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The fission yeast ptr1+ gene involved in nuclear mRNA export encodes a putative ubiquitin ligase
BBRC Biochemical and Biophysical Research Communications 317 (2004) 1138–1143 www.elsevier.com/locate/ybbrc
The fission yeast ptr1+ gene involved in nuclear mRNA export encodes a putative ubiquitin ligase Tomoko Andoh,a,1 Abul Kalam Azad,b,1,2 Asako Shigematsu,b Yasumi Ohshima,b and Tokio Tania,* a b
Department of Biological Science, Faculty of Science, Kumamoto University, Kurokami, Kumamoto 860-8555, Japan Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Hakozaki, Fukuoka 812-8581, Japan Received 5 March 2004
Abstract Fission yeast ptr1-1 is one of the mRNA transport mutants that accumulate poly(A)þ RNA in the nuclei at the nonpermissive temperature. We found that the ptr1þ gene encodes a homolog of Saccharomyces cerevisiae Tom1p, a hect type ubiquitin ligase. In ptr1-1, a conserved amino acid in the hect domain of Ptr1p is mutated. The ptr1þ gene is essential for growth and its mutation did not affect nuclear protein export. A ptr1-1 rae1-167 double mutant showed a synthetic effect on a growth defect, indicating that Ptr1p functionally interacts with an essential mRNA export factor Rae1p. We also isolated a multi-copy suppressor for ptr1-1 and found that it is the mpd2þ gene isolated as a multi-copy suppressor of cdc7-PD1. Ó 2004 Elsevier Inc. All rights reserved. Keywords: ptr1þ ; mRNA export; Ubiquitin ligase; Fission yeast
In eukaryotic cells, export of mRNA from the nucleus to the cytosol is an essential step for protein production. Several factors affecting nuclear mRNA export have been identified in yeast, using genetic approaches (for a review, [1]). Most of the identified factors are conserved among eukaryotes. For example, mRNA carrier proteins, Mex67p and Yra1p, and a component of the spliceosome, Sub2p, are involved in mRNA export in yeast cells, and their mammalian homologs also play important roles in mRNA export [1]. Yra1p and Sub2p were reported to be associated with the THO complex that plays a role in transcription elongation in yeast [2]. Mutations in mRNA 30 processing factors, including poly(A) polymerase, also affect mRNA transport [3]. Thus, it was suggested that the export system couples with transcription, splicing, and the 30 end formation of mRNA. *
Corresponding author. Fax: +81-96-342-3461. E-mail address: [email protected] (T. Tani). 1 Both authors contributed equally to this work. 2 Present address: University of Oxford, South Parks Road, Oxford OX13RE, UK. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.03.171
By the screening of a ts mutant bank with in situ hybridization, we isolated six mutants named ptr1 to ptr6, which are defective in mRNA export at the nonpermissive temperature [4,5]. The ptr3þ gene was found to encode the ubiquitin-activating enzyme E1, suggesting that the ubiquitin system functions in mRNA export from the nucleus in Schizosaccharomyces pombe [4]. Ubiquitination of a protein is known to be a signal for degradation by the proteasome and for internalization into the lysosomal system, as well as a modification of function of a target protein (for a review, [6]). The mechanism that the ubiquitin system affects the mRNA export machinery, however, remains unclear. Ubiquitin conjugation is mediated by three types of enzymes, E1, E2, and E3. Ubiquitin is activated by E1, transferred to E2 (ubiquitin-conjugating enzyme), and finally conjugated to a target protein by E3 (ubiquitin– protein ligase). Hect type ubiquitin ligase is one of the E3s categorized into the three types (hect, RING-finger, and U-box types) and possesses a domain homologous to the carboxyl-terminus of a human E3 enzyme, E6-AP (for a review, [7]). We have now characterized a ptr1 mutant and found that the ptr1þ gene encodes a putative
T. Andoh et al. / Biochemical and Biophysical Research Communications 317 (2004) 1138–1143
homolog of Saccharomyces cerevisiae Tom1p, a hect type E3 enzyme. Our results suggest that a hect type ubiquitin ligase plays an important role in the mRNA export system.
