TY - JOUR
AU - Nakamura, Kenzo
AB - Abstract TONSOKU(TSK)/MGOUN3/BRUSHY1 from Arabidopsis thaliana, which plays an important role in the maintenance of meristem organization, contains an LGN repeat motif similar to that found in animal proteins involved in asymmetric cell division. One protein that interacts with the LGN motif of TSK in a yeast two-hybrid screen, TSK-associating protein 1 (TSA1), contains a 10-fold repeat of a unique 41 amino acid sequence. The repeat sequence, with a glutamic acid–phenylalanine–glutamic acid (EFE) conserved core sequence, is enriched with acidic amino acids. TSA1 also contains an N-terminal putative signal peptide and it interacts with the LGN motif of TSK through a C-terminal region separated from the EFE repeats by a putative membrane-spanning region. The recombinant protein consisting of EFE repeats was rich in α-helical structure and possessed Ca2+-binding activity. Unlike nuclear localization of TSK, the TSA1 fused with green fluorescent protein (GFP) expressed in tobacco BY-2 cells was localized in small cytoplasmic vesicles during interphase. However, cellular localization of both TSA1–GFP and GFP–TSK changed dynamically during mitosis. In particular, both GFP–TSK and TSA1–GFP were concentrated in limited areas that are close to the ends of spindle microtubules ahead of separating chromatids. These results are discussed in terms of the possible involvement of TSK and TSA1 in mitosis. Introduction Higher plants continue organogenesis throughout their entire life, a process made possible by continued meristematic activity. After germination, the shoot apical meristem (SAM) and root apical meristem (RAM) are programmed to supply new cells for growth and organ formation, while some of the newly divided cells replace the cells of the meristem itself. Although meristems continue active cell division, the size and structure of meristems are kept essentially constant, indicating that the pattern, timing and direction of cell division in the meristem are tightly controlled. Recent progress in the genetic analysis of Arabidopsis thaliana mutants has identified several key genes with essential roles in cell division control in RAM and SAM. The TONSOKU/MGOUN3/BRUSHY1 (TSK/MGO3/BRU1) of Arabidopsis plays an important role in cell division control and plant morphogenesis (Guyomarc’h et al. 2004, Suzuki et al. 2004, Takeda et al. 2004). The SAM and RAM of the tsk mutants show disorganized cell arrangements, resulting in morphologically characteristic phenotypes including fasciated stems, disorganized phyllotaxy and short roots. Similar phenotypes were frequently observed in a class of mutants defective in factors involved in genome maintenance, including DNA replication, repair and recombination, such as chromatin assembly factor-1 (Kaya et al. 2001), Mre11 nuclease (Bundock and Hooykaas 2002) or subunits of condensin involved in chromatin condensation (Siddiqui et al. 2003). Takeda et al. (2004) demonstrated that the brushy1 mutant was sensitive to DNA-damaging agents and possessed reduced epigenetic gene silencing. These results suggest that TSK is also involved in genome maintenance (Suzuki et al. 2004, Takeda et al. 2004), despite the fact that TSK protein shares no homology with factors previously known to be involved in genome maintenance. We showed that NtTSK, a tobacco homolog of TSK, was specifically expressed during the S phase of the cell cycle, and the progression of the cell cycle from G2 to M phase was delayed in the tsk mutant (Suzuki et al. 2005). The mechanism whereby defects in some of the factors involved in genome maintenance cause defective meristem organization and function is not known. A large TSK protein contains two domains possibly involved in protein–protein interactions, leucine–glycine–asparagine (LGN) repeats and leucine-rich repeats (Suzuki et al. 2004). The LGN repeats (Mochizuki et al. 1996) have been identified in several animal proteins such as PARTNER OF INSCUTEABLE (PINS) of Drosophila (Yu et al. 2000) and mammalian LGN protein (Blumer et al. 2002). The LGN repeats of PINS and mammalian LGN protein have been shown to interact with INSCUTEABLE (INS; Kraut and Campos-Ortega 1996) and the nuclear mitotic apparatus protein NuMA (Du et al. 2001), respectively, and these proteins are involved in asymmetric cell division. To understand the cellular function of TSK, it is important to identify protein(s) that interact with it. In this study, we searched for an Arabidopsis protein that can interact with the LGN repeats of TSK by yeast two-hybrid assay and identified TSK-associating protein 1 (TSA1), that contained a novel calcium-binding repeat sequence. The TSA1 fused with green fluorescent protein (GFP) was associated with small cytoplasmic vesicles in interphase cells of the tobacco BY-2 cell line, which is different from the nuclear localization of GFP–TSK (Suzuki et al. 2004). However, in cells synchronized for cell division, both TSA1–GFP and GFP–TSK changed their localization dynamically and relocalized to ends of spindle microtubules that are ahead of separating chromatids during metaphase and anaphase of mitosis. It is suggested that TSK is involved in mitosis through its association with TSA1. Results Identification of TSK-associating protein 1 Yeast two-hybrid screening was used to identify proteins able to interact with the LGN repeats of TSK. The coding region of TSK corresponding to exons 2–5, which covers three of four LGN repeats (Suzuki et al. 2004), was fused with the coding sequence of LexA and used as