation sites. the data on ranbpm initiated an investigation of the role of the rangtpase pathway and its role in chromatin-mediated microtubule nucleation and spindle assembly. however, later it was found that antibody against the 55 kda form of ranbpm that was characterized by nakamura et al. did not recognize the full length 90 kda ranbpm protein. the truncated form of ranbpm was shown to be an incorrectly translated product of the ranbpm gene and moreover, only the truncated version but not the whole ranbpm molecule was active in microtubule nucleation. the complete ranbpm molecule thus does not show the same properties as the truncated version and it was depicted as a scaffold protein that links and modulates interactions between cell surface receptors and their intracellular signalling pathways. the molecular mass of plant atranbpm corresponds to that of the truncated ranbpm rather than to the whole human ranbpm molecule. however, the reason for the apparent discrepancy in size of the full length human and plant ranbpm molecules is an insertion of amino acids between ctlh and cra domains in the plant protein and the presence of a long stretch of proline and glutamine residues on the n-terminal part of human ranbpm molecule. we found that atranbpm maintained the properties of the full length ranbpm of mammals: while the truncated version of human ranbpm is present in centrosomal and ectopic microtubule nucleation sites, atranbpm protein in acentrosomal plant cells did not colocalize with microtubule nucleation sites positive for γ-tubulin; proteins of the γ-tubulin nucleating machinery were not identified by maldi-ms or lc-maldi-ms/ms among proteins interacting with atranbpm, nor were they found in coexpression databases and a biological role of atranbpm other than in processes relating to centrosomes and microtubules was thus suggested; the presence of highly conserved spry, lish, ctlh and cra domains in atranbpm indicated its function in mediating multiple protein interactions that were described for the whole molecule of human ranbpm. atranbpm protein was not specifically enriched with microtubules in dividing cells or localized in putative microtubule nucleation sites. subcellular localization of atranbpm corresponded to published data on the subcellular localization of the whole ranbpm molecule in mammalian cells. since a weak nuclear signal, observed for human ranbpm was suggested to be a consequence of over-expression of its tagged version, we analysed by immunofluorescence the nuclear localization for endogenous atranbpm protein. a smaller portion of atranbpm was present in nuclei and the possibility that the nuclear signal observed for gfp-atranbpm or atranbpm-gfp resulted from over expression of gfp fusions was thus excluded. we observed only partial colocalization of ran and atranbpm in nuclei. it would be interesting to address the question whether transport of the atranbpm complex between nucleus, perinuclear area and cytoplasm might be regulated by a weak interaction between atranbpm and ran as it was suggested for mammalian ranbpm. heterogeneity in the localization of mammalian ranbpm might depend on interacting proteins. conserved domains of ranbpm protein are responsible for interactions and complex formations with a physiologically divergent group of proteins. as reviewed by suresh et al., ranbpm is a modulator/protein stabilizer, a regulator of transcriptional activity, and has cell cycle and neurological functions. ranbpm interacts with a wide range of receptors, acts as a scaffolding protein, and is involved in signal transduction pathways, and the apoptotic pathways. plant homologues of ranbpm belong to genes with unknown functions. we found that the product of atranbpm is predominantly a cytoplasmic protein that is a part of protein complexes with a molecular mass of approximately – 500 kda. a large protein complex of ranbpm was described by nishitani et al. and ideguchi et al.. later the complex was designated as a ctlh complex. five from eleven ctlh domain-containing proteins that were identified in databases were described as being a part of the ctlh complex and an interaction within the complex via lish-ctlh domains was suggested. in saccharomyces cerevisiae all four ctlh domain-containing proteins present in the genome are part of the ctlh-like complex gid. the gid complex is suggested to be involved in proteasome-dependent glucose-induced degradation of fructose- -bisphosphatase. there are twenty one ctlh-domain containing proteins encoded in the arabidopsis genome and of these, six proteins were identified in our experiments to form ctlh-like complexes with atranbpm protein. though we identified the same spectrum of atranbpm interacting proteins in several independent experiments it cannot be ruled out that the members of ctlh complexes exist and remain to be identified. we found that the antibody raised against a peptide from the atranbpm sequence did not work in immunopurification experiments. the immunopurifications performed with a panel of antibodies against the atranbpm