Materials and methods Strains, media, and culture. rae1-167 (h rae1-167 leu1-32 ura4-D18) strain [8] was kindly provided by Dr. Ravi Dhar (NIH, Maryland). The other S. pombe strains used in this study were as follows: 972 (h ), HM123 (h leu1-32), UDP6 (h =hþ leu1-32/leu1-32 ura4-D18/ura4D18 ade6-M210/ade6-M216), UDP6Dptr1 (h =hþ ptr1::kanMX6/ptr1þ leu1-32/leu1-32 ura4-D18/ura4-D18 ade6-M210/ade6-M216), ptr1-1 (h ptr1-1 leu1-32), and ptr1-1 rae1-167 (h ptr1-1 rae1-167 leu1-32 ura4-D18). Standard cultures and the general genetic method used for S. pombe were as described [9]. Plasmids and a library. pSP1 and S. pombe genomic library constructed in pSS10 were provided by Dr. Tomohiro Matsumoto (Kyoto University, Japan). pR1GLFPA6 used for a protein export and import assay was obtained from Dr. Minoru Yoshida (RIKEN, Japan). Disruption of the ptr1þ gene. Disruption of ptr1þ was done by replacement of the 7.1-kb HindIII–StuI fragment (nucleotides +1010 to +8138) in the ORF of the ptr1þ gene with the kanMX6 gene of pFA6akanMX6 [10]. Disruption was confirmed by PCR using as a template genomic DNA of G418-resistant transformants [10]. Fluorescent in situ hybridization. To examine the intracellular distribution of poly(A)þ RNA in yeast, fluorescent in situ hybridization was done essentially as described [4]. Briefly, cells that shifted to the nonpermissive temperature of 37 °C for the indicated times were fixed with 4% paraformaldehyde and subjected to in situ hybridization with the biotin-labeled oligo(dT)50 probe, followed by treatment with FITC conjugated avidin. The samples were finally stained with DAPI and observed under an Olympus AX70 fluorescence microscope equipped with a Photometrics Quantix cooled CCD camera. Analysis of nuclear protein import and export. To determine if the ptr1-1 mutant has a defect in nucleocytoplasmic transport of proteins, pR1GLFPA6 [11] expressing a GFP-tagged fusion protein that contains a nuclear export signal (NES) and a nuclear localization signal (NLS) derived from Pap1p and SV40, respectively, was introduced into the ptr1-1 mutant. After culturing overnight at 26 °C in MM with thiamine, the cells were washed with sterilized water, transferred to fresh MM without thiamine, cultured for 18 h, then shifted to 37 °C for 4 h, and treated with leptomycin B (LMB) at a concentration of 200 ng/ ml for 30 min. Localization of the GFP-tagged protein was then examined using an Olympus AX70 fluorescence microscope equipped with a Photometrics Quantix cooled CCD camera.
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restriction site mapping, we found that those clones could be categorized into two groups represented by #c1 and #c15. The cosmid clones represented by #c15 suppressed the growth defect of the ptr1-1 mutant at 37 °C both on YPD and MM plates, whereas the #c1 clones suppressed it at 33 °C but not at 37 °C on a YPD plate (see Fig. 6B), suggesting that #c1 clones contain a suppressor for the mutation. After several steps of subcloning, the activity complementing the ptr1-1 mutation was detected in a 17-kb fragment in the cosmid clone #c15. To determine if the cosmid fragment includes the authentic ptr1þ gene, we did gap repair experiments to identify a mutation site in the genomic region corresponding to the cosmid fragment. As shown in Fig. 1, when the ptr1-1 cells were transformed with the plasmid with the XbaI–XbaI gap, no tsþ transformant was obtained among total 17 transformants. In contrast, plasmids with the XhoI– XhoI gap and with the AatII–AatII gap generated 19 tsþ transformants in total 56 and 9 tsþ in total 54, respectively, suggesting that there are no mutations causing ts between the left AatII site and the right XhoI site in Fig. 1. These results strongly suggest that the region between the right XhoI site and the right XbaI site contains a mutation site. In a database search, one large ORF (SPAC19D.5.04) was found to cover this region and this ORF encodes a putative hect type ubiquitin ligase which is homologous (29.5% overall identity) to S. cerevisiae Tom1p (Fig. 2A). For precise determination of the mutation site, sequence analysis was performed for the 1.8-kb XhoI– XbaI region of the ptr1-1 mutant. A comparison of the determined sequence with that of the ORF SPAC19D5.04 in the database of the Sanger Center revealed a single point mutation in the gene. The point mutation changes T to A at nucleotide position 8660, resulting in replacement of leucine 2887 in the hect domain in the C-terminal region to glutamine (Fig. 2B). We named the SPAC19D5.04 ptr1þ , since presence of a mutation site demonstrated that the SPAC19D5.04 gene is not a suppressor for the ptr1 mutation, but rather an authentic ptr1þ gene.
Results Cloning of the ptr1þ gene We isolated six mutants that showed defects in mRNA export in our previous work [4,5]. Of those, the ptr2þ , ptr3þ , ptr4þ , and ptr6þ genes were cloned [4,5,12]. To isolate the gene responsible for the ptr1-1 mutation, we transformed the mutant with an S. pombe wild-type genomic library constructed in a pSS10 cosmid vector. After transformation, we obtained more than ten clones that complemented the temperature-sensitive growth defect of the ptr1-1 mutant. When the cosmids were recovered from the transformants and subjected to
Fig. 1. Gap repair experiment. The ptr1-1 mutant was transformed with the cosmid clones #c15 cut by the indicated restriction enzymes. Numbers of tsþ clones among the total Leuþ transformants are shown. A, AatII; Xb, XbaI; and Xh, XhoI.