a bait (pBTE25). The pBTMSBEN without TSK cDNA served as a negative control. The yeast strain L40 (trp–, leu–, his–), with lacZ and HIS3 reporter genes under the control of promoter containing the LexA-binding sequence, was used as a host in our screening. The L40 strain was transformed with pBTE25, then further transformed with the Arabidopsis cDNA library in the pVP16S1 vector, in which the coding sequence of cDNA was fused to the activation domain of herpes simplex virus VP16 protein. Approximately 14 million transformants were plated on selection plates containing 1 mM 3-aminotriazole, and colonies that grew on selection plates were assayed for β-galactosidase activity. Plasmid DNA isolated from a positive clone was used to retransform yeast host cells with pBTMSBEN or pBTE25; clones that allowed the growth of cells with pBTE25, but not cells with pBTMSBEN, on selective plates, were chosen for further analysis. Among the positive clones, >30 clones contained partial cDNA derived from the same mRNA with varying lengths. The growth of yeast cells harboring one of these positive clones, pTSA1, on selective plates is shown in Fig. 1A. The pTSA1 enabled yeast host cells to grow on selective plates only with pBTE25. The transcript covered by pTSA1 and other clones was derived from the predicted Arabidopsis gene, At1g52410, which was named TSA1 (TSK-associating protein 1). Other cDNA clones contained the 3′-terminal portion of TSA1 mRNA in common, with varying lengths toward the 5′-terminus. TSA1 contains a novel repeat sequence The public database contained one full-length cDNA (accession No. AY048247) for TSA1, which was 2536 bp in length and coded for a protein with 755 amino acid residues. Comparison of the sequences of the full-length cDNA and genomic DNA indicated that the TSA1 gene was split into 26 exons interrupted by 25 introns (Fig. 1B). Another predicted cDNA for one splicing variant of TSA1 mRNA (accession No. NM_179466) was also present in the database. In this cDNA, selection of an alternative splice acceptor site of the 13th intron resulted in a 12 nucleotide extension of the 14th exon compared with AY048247. The occurrence of alternative splicing of the TSA1 transcript was verified by sequencing the reverse transcription (RT)–PCR products, with the longer transcripts being more abundant (data not shown). The amino acid sequence of TSA1 predicted from cDNA begins with the N-terminal hydrophobic signal peptide-like sequence, followed by 10 times repeats of similar sequences occupying the N-terminal half of the molecule (Fig. 1B). Most of the repeated sequences are 36–41 amino acids long, except for the C-terminal unit consisting of 30 amino acids (Fig. 1C). Each repeat sequence is encoded by two exons, and the intron insertion sites are highly conserved (indicated by the vertical line in Fig. 1C). The repeat sequence is enriched with acidic amino acids and the predicted pI of TSA1 is 4.30. The repeat sequence was named EFE repeat after the highly conserved core amino acid sequence of the repeats. Several computer programs indicated that the repeat sequence, in particular the sequence around the EFE core motif, exists as an α-helix. The consensus sequence of EFE repeats contained hydrophobic amino acids with intervals of 3–4 amino acids, which suggests the participation of EFE repeats in the formation of a coiled-coil structure (Fig. 1C). A database search for a homolog of TSA1 indicated the presence of another Arabidopsis gene (At3g15950) coding for a protein highly similar to TSA1. The TSA1-like protein encoded by this gene consisted of 722 amino acids and contained 10 EFE repeats similar to TSA1 (Fig. 1D). The intron–exon organization of the TSA1-like gene was also similar to that of TSA1 (data not shown), and TSA1 (At1g52410) and TSA1-like (At3g15950) were located on a large duplicated segment between chromosomes 1 and 3 (Arabidopsis Genome Initiative 2000). In addition to these two Arabidopsis proteins, only one expressed sequence tag (EST) sequence of Chinese cabbage coded for a protein containing at least three EFE repeats. These results suggest that the EFE repeat is conserved at least in Brassicaceae and can be considered to be a novel repeat sequence unique to plants. Expression of TSA1 in the shoot apex The TSK gene is expressed predominantly in the meristems (Suzuki et al. 2004). Expression of TSA1 and TSA1-like genes in various organs of Arabidopsis was examined and compared with that of TSK by quantitative real-time RT–PCR (Fig. 2). The expression levels of TSA1, TSA1-like and TSK were normalized to the level of actin mRNA. The expression of the TSK gene was most abundant in the flower, and slightly less abundant in the shoot apex. Similar to TSK, TSA1 was preferentially expressed in the flower and shoot apex. The expression of TSA1-like was different from that of TSK and TSA1 and was greatest in the roots. These results suggest that TSA1 and TSK are expressed under similar developmental control systems, while expression of TSA1 and TSA1-like is differentially controlled. Interaction of TSK with TSA1 in vitro To confirm the interaction between TSK and TSA1 in vitro, the LGN repeat region of TSK encoded by exons 2–5 used in two-hybrid screening was expressed in Escerichia coli as a fusion with the T7 tag at the N-terminus and with the His tag at the C-terminus (TSKE25). The C-terminal region of TSA1 encoded by exon 26 was similarly expressed in E. coli as a fusion with the His tag at the C-terminus (TSA1E26). TSKE25 and TSA1E26 proteins, partially purified