protein might help to disclose the presence of other putative members of plant ctlh complexes. alternatively, immunopurification of the ctlh complex or pull down experiments via proteins that copurified with atranbpm might extend our knowledge of the composition and protein interactions of newly identified ctlh complexes. all the members of the ctlh complexes that we identified belong to yet uncharacterized plant lish-ctlh domain-containing proteins. protein at1g is a homologue of the human twa protein found in yeast by two-hybrid analysis to interact with ranbpm. lish, cra, and ring/u-box domain-containing proteins of unknown function, copurified with atranbpm, are homologous to human maea and rmd respectively, that are members of the ctlh complex annotated by kobayashi et al.. similarly, lish and ctlh domain-containing proteins such as ranbp or the wd repeat domain, were suggested to be putative members of the ctlh complex. we found that uncharacterized arabidopsis transducin/wd- domain-containing proteins at5g and at5g which copurified with atranbpm, are homologues of human wdr protein and thus belong to components of the plant ctlh complex. corresponding to our microscopic data on cytoplasmic and nuclear localizations of atranbpm, the members of the human ctlh complex, proteins ranbpm, rmd wdr twa and maea, were shown to have predominantly cytoplasmic and a weaker nuclear localization. a component of the human ctlh complex, the armadillo repeat-containing protein armc when exogenously expressed, upregulates the proteolytic degradation of ectopically expressed α-catenin, and thus was suggested to play an important role in the ubiquitin-independent and proteasome-dependent degradation of α-catenin. α-catenin is known to function as a link protein between cadherins and actin-containing filaments of the cytoskeleton; thus a complex might regulate molecular adhesion. we identified armadillo-repeat-containing protein among proteins copurified with atranbpm but its homology with armadillo repeat armc a member of the human ctlh complex, was not proven. however, we found that expression of copurified protein at4g a homologue of rmd protein, correlated strongly with expression of the armadillo repeat-containing protein at3g one of the homologues of armc and with expression of the plant adhesion molecule rabgap/tbc domain-containing protein at3g. it is tempting to speculate whether similar function in connecting external signals in adhesion with the cell centre analogous to the cadherin and catenin pathway in animals might exist for plant ctlh complexes. however, further experimental data are needed to prove this hypothetical function of the ctlh complexes in plants. besides copurification with ctlh complex proteins, we found that plant atranbpm interacts with yippee proteins and yippee-like protein. it was shown in human cells that ranbpm binds members of ypel family of proteins involved in a cell division-related functions. our data showed that binding with yippee proteins might present one of the atranbpm multiple protein interactions in plant cells too.  ranbpm protein was shown to have numerous apparently functionally unrelated protein interactors, including membrane receptors, cytoplasmic and nuclear proteins. this suggests its role in a variety of cellular processes. our biochemical and proteomic studies showed that atranbpm is a component of plant homologues of ctlh complexes. the fact that ctlh complexes are present in mammals, yeast and in plants suggests their structural conservation in evolution. ctlh complexes thus might represent a module with important but not fully understood biological functions. 
however, ranbpm protein containing highly conserved spry, lish, ctlh and cra domains is currently considered as a scaffolding protein with multiple cellular functions. atranbpm protein has highly conserved spry, lish, ctlh and cra domains. immunopurification of atranbpm followed by mass spectrometric analysis identified proteins containing lish and cra domains; lish, cra, ring-u-box domains and a transducin/wd repeats in a complex with atranbpm. 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. we showed that as yet uncharacterized arabidopsis ranbpm protein physically interacts with lish-ctlh domain-containing proteins. 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. ranbpm was originally reported as a centrosome-associated protein in human cells. based on sequence similarity, we identified a homologue of the human ranbpm in arabidopsis thaliana. arabidopsis homologue of ranbpmctlh-complexlish-ctlh domain proteins
 ranbpm 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.  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 ~ – 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/wd repeats in a complex with atranbpm. homologues of identified proteins are known to be components of the c-terminal to the lish motif 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.  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. arabidopsis homologue of ranbpmctlh-complexlish-ctlh domain proteins 