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Fig. 2. The mutation site in the hect domain of Ptr1p. (A) Schematic representation of structures of Ptr1p and S. cerevisiae Tom1p. The striped box shows a hect domain. (B) Comparison of amino acid sequences of the hect domains between Ptr1p and Tom1p. The highlighted residue in the black background was changed from L to Q in the ptr1-1 mutant. Asterisks indicate identical amino acids.
Disruption of the ptr1þ gene To determine if the ptr1þ gene is essential for growth in S. pombe, we prepared a ptr1 disruptant by replacement of the 7.1-kb HindIII–StuI region in the ORF with a kanMX6 marker gene [10]. ptr1þ /ptr1::kanMX6 diploid cells were prepared and subjected to tetrad analysis. As shown in Fig. 3, among four spores, only two formed normal colonies. None of the segregants that formed colonies could grow on a G418 plate, suggesting that they were wild-type cells (data not shown). The other two, ptr1::kanMX6 strains, formed very small colonies
Fig. 3. Tetrad analysis of the ptr1 disruptants. Spores derived from ptr1þ /ptr1::kanMX6 diploid cells were incubated on a YPD plate at 26 °C for 5 days. Each lane contains four spores from one diploid cell.
microscopically observed, but the colonies never became large. Therefore, the ptr1::kanMX6 spores can germinate but divide only several times. Protein import and export in the ptr1-1 mutant In our previous work [4], we showed that the ptr1-1 mutant has no defects in protein import, since a GFP– nucleoplasmin fusion protein localized in the nucleus in the ptr1-1 mutant both at the nonpermissive and permissive temperatures. We further examined whether the ptr1-1 mutant has defects in protein export. Wild-type and the ptr1-1 mutant cells transformed with the pR1GLFPA6 plasmid [11], which carries a gene for GST–NES–NLS–GFP fusion protein, were cultured at 26 °C and shifted to 37 °C for 4 h. The intracellular distribution of the fusion protein was then observed. In both the ptr1-1 mutant and the wild-type cells, the fusion protein distributed throughout the cells at 37 °C (Fig. 4). However, addition of LMB led to the accumulation of the fusion protein in the nucleus in both cells, again demonstrating no defects in protein import in the ptr1-1 mutant (Fig. 4). The cytoplasmic distribution of the fusion protein in the absence of LMB also
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Multi-copy suppressor of the ptr1-1 mutation We next analyzed another cosmid clone that suppressed the growth defect of the ptr1-1 mutant at 33 °C on a YPD plate. Subcloning and partial sequence analysis revealed that the mpd2þ gene in the cosmid clone possesses suppressor activity (Fig. 6A). The multicopy of mpd2þ suppressed the growth defect of the ptr11 mutant at 33 °C on a YPD plate and at 37 °C on an MM plate, on which the ptr1-1 mutant could grow at 33 °C (Fig. 6B). The mpd2þ gene was originally isolated as a multi-copy suppressor of the cdc7-D1 mutant [15].
Fig. 4. Protein import and export in the ptr1-1 mutant. Wild-type and the ptr1-1 mutant cells transformed with pR1GLFPA6 plasmid were cultured at 26 °C, shifted to 37 °C for 4 h, and then observed under a fluorescence microscope. In the LMB column, cells were further incubated with LMB at 37 °C for 30 min after the 4 h-incubation. GFP denotes distribution of the GFP fusion protein, and DNA shows the cells stained with Hoechst 33342.
implies no major defects in protein export. Thus, we concluded that protein export and import are normal in the ptr1-1 mutant. Growth defect of the ptr1-1 rae1-167 double mutant As described above, we showed that blockage of nuclear export in the ptr1-1 mutant is specific for poly(A)þ mRNA. To determine the role of Ptr1p in mRNA export, we examined genetic interaction between ptr1 and rae1 by preparing a ptr1-1 rae1-167 double mutant. Rae1p is known as one of the essential mRNA export factors in S. pombe and is localized in the periphery of the nucleus [13,14]. As shown in Fig. 5, the ptr1-1 and rae1-167 single mutants grew at 28 °C and formed colonies after 3-day incubation. However, compared to the single mutants, the ptr1-1 rae1-167 double mutant showed very slow growth at the same temperature. Such synthetic growth defect indicates that Ptr1p functionally interacts with Rae1p in S. pombe.
Fig. 5. Synthetic growth defect of the ptr1-1 rae1-167 double mutant. Wild-type, ptr1-1, rae1-167, and ptr1-1 rae1-167 cells were incubated on a YPD plate at 28 °C for 3 days.