from cell lysates with an Ni-NTA resin column, were separated by SDS–PAGE, blotted onto the membrane and detected with anti-His tag antibody. The TSA1E26 appeared as a single band of 27 kDa, as expected from the sequence (Fig. 3, lane 4). In contrast, the preparation of TSKE25 showed a major band of 42 kDa, which is similar to the size expected from the sequence, and an additional band of 30 kDa, which could be a degradation product (Fig. 3, lane 5). In the co-precipitation assay, TSKE25 and TSA1E26 were mixed and incubated, followed by immunoprecipitation of TSKE25 with anti-T7 tag antibody conjugated to agarose. The precipitated fraction contained, in addition to TSKE25, the 27 kDa band of TSA1E26 (Fig. 3, lane 1). TSA1E26 was not detected in the immunoprecipitated fraction when either TSKE25 or TSA1E26 was omitted from the reaction mixture. These results indicate that the C-terminal region of TSA1 interacts with the LGN repeat region of TSK in vitro. LGN repeats of TSK interact with the C-terminal region of TSA1 To define further the domains of TSK and TSA1 that participate in the interaction, a yeast two-hybrid assay was performed. First, various deletions from the N-terminal region of TSK were created and fused with the LexA DNA-binding domain (BD), followed by an examination of their ability to interact with the C-terminal region of TSA1 fused with the VP16 transcription activation domain (AD) by the growth of yeast cells on selective medium (Fig. 4A). The expression of various deletion derivatives of the TSKE25-BD fusion protein was verified by Western blotting of the yeast cell lysate with anti-LexA antibody (Fig. 4B). TSK contained four LGN repeats, and three of the four LGN repeats are covered in TSKE25-BD. Deletion of the region outside of the LGN repeats did not abolish interaction with TSA1E26-AD (TSKE25N, TSKE25N2). In contrast, TSK with partial deletions of the LGN repeats did not bind to TSA1. These results indicate that the LGN repeats of the TSK protein are necessary and sufficient to interact with TSA1. The C-terminal region of TSA1 was cloned into the pBTMSBEN vector to be expressed as a fusion with BD in yeast (TSA1E26-BD), and the region of TSK used in the yeast two-hybrid screening was cloned into the pVP16S1 vector to be expressed as a fusion with AD (TSKE25-AD). Various deletions from TSA1E26-BD were examined for the ability to interact with TSKE25-AD (Fig. 4C). Western blotting of the yeast cell lysate with anti-LexA antibody verified expression of these deletion derivatives of TSA1E26-BD fusion protein in yeast cells. In addition to TSAE26, two deletion derivatives (TSA1E26C and TSA1E26C2) interacted with TSKE25-AD. All the other deletion derivatives of TSA1E26-BD failed to interact with TSKE25-AD. These results indicate that the C-terminal 92 amino acid sequence from 616 to 707 of TSA1 is necessary and sufficient to interact with the LGN repeats of TSK. TSA1 binds calcium and forms a multimer The most characteristic feature of TSA1 is the EFE motif repeated 10 times consecutively (Fig. 1C). The EFE repeat sequence is highly enriched with charged amino acids; 29 and 16% of amino acid residues in the repeat region (from Ser to Glu; Fig. 1C) are acidic and basic amino acids, respectively. This high ratio of acidic amino acids in the EFE repeats and the localization of TSA1 to small cytoplasmic vesicles as described below suggested that the EFE repeats may bind cations, in particular Ca2+. The portion of TSA1 corresponding to the sequence covered by exons 2–25 (TSA1E225; Fig. 1B) that encompassed all 10 EFE repeats was fused with a T7 tag at its N-terminus and a His tag at its C-terminus, and expressed in E. coli. TSA1E225 was purified from the cell lysate with Ni-NTA resin and anti-T7 ag antibody. Upon SDS–PAGE and staining with Coomassie brilliant blue, TSA1E225 migrated as a single band with an apparent size of 76 kDa, which is larger than the molecular mass of 58 kDa predicted from the sequence (Fig. 5A, lane 2). The band of TSA1E225 was clearly stained blue by Stains-all (lane 4), which specifically stains calcium-binding protein blue (Campbell et al. 1983). Bovine serum albumin (BSA), used as a negative control (lanes 1 and 3), was not stained by Stains-all. The calcium-binding activity of TSA1 was directly examined by 45Ca2+ overlay assay. TSA1E225 was blotted on the membrane and incubated with a solution containing 45Ca2+. After washing with 50% ethanol, 45Ca2+ bound to the membrane was detected by autoradiography (Fig. 5B). TSA1E225 gave a clear positive signal of 45Ca2+. Calmodulin blotted on the membrane also showed a strong positive signal, while BSA did not show significant binding. Binding of 45Ca2+ to TSA1E225 was abolished by the addition of 5 mM Ca2+ in the reaction mixture, while the addition of 5 mM Mg2+, Mn2+, Zn2+, Na+ and K+ did not abolish the binding (data not shown). These results indicate that the EFE repeat is a novel motif that specifically binds calcium. The secondary structure prediction program (EMBOSS garnier) suggested that a large part of the EFE repeat sequence assumes an α-helix with a spacer of variable length (Fig. 1C). The circular dichroism (CD) spectrum of purified TSA1E225 showed a peak at 205 nm with a shoulder near 225 nm (Fig. 5C), supporting that the EFE repeat region of TSA1 is rich in α-helical structure. Under the conditions employed, the CD spectrum of TSA1E225 did not change significantly in the presence or absence of calcium (data not shown). To examine whether TSA1 forms a multimer, which was anticipated