Nuclear Import of Spo12p, a Protein Essential for Meiosis

In Saccharomyces cerevisiae, Spo12p is involved in mitosis and is essential for meiosis. We found that Spo12p is imported into the nucleus by the karyopherin Kap121p. A complex containing Spo12p and Kap121p was isolated from cytosol and was also reconstituted with recombinant proteins, indicating that this interaction is direct. Spo12p was mislocalized to the cytosol inpse1–1, a temperature-sensitive strain harboring a mutation of Kap121p, at the permissive temperature, confirming an essential role for Kap121p in Spo12p import. Spo12p was also mislocalized in a pse1–1/pse1–1 homozygous strain, suggesting it is imported via the same pathway in diploid cells. Furthermore, we found that pse1–1/pse1–1 shows a sporulation defect similar to that ofspo12Δ/spo12Δ. In addition, we have characterized the Spo12p nuclear localization signal, mapped it to residues 76–130, and identified residues within this region that are important for nuclear localization signal function. In Saccharomyces cerevisiae, Spo12p is involved in mitosis and is essential for meiosis. We found that Spo12p is imported into the nucleus by the karyopherin Kap121p. A complex containing Spo12p and Kap121p was isolated from cytosol and was also reconstituted with recombinant proteins, indicating that this interaction is direct. Spo12p was mislocalized to the cytosol inpse1–1, a temperature-sensitive strain harboring a mutation of Kap121p, at the permissive temperature, confirming an essential role for Kap121p in Spo12p import. Spo12p was also mislocalized in a pse1–1/pse1–1 homozygous strain, suggesting it is imported via the same pathway in diploid cells. Furthermore, we found that pse1–1/pse1–1 shows a sporulation defect similar to that ofspo12Δ/spo12Δ. In addition, we have characterized the Spo12p nuclear localization signal, mapped it to residues 76–130, and identified residues within this region that are important for nuclear localization signal function. nuclear pore complex karyopherin nuclear localization signal nuclear export sequence classical NLS Protein A fromS. aureus matrix-assisted laser desorption ionization/time of flight mass spectrometry matrix-assisted laser desorption ionization/time of flight polyacrylamide gel electrophoresis glutathione S-transferase green fluorescent protein polymerase chain reaction transport buffer differential interference contrast Bidirectional exchange of macromolecules between the nucleus and the cytoplasm occurs through a large complex embedded in the nuclear envelope, the nuclear pore complex (NPC).1 InSaccharomyces cerevisiae (herein referred to as yeast), the NPC is composed of about 30 proteins (nucleoporins), which provide docking sites for a family of soluble and structurally related transport factors, karyopherins (also known as importins/exportins/transportins). In addition to docking at the NPC, karyopherins bind cargo to be transported into and out of the nucleus (see Refs. 1Görlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1674) Google Scholar and 2Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (212) Google Scholar for a review). They recognize specific sequence elements in their cognate transport substrates that constitute the signal for import (nuclear localization sequence, or NLS) or export (nuclear export sequence, or NES). Kapβ1 needs an adapter (Kapα) in order to bind to most substrates. Kapα recognizes proteins containing a classical NLS (cNLS), which consists of one or two stretches of basic amino acid residues (3Fontes M.R. Teh T. Kobe B. J. Mol. Biol. 2000; 297: 1183-1194Crossref PubMed Scopus (313) Google Scholar, 4Conti E. Kuriyan J. Struct. Fold. Des. 2000; 8: 329-338Abstract Full Text Full Text PDF Scopus (254) Google Scholar). In yeast, there is one Kapα and 14 Kapβs (herein referred to as kaps). The kaps share a low but significant identity (about 20%), most striking at the amino terminus (1Görlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1674) Google Scholar, 2Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (212) Google Scholar, 5Görlich D. Dabrowski M. Bischoff F.R. Kutay U. Bork P. Hartmann E. Prehn S. Izaurralde E. J. Cell Biol. 1997; 138: 65-80Crossref PubMed Scopus (350) Google Scholar). To date, all yeast kaps except Kap120p have been shown to function in import or in export. The involvement of a kap in both processes has recently been discovered (6Yoshida K. Blobel G. J. Cell Biol. 2001; 152: 729-740Crossref PubMed Scopus (109) Google Scholar), and it remains to be elucidated whether more share the same ability. There are hundreds of yeast nuclear proteins or proteins with a nuclear life style, but only 14 kaps. This necessarily means that some kaps will be responsible for the transport of a large number of cargoes. Sequences of the NLSs that have been mapped so far do not resemble the cNLS or each other. Hence, the basis for kap/substrate specificity and the overall mechanism of substrate recognition by