Fig. 6. Multi-copy suppressor of the ptr1-1 mutation. (A) Schematic representation of a 4.3-kb fragment including the mpd2þ gene in the pSP1-mpd2þ plasmid. The black box and the striped box indicate an intron and a sequence coding for a GYF domain, respectively. In the pSP1-mpd2þ plasmid, the fragment of nucleotides )692 to +3561 of the mpd2þ gene is inserted between BamHI and PstI sites of the pSP1 vector. (B) Suppression of the temperature-sensitive growth of the ptr1-1 mutant. The ptr1-1 mutant cells were transformed with pSP1ptr1þ , pSP1-mpd2þ , or pSP1. Transformants were incubated on YPD and MM plates at indicated temperatures for 3 (33 and 37 °C) or 6 days (26 °C). (C) In situ hybridization of the transformants in (B). Cells were cultured at 26 °C, shifted to 37 °C for 4 h, and then subjected to in situ hybridization with the biotin-labeled oligo(dT)50 probe. The poly(A)þ RNA was visualized using FITC-avidin. DNA was stained with DAPI.
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Cdc7p is a Dbf4p-dependent protein kinase that regulates replication initiation and heterochromatinmediated cohesion [16]. Mpd2p is homologous to S. cerevisiae Smy2p, overexpression of which suppresses a growth defect of the myo2-66 mutant [17]. Both Mpd2p and Smy2p have the GYF domain (Fig. 6A), which is well conserved among eukaryotes [18]. However, precise functions of Mpd2p and Smy2p are unknown. The mpd2þ gene also suppressed the defect of mRNA export of the ptr1-1 mutant (Fig. 6C). Our observations suggest the involvement of mpd2þ in the mRNA export pathway regulated by ptr1þ , although the mpd2 disruptant showed normal growth at any temperature and normal export of mRNA (data not shown).
Discussion Comparison between Ptr1p and other hect type ubiquitin ligases The ptr1þ gene encodes a large protein with a hect domain that is well conserved among eukaryotes, including mammals. Tom1p, known as a ubiquitin ligase E3 in S. cerevisiae, has an overall homology to Ptr1p (Fig. 2). While investigating the ptr1-1 mutant, other authors reported that the tom1 mutant is deficient in mRNA export [19,20]. As the leucine 2887 residue, which is mutated in the ptr1-1 mutant, is also conserved in Tom1p, this residue may be important for functions of these ubiquitin ligases. Recently, Rsp5p, another hect type ubiquitin ligase of S. cerevisiae, was also seen to play a role in mRNA export [21,22], hence hect type ubiquitin ligases are important for mRNA transport. Possible functions of Ptr1p in mRNA export As cellular distribution of GST–NLS–NES–GFP fusion protein was normal in the ptr1-1 mutant, Ptr1p does not seem to be involved in general protein import and export. The tom1 mutant is reported to have a specific defect in the export of Nab2p, an hnRNP protein shuttling between the nucleus and the cytoplasm [20]. Although it is not known how Tom1p regulates the export of Nab2p, control of hnRNP carrying mRNA may be one of the functions of Tom1p, and Ptr1p may have the same function. In this study, we found that the ptr1-1 rae1-167 double mutant caused a synthetic growth defect, suggesting that Ptr1p interacts with Rae1p, an essential export factor in S. pombe. As the rae1-167 mutation is known to be synthetically lethal with mex67 null mutation [8], Mex67p and Rae1p are also functionally related. In S. pombe, overexpression of Mex67p inhibits mRNA export [8]. Interestingly, Mex67p possesses a motif homologous to an UBA domain, which is known to bind
ubiquitin, in its C terminus. Some functions of Ptr1p in mRNA export may be operative through Mex67p, although the mex67 null mutation does not cause defects in the nuclear export of mRNA in S. pombe [8]. Multi-copy suppressor of the ptr1-1 defect The mpd2þ gene is a multi-copy suppressor for growth and mRNA export defects of the ptr1-1 mutant. Although the mechanism of suppression is not clear, the S. cerevisiae two-hybrid database shows two proteins which interact with both Tom1p and Smy2p, a homolog of Mpd2p. Those proteins, Msl5p and Mud2p, are splicing factors. Thus, Smy2p/Mpd2p might associate with Tom1p/Ptr1p via those splicing factors. It is noteworthy that Mud2p is functionally interacting with Sub2p essential for the coupling of pre-mRNA splicing and mRNA export [23]. To elucidate the precise function of Ptr1p in mRNA export, identification of substrates ubiquitinated by Ptr1p in mRNA export system is necessary. Searching of such substrates for Ptr1p is now in progress.
Acknowledgments We thank Dr. M. Yoshida for providing the pR1GLFPA6 plasmid and leptomycin B. Language assistance was provided by M. Ohara (Fukuoka). This work was supported by grants from the Ministry of Education, Science, Technology, Sports and Culture of Japan to T.A. and T.T.