from the regularly spaced hydrophobic amino acids in the α-helix of the EFE repeat (Fig. 1C, D), the N-terminally truncated TSA1 corresponding to exons 2–26 was expressed in E. coli as a fusion protein with a T7 tag at its N-terminus and a His tag at its C-terminus (TSAE226). TSAE226 purified from the cell lysate was applied to a Superose 6 gel filtration column. Although the molecular mass of TSA1E226 estimated from the sequence was 84 kDa, it migrated on SDS–PAGE as a band of 105 kDa (Fig. 5D), probably due to a high proportion of acidic amino acids. Upon column chromatography, the TSA1E226 band eluted in fractions 12 and 13. From elution profiles of size marker proteins, the molecular mass of the eluted protein was estimated to be 570 kDa, suggesting that TSA1E226 forms a hexamer or heptamer. Under these conditions, calcium did not affect the multimerization of TSA1E226 (data not shown). Similar results were obtained from the gel filtration assay using TSA1E225 (data not shown), indicating that multimerization was mediated by the EFE repeats. Association of TSA1–GFP with small vesicles in interphase cells In plant cells, the major calcium storage organelles are the endoplasmic reticulum (ER) and vacuole. Since the TSA1 protein was expected to have a signal peptide and bind calcium, the cellular localization of TSA1 was examined by fusion with GFP. The full-length cDNA of TSA1 was cloned into pGWB5 binary vector to express TSA1 fused with GFP at its C-terminus under the 35S promoter. Arabidopsis plant and tobacco cell culture line BY-2 was transformed by Agrobacterium-mediated transformation. Transformed Arabidopsis plant and tobacco BY-2 cells expressing TSA1–GFP were selected and observed under confocal laser scanning microscopy (CLMS). In both cells, the fluorescence of TSA1–GFP appeared as many spots in the cytoplasm, suggesting that TSA1 is associated with cytoplasmic vesicles, possibly in the ER or the Golgi, but is not localized in vacuoles, nucleus or the plasma membrane (Fig. 6B, C). The fluorescence of GFP alone was observed in the cytoplasm and nucleus (Fig. 6A). Both TSA1 and TSK change their location during mitosis Since the TSK protein was localized to the nucleus (Suzuki et al. 2004), the association of TSA1 with cytoplasmic vesicles is not readily consistent with an interaction between TSK and TSA1. To investigate this apparent contradiction, the cellular localization of TSK and TSA1 during cell division cycle was examined in tobacco BY-2 cells. Cell division of tobacco BY-2 cells expressing TSA1–GFP was synchronized by treatment with aphidicolin followed by propyzamide (Nagata and Kumagai 1999). Cells at each stage of the cell cycle were fixed, stained with anti-α-tubulin antibody and observed under CLSM. In cells arrested in interphase by propyzamide (Fig. 7A), many spots of fluorescence of TSA1–GFP were observed in the cytoplasm around the nuclear envelope, along the cytoplasmic strand and near the cell periphery. At this stage, microtubules were also observed around the nuclear envelope, and they extended from the nucleus to the cell cortex. In the prophase cells (Fig. 7B), when the nuclear envelope disappears and chromatins appear, both TSA1–GFP and microtubules were assembled around the periphery of chromatids. When the cell cycle proceeds to the metaphase (Fig. 7C), TSA1–GFP was largely concentrated at external ends of spindle microtubules where spindle poles are supposed to be formed. The other ends of spindle microtubules surrounded chromatins. In the anaphase cells (Fig. 7D), TSA1–GFP appeared as clear disc-like structures ahead of separated chromatids, while microtubules appeared between two chromatids where phragmoplast are to be formed. During the telophase (Fig. 7E), fluorescence of TSA1–GFP resumed its spotty appearance around the nuclear envelope. Cell division of tobacco BY-2 cells expressing GFP–TSK was also synchronized with aphidicolin and propyzamide, stained with propidium iodide for the cell wall and DNA, and observed under CLSM. In interphase cells, fluorescence of GFP–TSK appeared uniformly in the nucleoplasm (data not shown; Suzuki et al. 2004). However, cellular localization of GFP–TSK also changed dynamically during the cell division cycle. In metaphase cells, most of the fluorescence of GFP–TSK appeared as spots (data not shown) and these spots gathered ahead of the separated chromatids in anaphase cells (Fig. 7G). Similar to TAS1–GFP, fluorescence of GFP–TSK was not detected between the separated chromatids. This appearance of fluorescence of GFP–TSK was quite different from that of free GFP in anaphase cells (Fig. 7F). The spotty appearance of GFP–TSK dissappered when cells entered telophase (data not shown). It was noteworthy that both TSA1–GFP and GFP–TSK showed dynamic relocalization during the cell cycle and a similar localization ahead of the separation of chromatids during anaphase. Discussion LGN repeats of TSK participate in protein–protein interaction The TSK protein contains two putative protein–protein interaction motifs, the LGN repeats and leucine-rich repeats (Suzuki et al. 2004). The LGN repeat (Mochizuki et al. 1996) is a subfamily of the tetratricopeptide repeat (TPR) motif involved in protein–protein interactions, and it has been found only in animals and plants. Previously, PINS of Drosophila, which is required for asymmetric cell division of neuroblasts, has been shown to interact with the INSCUTEALE (INSC) protein via the LGN repeat region (Yu et al. 2000). The human homolog of PINS, human LGN