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The fission yeast ptr1+ gene involved in nuclear mRNA export encodes a putative ubiquitin ligase Tomoko Andoh,a,1 Abul Kalam Azad,b,1,2 Asako Shigematsu,b Yasumi Ohshima,b and Tokio Tania,* a b
Department of Biological Science, Faculty of Science, Kumamoto University, Kurokami, Kumamoto 860-8555, Japan Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Hakozaki, Fukuoka 812-8581, Japan Received 5 March 2004
Abstract Fission yeast ptr1-1 is one of the mRNA transport mutants that accumulate poly(A)þ RNA in the nuclei at the nonpermissive temperature. We found that the ptr1þ gene encodes a homolog of Saccharomyces cerevisiae Tom1p, a hect type ubiquitin ligase. In ptr1-1, a conserved amino acid in the hect domain of Ptr1p is mutated. The ptr1þ gene is essential for growth and its mutation did not affect nuclear protein export. A ptr1-1 rae1-167 double mutant showed a synthetic effect on a growth defect, indicating that Ptr1p functionally interacts with an essential mRNA export factor Rae1p. We also isolated a multi-copy suppressor for ptr1-1 and found that it is the mpd2þ gene isolated as a multi-copy suppressor of cdc7-PD1. Ó 2004 Elsevier Inc. All rights reserved. Keywords: ptr1þ ; mRNA export; Ubiquitin ligase; Fission yeast
In eukaryotic cells, export of mRNA from the nucleus to the cytosol is an essential step for protein production. Several factors affecting nuclear mRNA export have been identified in yeast, using genetic approaches (for a review, [1]). Most of the identified factors are conserved among eukaryotes. For example, mRNA carrier proteins, Mex67p and Yra1p, and a component of the spliceosome, Sub2p, are involved in mRNA export in yeast cells, and their mammalian homologs also play important roles in mRNA export [1]. Yra1p and Sub2p were reported to be associated with the THO complex that plays a role in transcription elongation in yeast [2]. Mutations in mRNA 30 processing factors, including poly(A) polymerase, also affect mRNA transport [3]. Thus, it was suggested that the export system couples with transcription, splicing, and the 30 end formation of mRNA. *
Corresponding author. Fax: +81-96-342-3461. E-mail address: [email protected] (T. Tani). 1 Both authors contributed equally to this work. 2 Present address: University of Oxford, South Parks Road, Oxford OX13RE, UK. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.03.171
By the screening of a ts mutant bank with in situ hybridization, we isolated six mutants named ptr1 to ptr6, which are defective in mRNA export at the nonpermissive temperature [4,5]. The ptr3þ gene was found to encode the ubiquitin-activating enzyme E1, suggesting that the ubiquitin system functions in mRNA export from the nucleus in Schizosaccharomyces pombe [4]. Ubiquitination of a protein is known to be a signal for degradation by the proteasome and for internalization into the lysosomal system, as well as a modification of function of a target protein (for a review, [6]). The mechanism that the ubiquitin system affects the mRNA export machinery, however, remains unclear. Ubiquitin conjugation is mediated by three types of enzymes, E1, E2, and E3. Ubiquitin is activated by E1, transferred to E2 (ubiquitin-conjugating enzyme), and finally conjugated to a target protein by E3 (ubiquitin– protein ligase). Hect type ubiquitin ligase is one of the E3s categorized into the three types (hect, RING-finger, and U-box types) and possesses a domain homologous to the carboxyl-terminus of a human E3 enzyme, E6-AP (for a review, [7]). We have now characterized a ptr1 mutant and found that the ptr1þ gene encodes a putative
T. Andoh et al. / Biochemical and Biophysical Research Communications 317 (2004) 1138–1143
homolog of Saccharomyces cerevisiae Tom1p, a hect type E3 enzyme. Our results suggest that a hect type ubiquitin ligase plays an important role in the mRNA export system.