protein, interacts with the nuclear mitotic apparatus protein NuMA (Du et al. 2001). The PINS of Drosophila and mammalian LGN protein contain seven LGN repeats in the N-terminal region and GoLoco motifs involved in the interaction with Gα in the C-terminal region. Unlike these proteins, TSK contains only four LGN repeats in the N-terminal region and leucine-rich repeats in the C-terminal region. In this study, we used a yeast two-hybrid method to screen for proteins that interacted with the N-terminal region of TSK with three of the four LGN repeats. Many of the positive clones contained partial cDNAs for TSA1 (Fig. 1). The interaction of TSK with TSA1 was confirmed in vitro with bacterially expressed recombinant protein (Fig. 3), and the specific interaction of TSK with TSA1 via the LGN repeats was confirmed in yeast (Fig. 4). These results indicate that the LGN repeats of TSK can participate in specific protein–protein interaction. Since we could not express a large TSK protein with an immnologically detectable tag in plants, the interaction between TSK and TSA1 in plant cells could not be confirmed. TSA1 is a novel calcium-binding protein associated with the cytoplasmic vesicle TSA1 contained 10 repeats of a previously uncharacterized sequence of 36–41 amino acids designated the EFE repeat (Fig. 1). Each EFE repeat unit was encoded precisely by two separate exons. The Arabidopsis genome contained another gene (At3g15950) encoding a protein similar to TSA1 (Fig. 1C, D). At present, a sequence similar to the EFE repeat has only been found in a Chinese cabbage EST, apart from the two Arabidopsis proteins in the public database. The EFE repeat of TSA1 was enriched with charged amino acids (Fig. 1), enriched in α-helical structure (Fig. 5C) and could specifically bind Ca2+ (Fig. 5A, B). TSA1 may assume a coiled coil-like structure and was able to form a multimer (Fig. 5D). The TSA1 contains the signal peptide-like hydrophobic stretch at its N-terminus, and the TSA1–GFP fusion protein appeared to be specifically associated with the small organelle or vesicles dispersed in the cytoplasm of root cells of Arabidopsis and tobacco BY-2 cells (Fig. 6B, C). In tobacco BY-2 cells expressing TSA1 with a myc tag at its C-terminus, TSA1-myc was recovered in the microsomal fraction, although it was detected in other subcellular fractions as well (data not shown). Thus, TSA1 seems to be localized to the ER or the small organelle derived from the ER. The LGN repeats of TSK interacted with the C-terminal region of TSA1 (Fig. 3, 4) and the sequence from 595 to 617 of TSA1, which is located between the EFE repeat region and the C-terminal TSK-interacting domain, is rich in hydrophobic amino acids and devoid of basic amino acids. Thus, TSA1 could be a transmembrane protein with the N-terminal Ca2+-binding EFE repeat region in the luminal side and it interacts with TSK through the C-terminal domain exposed to the cytoplasm. Relocalization of TSA1–GFP and GFP–TSK during the cell cycle In contrast to TSA1–GFP associated with cytoplasmic vesicles, GFP–TSK was localized exclusively and evenly in the nucleoplasm of interphase cells of tobacco BY-2 (Suzuki et al. 2004). Despite these differences in subcellular localization, the mRNAs for TSA1 and TSK were both enriched in meristems (Fig. 2). We explored the possibility that the interaction between TSK and TSA1 might be spatially and temporally limited during cell division. Although TSA1–GFP appeared as many spots dispersed in the cytoplasm in interphase cells (Fig. 6B, C), it was concentrated in the perinuclear region in prophase cells. From metaphase to anaphase, fluorescence of TSA1–GFP was concentrated at ends of spindle microtubules that are ahead of separating chromatids. As the cell cycle proceeds to telophase, TSA1–GFP resumed localization to the nuclear envelope region. On the other hand, fluorescence of GFP–TSK uniformly dispersed in the nucleoplasm of the interphase cells appeared as spots during mitosis. In particular, the spotty appearance of GFP–TSK was concentrated around the ends of spindle microtubules that are ahead of separating chromatids in metaphase and anaphase cells. The spotty appearance of the fluorescence of GFP–TSK during mitosis could be caused by association with a specific structure or vesicle. Since the localization of GFP–TSK in metaphase and anaphase cells overlapped with the area of the localization of TSA–GFP, the spotty appearance of the fluorescence of GFP–TSK during mitosis might be caused by its interaction with the C-terminal region of TSA1 exposed on the surface of membranous vesicles. The spotty appearance of GFP–TSK is lost when the cell cycle proceeds to telophase, suggesting that the interaction of TSK and TSA1 might be regulated in a cell cycle-dependent manner. During metaphase and anaphase, both GFP–TSK and TSA1–GFP were concentrated in the region where the spindle poles would be formed prior to the separation of duplicated chromatids. In animal cells, microtubular arrays of mitotic spindle involved in chromosome separation are nucleated at the centrosomes. In contrast, plant mitotic cells do not contain centrosomes, and nuclear and cortical membranes are probably sites of microtubule nucleation (Kumagai et al. 2001, Dryková et al. 2003). In addition, microtubule-organizing centers (MTOCs) in plant cells were localized to the nuclear envelope (Lambert 1993, Mizuno 1993, Stoppin et al. 1994). The γ-tubulin participates in the nucleation of microtubules from MTOCs in animal cells (Oakley et al. 1990, Joshi et al. 