Materials and methods Strains, media, and culture. rae1-167 (h rae1-167 leu1-32 ura4-D18) strain [8] was kindly provided by Dr. Ravi Dhar (NIH, Maryland). The other S. pombe strains used in this study were as follows: 972 (h ), HM123 (h leu1-32), UDP6 (h =hþ leu1-32/leu1-32 ura4-D18/ura4D18 ade6-M210/ade6-M216), UDP6Dptr1 (h =hþ ptr1::kanMX6/ptr1þ leu1-32/leu1-32 ura4-D18/ura4-D18 ade6-M210/ade6-M216), ptr1-1 (h ptr1-1 leu1-32), and ptr1-1 rae1-167 (h ptr1-1 rae1-167 leu1-32 ura4-D18). Standard cultures and the general genetic method used for S. pombe were as described [9]. Plasmids and a library. pSP1 and S. pombe genomic library constructed in pSS10 were provided by Dr. Tomohiro Matsumoto (Kyoto University, Japan). pR1GLFPA6 used for a protein export and import assay was obtained from Dr. Minoru Yoshida (RIKEN, Japan). Disruption of the ptr1þ gene. Disruption of ptr1þ was done by replacement of the 7.1-kb HindIII–StuI fragment (nucleotides +1010 to +8138) in the ORF of the ptr1þ gene with the kanMX6 gene of pFA6akanMX6 [10]. Disruption was confirmed by PCR using as a template genomic DNA of G418-resistant transformants [10]. Fluorescent in situ hybridization. To examine the intracellular distribution of poly(A)þ RNA in yeast, fluorescent in situ hybridization was done essentially as described [4]. Briefly, cells that shifted to the nonpermissive temperature of 37 °C for the indicated times were fixed with 4% paraformaldehyde and subjected to in situ hybridization with the biotin-labeled oligo(dT)50 probe, followed by treatment with FITC conjugated avidin. The samples were finally stained with DAPI and observed under an Olympus AX70 fluorescence microscope equipped with a Photometrics Quantix cooled CCD camera. Analysis of nuclear protein import and export. To determine if the ptr1-1 mutant has a defect in nucleocytoplasmic transport of proteins, pR1GLFPA6 [11] expressing a GFP-tagged fusion protein that contains a nuclear export signal (NES) and a nuclear localization signal (NLS) derived from Pap1p and SV40, respectively, was introduced into the ptr1-1 mutant. After culturing overnight at 26 °C in MM with thiamine, the cells were washed with sterilized water, transferred to fresh MM without thiamine, cultured for 18 h, then shifted to 37 °C for 4 h, and treated with leptomycin B (LMB) at a concentration of 200 ng/ ml for 30 min. Localization of the GFP-tagged protein was then examined using an Olympus AX70 fluorescence microscope equipped with a Photometrics Quantix cooled CCD camera.
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restriction site mapping, we found that those clones could be categorized into two groups represented by #c1 and #c15. The cosmid clones represented by #c15 suppressed the growth defect of the ptr1-1 mutant at 37 °C both on YPD and MM plates, whereas the #c1 clones suppressed it at 33 °C but not at 37 °C on a YPD plate (see Fig. 6B), suggesting that #c1 clones contain a suppressor for the mutation. After several steps of subcloning, the activity complementing the ptr1-1 mutation was detected in a 17-kb fragment in the cosmid clone #c15. To determine if the cosmid fragment includes the authentic ptr1þ gene, we did gap repair experiments to identify a mutation site in the genomic region corresponding to the cosmid fragment. As shown in Fig. 1, when the ptr1-1 cells were transformed with the plasmid with the XbaI–XbaI gap, no tsþ transformant was obtained among total 17 transformants. In contrast, plasmids with the XhoI– XhoI gap and with the AatII–AatII gap generated 19 tsþ transformants in total 56 and 9 tsþ in total 54, respectively, suggesting that there are no mutations causing ts between the left AatII site and the right XhoI site in Fig. 1. These results strongly suggest that the region between the right XhoI site and the right XbaI site contains a mutation site. In a database search, one large ORF (SPAC19D.5.04) was found to cover this region and this ORF encodes a putative hect type ubiquitin ligase which is homologous (29.5% overall identity) to S. cerevisiae Tom1p (Fig. 2A). For precise determination of the mutation site, sequence analysis was performed for the 1.8-kb XhoI– XbaI region of the ptr1-1 mutant. A comparison of the determined sequence with that of the ORF SPAC19D5.04 in the database of the Sanger Center revealed a single point mutation in the gene. The point mutation changes T to A at nucleotide position 8660, resulting in replacement of leucine 2887 in the hect domain in the C-terminal region to glutamine (Fig. 2B). We named the SPAC19D5.04 ptr1þ , since presence of a mutation site demonstrated that the SPAC19D5.04 gene is not a suppressor for the ptr1 mutation, but rather an authentic ptr1þ gene.
Results Cloning of the ptr1þ gene We isolated six mutants that showed defects in mRNA export in our previous work [4,5]. Of those, the ptr2þ , ptr3þ , ptr4þ , and ptr6þ genes were cloned [4,5,12]. To isolate the gene responsible for the ptr1-1 mutation, we transformed the mutant with an S. pombe wild-type genomic library constructed in a pSS10 cosmid vector. After transformation, we obtained more than ten clones that complemented the temperature-sensitive growth defect of the ptr1-1 mutant. When the cosmids were recovered from the transformants and subjected to
Fig. 1. Gap repair experiment. The ptr1-1 mutant was transformed with the cosmid clones #c15 cut by the indicated restriction enzymes. Numbers of tsþ clones among the total Leuþ transformants are shown. A, AatII; Xb, XbaI; and Xh, XhoI.
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Fig. 2. The mutation site in the hect domain of Ptr1p. (A) Schematic representation of structures of Ptr1p and S. cerevisiae Tom1p. The striped box shows a hect domain. (B) Comparison of amino acid sequences of the hect domains between Ptr1p and Tom1p. The highlighted residue in the black background was changed from L to Q in the ptr1-1 mutant. Asterisks indicate identical amino acids.
Disruption of the ptr1þ gene To determine if the ptr1þ gene is essential for growth in S. pombe, we prepared a ptr1 disruptant by replacement of the 7.1-kb HindIII–StuI region in the ORF with a kanMX6 marker gene [10]. ptr1þ /ptr1::kanMX6 diploid cells were prepared and subjected to tetrad analysis. As shown in Fig. 3, among four spores, only two formed normal colonies. None of the segregants that formed colonies could grow on a G418 plate, suggesting that they were wild-type cells (data not shown). The other two, ptr1::kanMX6 strains, formed very small colonies
Fig. 3. Tetrad analysis of the ptr1 disruptants. Spores derived from ptr1þ /ptr1::kanMX6 diploid cells were incubated on a YPD plate at 26 °C for 5 days. Each lane contains four spores from one diploid cell.
microscopically observed, but the colonies never became large. Therefore, the ptr1::kanMX6 spores can germinate but divide only several times. Protein import and export in the ptr1-1 mutant In our previous work [4], we showed that the ptr1-1 mutant has no defects in protein import, since a GFP– nucleoplasmin fusion protein localized in the nucleus in the ptr1-1 mutant both at the nonpermissive and permissive temperatures. We further examined whether the ptr1-1 mutant has defects in protein export. Wild-type and the ptr1-1 mutant cells transformed with the pR1GLFPA6 plasmid [11], which carries a gene for GST–NES–NLS–GFP fusion protein, were cultured at 26 °C and shifted to 37 °C for 4 h. The intracellular distribution of the fusion protein was then observed. In both the ptr1-1 mutant and the wild-type cells, the fusion protein distributed throughout the cells at 37 °C (Fig. 4). However, addition of LMB led to the accumulation of the fusion protein in the nucleus in both cells, again demonstrating no defects in protein import in the ptr1-1 mutant (Fig. 4). The cytoplasmic distribution of the fusion protein in the absence of LMB also
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Multi-copy suppressor of the ptr1-1 mutation We next analyzed another cosmid clone that suppressed the growth defect of the ptr1-1 mutant at 33 °C on a YPD plate. Subcloning and partial sequence analysis revealed that the mpd2þ gene in the cosmid clone possesses suppressor activity (Fig. 6A). The multicopy of mpd2þ suppressed the growth defect of the ptr11 mutant at 33 °C on a YPD plate and at 37 °C on an MM plate, on which the ptr1-1 mutant could grow at 33 °C (Fig. 6B). The mpd2þ gene was originally isolated as a multi-copy suppressor of the cdc7-D1 mutant [15].
Fig. 4. Protein import and export in the ptr1-1 mutant. Wild-type and the ptr1-1 mutant cells transformed with pR1GLFPA6 plasmid were cultured at 26 °C, shifted to 37 °C for 4 h, and then observed under a fluorescence microscope. In the LMB column, cells were further incubated with LMB at 37 °C for 30 min after the 4 h-incubation. GFP denotes distribution of the GFP fusion protein, and DNA shows the cells stained with Hoechst 33342.
implies no major defects in protein export. Thus, we concluded that protein export and import are normal in the ptr1-1 mutant. Growth defect of the ptr1-1 rae1-167 double mutant As described above, we showed that blockage of nuclear export in the ptr1-1 mutant is specific for poly(A)þ mRNA. To determine the role of Ptr1p in mRNA export, we examined genetic interaction between ptr1 and rae1 by preparing a ptr1-1 rae1-167 double mutant. Rae1p is known as one of the essential mRNA export factors in S. pombe and is localized in the periphery of the nucleus [13,14]. As shown in Fig. 5, the ptr1-1 and rae1-167 single mutants grew at 28 °C and formed colonies after 3-day incubation. However, compared to the single mutants, the ptr1-1 rae1-167 double mutant showed very slow growth at the same temperature. Such synthetic growth defect indicates that Ptr1p functionally interacts with Rae1p in S. pombe.
Fig. 5. Synthetic growth defect of the ptr1-1 rae1-167 double mutant. Wild-type, ptr1-1, rae1-167, and ptr1-1 rae1-167 cells were incubated on a YPD plate at 28 °C for 3 days.