1992). The plant γ-tubulin may also be a subunit of the MTOC (Liu et al. 1994), and the large γ-tubulin complexes active in microtubule nucleation have been shown to be associated with membranes (Dryková et al. 2003). The fluorescence of TSA1–GFP appears in the perinuclear region in prophase cells, and it relocates to external ends of microtubules in metaphase cells. The other ends of microtubules at this stage are attached to separating chromatids The fluorescence of GFP–TSK also appears as spots in a similar localization to TSA1–GFP (Fig. 7). These results suggest an interesting possibility that TSA1 and TSK might be involved in the organization of spindle microtubules. TSK is expressed during the S phase and is suggested to be required for genome maintenance (Takeda et al. 2004, Suzuki et al. 2005), and the delayed cell cycle progression from G2 to M in tsk mutants could be due to activation of the G2/M checkpoint (Suzuki et al. 2005). However, the delayed cell cycle progression in the tsk mutant could also be due to defects in mitosis as suggested herein. In addition, possible involvement of TSK in mitosis may be somehow related to the irregular cell division planes and disorganized cell arrangements frequently observed in the meristems of the tsk mutant (Suzuki et al. 2004). To examine further the role of TSA1 and TSK in mitosis, the interaction between TSK and TSA1 during mitosis needs to be confirmed in plants and we need to examine whether the dynamic relocalization of TSK during mitosis is dependent on TSA1, and vice versa. Analysis of disruptants of TSA1 and TSA1-like genes are also expected to give us some clues to the function of these novel calcium-binding proteins in mitosis and other aspects of plant growth and development. Materials and Methods Yeast two-hybrid screening Saccharomyces cerevisiae strain L40, pBTMSBEN used as bait vector and pVP16S1 used as prey vector were generously donated by Dr. M. Ishikawa and Dr. Y. Machida of the Graduate School of Science, Nagoya University, Japan. The Arabidopsis cDNA library cloned into pVP16S1 was a gift from Dr. Y. Yoshioka of the Graduate School of Science, Nagoya University, Japan. L40 cells were transformed with pBTE25 to produce the fusion protein of LexA and partial protein of TSK encoded in exons 2–5, followed by transformation of L40 carrying pBTE25 with the cDNA library. Approximately 14 million transformants were screened on selection plates without tryptophan, leucine and histidine supplemented with 1 mM 3-aminotriazole for 1 week at 22°C. Positive colonies were replicated on filter paper, and frozen in liquid nitrogen. After thawing, the filter was incubated with 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal; 0.33 mg ml–1) in 100 mM sodium phosphate buffer (pH 7.0) containing 10 mM potassium chloride, 1 mM magnesium sulfate and 35 mM β-mercaptoethanol. The derivatives of pVP16S1 plasmids were recovered from β-galactosidase-positive colonies and the cDNA inserts were sequenced. Real-time RT–PCR Total RNA was extracted from each organ of Arabidopsis with TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) and used as a template for reverse transcription with oligo(dT) primer. Real-time PCR was performed with iQ SYBR Green Supermix and iCycler (BIO-RAD, Hercules, CA, USA). Expression and purification of recombinant protein Target sequences were cloned into pET24b (Novagen, Darmstadt, Germany) to produce proteins tagged with His at the C-terminus. Some of them were tagged further with the T7 epitope at the N-terminus. Recombinant fusion proteins were produced in E. coli BL21 (DE3) pLysS cells. The extracted proteins were affinity purified on Ni-NTA Superflow (Qiagen, Tokyo, Japan) and some of them were purified further on T7 tag antibody agarose as described in the manufacturer’s instructions (Novagen, Darmstadt, Germany). Eluates were dialyzed against phosphate-buffered saline and concentrated with an Ultrafree-4 centrifugal filter unit (Nihon Millipore, Tokyo, Japan). The assay to detect interactions between TSKE25 and TSA1E26 in vitro was performed as described previously (Kitakura et al. 2002). Biochemical assays SDS–PAGE with a 10 or 12% (w/v) polyacrylamide gel was conducted by the method of Laemmli (1970). Coomassie brilliant blue staining was performed with Blue Stain Reagent (Pierce, Rockford, IL, USA) as described in the manufacturer’s instructions. To detect the Ca2+-binding ability of TSA1E225, the polyacrylamide gel was stained with Stains-all (Sigma Aldrich, Tokyo, Japan) as described (Yuasa and Maeshima 2000). The Ca2+ overlay assay was also conducted as described (Yuasa and Maeshima 2000). The CD spectrum of TSAE225 was measured using a JASCO J-720WI spectropolarimeter (Nihonbunko, Tokyo, Japan). Gel filtration chromatography was performed on a Superose 6 column with the ÄKTA Explorer system (Amersham Biosciences, Tokyo, Japan). Observation of sGFP fusion proteins The construction of plasmids and transformation of tobacco BY-2 cells were performed as described previously (Suzuki et al. 2004). Synchronization of cell division was performed as described previously (Suzuki et al. 2005). Agrobacterium-mediated transformation of Arabidopsis was carried out by the vacuum infiltration method. Seeds were sown on selective plates containing appropriate actibiotics. Immunofluorescence analysis was performed as described (Nishihama et al. 2001). Acknowledgments The authors thank Dr. Y. Machida, Dr. M. Ishikawa and Dr. Y. Yoshioka of the Graduate School of Science, Nagoya University for their technical advice and donation of the yeast strain and plasmids used for yeast two-hybrid screening; Dr. M. Maeshima and Mr. T. Kamiya of the Graduate School of Bioagricultural Sciences, Nagoya University, for their technical advice on the Ca2+ overlay assay; and Dr. T. Nakagawa of the Research Institute of Molecular Genetics, Shimane University, for the sGFP binary vectors. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (grant No. 14036101; Molecular Basis of Axis and Signals in Plant Development) from the Japan Society for the Promotion of Science to A.M. and K.N., and the 21st Century COE program from the Ministry of Education, Science, Sports and Culture of Japan 3 Present address: Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, 464-8602 Japan. View largeDownload slide Fig. 1 TSA1 identified by yeast two-hybrid assay encodes a protein with a novel repeat sequence. (A) Yeast strain L40 carrying pBTMSBEN (vector alone) or pBTE25 (N-terminal region of TSK fused with BD) as a bait and pVP16S1 (vector alone) or pTSA1 (TSA1 fused with AD) as a prey were grown on +His (left) or –His (right) selection plates. (B) Structure of the TSA1 gene and encoded protein. Boxes in the upper drawing represent exons. The white, shaded and black boxes represent untranslated, coding and EFE repeat-coding regions, respectively. The features of TSA1 protein including signal peptide (SP), EFE repeats, putative transmembrane domain (TM) and TSK-interacting domain (TID) are indicated in the lower drawing. (C) and (D) Alignment of the amino acid sequence of the EFE repeats of TSA1 and TSA1-like proteins, respectively. Amino acid residues conserved in more than half of the repeat sequences are shaded. A vertical line indicates sites of intron insertions. The four amino acid sequence removed by alternative splicing in NM_179466 is underlined in (C). In the consensus sequence, the region of the repeat sequence predicted to assume an α-helix is indicated by the underline, and amino acid residues assumed to form a leucine zipper-like structure are shaded. View largeDownload slide Fig. 1 TSA1 identified by yeast two-hybrid assay encodes a protein with a novel repeat sequence. (A) Yeast strain L40 carrying pBTMSBEN (vector alone) or pBTE25 (N-terminal region of TSK fused with BD) as a bait and pVP16S1 (vector alone) or pTSA1 (TSA1 fused with AD) as a prey were grown on +His (left) or –His (right) selection plates. (B) Structure of the TSA1 gene and encoded protein. Boxes in the upper drawing represent exons. The white, shaded and black boxes represent untranslated, coding and EFE repeat-coding regions, respectively. The features of TSA1 protein including signal peptide (SP), EFE repeats, putative transmembrane domain (TM) and TSK-interacting domain (TID) are indicated in the lower drawing. (C) and (D) Alignment of the amino acid sequence of the EFE repeats of TSA1 and TSA1-like proteins, respectively. Amino acid residues conserved in more than half of the repeat sequences are shaded. A vertical line indicates sites of intron insertions. The four amino acid sequence removed by alternative splicing in NM_179466 is underlined in (C). In the consensus sequence, the region of the repeat sequence predicted to assume an α-helix is indicated by the underline, and amino acid residues assumed to form a leucine zipper-like structure are shaded. View largeDownload slide Fig. 2 The mRNA expression profile of TSK and TSA1 in various organs. The levels of mRNA for TSK, TSA1 and TSA1-like in different organs were assayed by quantitative real-time RT–PCR. Each bar from left to right represents the expression level of each gene in shoot apex, root, leaf, stem and flower. View largeDownload slide Fig. 2 The mRNA expression profile of TSK and TSA1 in various organs. The levels of mRNA for TSK, TSA1 and TSA1-like in different organs were assayed by quantitative real-time RT–PCR. Each bar from left to right represents the expression level of each gene in shoot apex, root, leaf, stem and flower. View largeDownload slide Fig. 3 Interaction of TSK with TSA1 in vitro. Partially purified TSKE25 and TSA1E26 were incubated and subjected to immunoprecipitation (IP) with anti-T7 tag antibody. Immunoprecipitates were separated by SDS–PAGE, blotted onto the membrane and reacted with anti-His-tag antibody (lane 1). The reaction mixture of the control contained only TSKE25 (lane 2) or TSA1E26 (lane 3). Partially purified TSA1E26 (lane 4) and TSKE25 (lane 5) were subjected to SDS–PAGE as markers. View largeDownload slide Fig. 3 Interaction of TSK with TSA1 in vitro. Partially purified TSKE25 and TSA1E26 were incubated and subjected to immunoprecipitation (IP) with anti-T7 tag antibody. Immunoprecipitates were separated by SDS–PAGE, blotted onto the membrane and reacted with anti-His-tag antibody (lane 1). The reaction mixture of the control contained only TSKE25 (lane 2) or TSA1E26 (lane 3). Partially purified TSA1E26 (lane 4) and TSKE25 (lane 5) were subjected to SDS–PAGE as markers. View largeDownload slide Fig. 4 Mapping of TSK and TSA1-interacting domains. (A) Mapping of the TSA1-interacting domain in TSK protein. Each bar represents a part of TSK fused with BD as a bait; amino acid residues are indicated on the right. Black bars indicate the region that enabled L40 cells to grow on selection plates with TSA1E26-AD. The LGN repeat region is underlined in the top bar. (B) Proteins in the lysate of yeast L40 cells expressing TSK-BD fusion proteins were subjected to SDS–PAGE, blotted onto the membrane and reacted with anti-LexA antibody. Arrowheads