Fig. 6. Multi-copy suppressor of the ptr1-1 mutation. (A) Schematic representation of a 4.3-kb fragment including the mpd2þ gene in the pSP1-mpd2þ plasmid. The black box and the striped box indicate an intron and a sequence coding for a GYF domain, respectively. In the pSP1-mpd2þ plasmid, the fragment of nucleotides )692 to +3561 of the mpd2þ gene is inserted between BamHI and PstI sites of the pSP1 vector. (B) Suppression of the temperature-sensitive growth of the ptr1-1 mutant. The ptr1-1 mutant cells were transformed with pSP1ptr1þ , pSP1-mpd2þ , or pSP1. Transformants were incubated on YPD and MM plates at indicated temperatures for 3 (33 and 37 °C) or 6 days (26 °C). (C) In situ hybridization of the transformants in (B). Cells were cultured at 26 °C, shifted to 37 °C for 4 h, and then subjected to in situ hybridization with the biotin-labeled oligo(dT)50 probe. The poly(A)þ RNA was visualized using FITC-avidin. DNA was stained with DAPI.
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Cdc7p is a Dbf4p-dependent protein kinase that regulates replication initiation and heterochromatinmediated cohesion [16]. Mpd2p is homologous to S. cerevisiae Smy2p, overexpression of which suppresses a growth defect of the myo2-66 mutant [17]. Both Mpd2p and Smy2p have the GYF domain (Fig. 6A), which is well conserved among eukaryotes [18]. However, precise functions of Mpd2p and Smy2p are unknown. The mpd2þ gene also suppressed the defect of mRNA export of the ptr1-1 mutant (Fig. 6C). Our observations suggest the involvement of mpd2þ in the mRNA export pathway regulated by ptr1þ , although the mpd2 disruptant showed normal growth at any temperature and normal export of mRNA (data not shown).
Discussion Comparison between Ptr1p and other hect type ubiquitin ligases The ptr1þ gene encodes a large protein with a hect domain that is well conserved among eukaryotes, including mammals. Tom1p, known as a ubiquitin ligase E3 in S. cerevisiae, has an overall homology to Ptr1p (Fig. 2). While investigating the ptr1-1 mutant, other authors reported that the tom1 mutant is deficient in mRNA export [19,20]. As the leucine 2887 residue, which is mutated in the ptr1-1 mutant, is also conserved in Tom1p, this residue may be important for functions of these ubiquitin ligases. Recently, Rsp5p, another hect type ubiquitin ligase of S. cerevisiae, was also seen to play a role in mRNA export [21,22], hence hect type ubiquitin ligases are important for mRNA transport. Possible functions of Ptr1p in mRNA export As cellular distribution of GST–NLS–NES–GFP fusion protein was normal in the ptr1-1 mutant, Ptr1p does not seem to be involved in general protein import and export. The tom1 mutant is reported to have a specific defect in the export of Nab2p, an hnRNP protein shuttling between the nucleus and the cytoplasm [20]. Although it is not known how Tom1p regulates the export of Nab2p, control of hnRNP carrying mRNA may be one of the functions of Tom1p, and Ptr1p may have the same function. In this study, we found that the ptr1-1 rae1-167 double mutant caused a synthetic growth defect, suggesting that Ptr1p interacts with Rae1p, an essential export factor in S. pombe. As the rae1-167 mutation is known to be synthetically lethal with mex67 null mutation [8], Mex67p and Rae1p are also functionally related. In S. pombe, overexpression of Mex67p inhibits mRNA export [8]. Interestingly, Mex67p possesses a motif homologous to an UBA domain, which is known to bind
ubiquitin, in its C terminus. Some functions of Ptr1p in mRNA export may be operative through Mex67p, although the mex67 null mutation does not cause defects in the nuclear export of mRNA in S. pombe [8]. Multi-copy suppressor of the ptr1-1 defect The mpd2þ gene is a multi-copy suppressor for growth and mRNA export defects of the ptr1-1 mutant. Although the mechanism of suppression is not clear, the S. cerevisiae two-hybrid database shows two proteins which interact with both Tom1p and Smy2p, a homolog of Mpd2p. Those proteins, Msl5p and Mud2p, are splicing factors. Thus, Smy2p/Mpd2p might associate with Tom1p/Ptr1p via those splicing factors. It is noteworthy that Mud2p is functionally interacting with Sub2p essential for the coupling of pre-mRNA splicing and mRNA export [23]. To elucidate the precise function of Ptr1p in mRNA export, identification of substrates ubiquitinated by Ptr1p in mRNA export system is necessary. Searching of such substrates for Ptr1p is now in progress.
Acknowledgments We thank Dr. M. Yoshida for providing the pR1GLFPA6 plasmid and leptomycin B. Language assistance was provided by M. Ohara (Fukuoka). This work was supported by grants from the Ministry of Education, Science, Technology, Sports and Culture of Japan to T.A. and T.T.
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