indicate the bands of TSK-BD fusion proteins. (C) Mapping of the TSK-interacting domain in TSA1. Each bar represents a portion of TSA1 fused with the AD; amino acid residues are indicated on the right. Black bars indicate the region that enabled L40 cells to grow on selection plates with TSKE25-AD. The EFE repeat region is underlined in the top bar. (D) Expression of partial TSA1E26-BD fusion proteins in yeast L40 cells was confirmed as in (B). Arrowheads indicate the bands of TSA1E26-AD fusion proteins. View largeDownload slide Fig. 4 Mapping of TSK and TSA1-interacting domains. (A) Mapping of the TSA1-interacting domain in TSK protein. Each bar represents a part of TSK fused with BD as a bait; amino acid residues are indicated on the right. Black bars indicate the region that enabled L40 cells to grow on selection plates with TSA1E26-AD. The LGN repeat region is underlined in the top bar. (B) Proteins in the lysate of yeast L40 cells expressing TSK-BD fusion proteins were subjected to SDS–PAGE, blotted onto the membrane and reacted with anti-LexA antibody. Arrowheads indicate the bands of TSK-BD fusion proteins. (C) Mapping of the TSK-interacting domain in TSA1. Each bar represents a portion of TSA1 fused with the AD; amino acid residues are indicated on the right. Black bars indicate the region that enabled L40 cells to grow on selection plates with TSKE25-AD. The EFE repeat region is underlined in the top bar. (D) Expression of partial TSA1E26-BD fusion proteins in yeast L40 cells was confirmed as in (B). Arrowheads indicate the bands of TSA1E26-AD fusion proteins. View largeDownload slide Fig. 5 Calcium-binding activity and multimer formation of TSA1 protein. (A) A 2 µg aliquot each of purified TSA1E225 (lanes 2 and 4) and BSA (lanes 1 and 3) was separated by SDS–PAGE. The gel was stained with Coomassie brilliant blue (left) or Stains-all (right). (B) 45Ca2+ overlay assay. TSA1E225, BSA and calmodulin (0.5 µg each) were blotted on a PVDF membrane. The membrane was reacted with 45Ca2+, washed with 50% ethanol and exposed to autoradiography. (C) The CD spectrum of TSA1E225 (0.37 mg ml–1). (D) Elution profile of TSA1E226 on Superose 6 column chromatography. Purified TSA1E226 was separated on a Superose 6 column. Size markers (Amersham Biosciences, Tokyo, Japan) were separated on the same column and detected by absorbance at 280 nm. Elution of size markers is indicated on the upper panel. Proteins in each fraction were subjected to SDS–PAGE and immunoblotted with anti-His tag antibody to detect TSA1E226 (lower panel). View largeDownload slide Fig. 5 Calcium-binding activity and multimer formation of TSA1 protein. (A) A 2 µg aliquot each of purified TSA1E225 (lanes 2 and 4) and BSA (lanes 1 and 3) was separated by SDS–PAGE. The gel was stained with Coomassie brilliant blue (left) or Stains-all (right). (B) 45Ca2+ overlay assay. TSA1E225, BSA and calmodulin (0.5 µg each) were blotted on a PVDF membrane. The membrane was reacted with 45Ca2+, washed with 50% ethanol and exposed to autoradiography. (C) The CD spectrum of TSA1E225 (0.37 mg ml–1). (D) Elution profile of TSA1E226 on Superose 6 column chromatography. Purified TSA1E226 was separated on a Superose 6 column. Size markers (Amersham Biosciences, Tokyo, Japan) were separated on the same column and detected by absorbance at 280 nm. Elution of size markers is indicated on the upper panel. Proteins in each fraction were subjected to SDS–PAGE and immunoblotted with anti-His tag antibody to detect TSA1E226 (lower panel). View largeDownload slide Fig. 6 Cellular localization of TSA1–GFP. Roots of Arabidopsis plants expressing GFP (A) or TSA1–GFP (B) were stained with propidium iodide (PI) and observed under CLMS. Tobacco BY-2 cells expressing TSA1–GFP (C) were observed under CLMS. Scale bars = 50 µm (A, B) and 20 µm (C). View largeDownload slide Fig. 6 Cellular localization of TSA1–GFP. Roots of Arabidopsis plants expressing GFP (A) or TSA1–GFP (B) were stained with propidium iodide (PI) and observed under CLMS. Tobacco BY-2 cells expressing TSA1–GFP (C) were observed under CLMS. Scale bars = 50 µm (A, B) and 20 µm (C). View largeDownload slide Fig. 7 Relocalization of TSA1–GFP and GFP–TSK during mitosis. Tobacco BY-2 cells expressing TSA1–GFP were synchronized for the cell cycle by treatment with aphidicolin and propyzamide. Cells were fixed, immunostained with anti-α-tubulin antibody and observed under CLMS. Cells at interphase (A), prophase (B), metaphase (C), anaphase (D) and telophase (E) are shown. Blue, green and red indicate signals of 4′,6-diamidino-2-phenylindole (DAPI), GFP and α-tubulin, respectively. Arrows indicate the chromosomes. Tobacco BY-2 cells expressing GFP (F) or GFP–TSK (G) were stained with propidium iodide (PI) and observed under CLSM. Arrows indicate the chromosomes. Green and red indicate signals of GFP and PI, respectively. Scae bars = 20 µm. View largeDownload slide Fig. 7 Relocalization of TSA1–GFP and GFP–TSK during mitosis. Tobacco BY-2 cells expressing TSA1–GFP were synchronized for the cell cycle by treatment with aphidicolin and propyzamide. 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TI - An Arabidopsis Protein with a Novel Calcium-binding Repeat Sequence Interacts with TONSOKU/MGOUN3/BRUSHY1 Involved in Meristem Maintenance
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pci155
DA - 2005-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/an-arabidopsis-protein-with-a-novel-calcium-binding-repeat-sequence-KlTE7ZQ08k
SP - 1452
EP - 1461
VL - 46
IS - 9
DP - DeepDyve
ER -