karyopherins remain to be further elucidated. Meiosis is one of the few differentiation pathways in yeast, and its regulation necessarily depends on the expression control of subsets of genes. Regulation of nuclear transport has been increasingly recognized as an important mechanism for the control of gene expression (see Ref.7Kaffman A. O'Shea E.K. Annu. Rev. Cell Dev. Biol. 1999; 15: 291-339Crossref PubMed Scopus (261) Google Scholar for a review). It is not known how most of the proteins involved in meiosis are transported into the nucleus to participate in its regulation. Spo12p was first identified as a protein whose absence causes sporulating diploid cells to skip one meiotic division, leading to the formation of two diploid spores (8Klapholz S. Esposito R.E. Genetics. 1980; 96: 567-588Crossref PubMed Google Scholar). More recently, its overexpression has been found to suppress a number of late mitotic mutants (9Jaspersen S.L. Charles J.F. Tinker-Kulberg R.L. Morgan D.O. Mol. Biol. Cell. 1998; 9: 2803-2817Crossref PubMed Scopus (263) Google Scholar, 10Aerne B.L. Johnson A.L. Toyn J.H. Johnston L.H. Mol. Biol. Cell. 1998; 9: 945-956Crossref PubMed Scopus (51) Google Scholar). Consistent with a role in exit from mitosis, cells in G2/M are over-represented in a spo12Δpopulation (11Parkes V. Johnston L.H. Nucleic Acids Res. 1992; 20: 5617-5623Crossref PubMed Scopus (30) Google Scholar). Spo12p is synthetically lethal with Dbf2p, a kinase required for events in anaphase/telophase and has been suggested by genetic studies to be an activator of Dbf2p and its relative Dbf20p (12Toyn J.H. Johnston L.H. Genetics. 1993; 135: 963-971Crossref PubMed Google Scholar). Its biological function remains unknown, and its functional differences in mitosis and meiosis await further clarification. Spo12p has been shown to be nuclear in both haploid and diploid cells (13Grether M.E. Herskowitz I. Mol. Biol. Cell. 1999; 10: 3689-3703Crossref PubMed Scopus (24) Google Scholar). Here we report that in haploid cells Spo12p is imported into the nucleus by Kap121p. Kap121p is a previously characterized karyopherin, first implicated in ribosomal protein import (14Rout M.P. Blobel G. Aitchison J.D. Cell. 1997; 89: 715-725Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar) and mRNA export (15Seedorf M. Silver P.A Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8590-8595Crossref PubMed Scopus (116) Google Scholar) and later shown to be responsible for import of the transcription factors Pho4p and Pdr1p (16Kaffman A. Rank N.M. O'Shea E. Genes Dev. 1998; 12: 2673-2683Crossref PubMed Scopus (205) Google Scholar, 17Delahodde A. Pandjaitan R. Corral-Debrinski M. Jacq C. Mol. Microbiol. 2001; 39: 304-313Crossref PubMed Scopus (39) Google Scholar) (see “Discussion”). Evidence for import of Spo12p by Kap121p includes binding to Kap121p both in vivo and in vitro and mislocalization of Spo12p to the cytosol in pse1–1, a temperature-sensitive strain of Kap121p. We have also characterized the Spo12p NLS and identified residues within this region that are important for NLS function. Since Spo12p has a role in both mitosis and meiosis, we sought to determine whether the import pathway differs in diploid cells. In this case, we found that Spo12p was mislocalized in apse1–1/pse1–1 homozygous strain, indicating that it is imported using the same pathway in both haploid and diploid cells. The procedures for yeast manipulation were as described (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A diploid strain expressing Spo12p-Protein A (PrA) was obtained by integrative transformation of DF5 (19Finley D. Ozkaynak E. Varshavsky A. Cell. 1987; 48: 1035-1046Abstract Full Text PDF PubMed Scopus (633) Google Scholar) with the coding sequence of four and a half IgG binding repeats of Staphylococcus aureus protein A immediately upstream of the SPO12 stop codon as described (20Titov A. Blobel G. J. Cell Biol. 1999; 147: 235-245Crossref PubMed Scopus (39) Google Scholar). Haploid strains were obtained by sporulation and tetrad dissection. Proper integration was assessed by PCR and Western blot analysis.kap142Δ (6Yoshida K. Blobel G. J. Cell Biol. 2001; 152: 729-740Crossref PubMed Scopus (109) Google Scholar), kap123Δ (14Rout M.P. Blobel G. Aitchison J.D. Cell. 1997; 89: 715-725Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar),pse1–1, PSY580, and PSY581(15), kap122Δ (20Titov A. Blobel G. J. Cell Biol. 1999; 147: 235-245Crossref PubMed Scopus (39) Google Scholar),kap114Δ (21Pemberton L.F. Rosenblum J.S. Blobel G. J. Cell Biol. 1999; 145: 1407-1417Crossref PubMed Scopus (99) Google Scholar), kap119Δ (22Albertini M. Pemberton L.F. Rosenblum J.S. Blobel G. J. Cell Biol. 1998; 143: 14747Crossref Scopus (53) Google Scholar),kap104Δ (23Aitchison J.D. Blobel G. Rout M.P. Science. 1996; 274: 624-627Crossref PubMed Scopus (270) Google Scholar), kap111Δ (24Pemberton L.F. Rosenblum J.S. Blobel G. J. Cell Biol. 1997; 139: 1645-1653Crossref PubMed Scopus (92) Google Scholar), andkap108Δ (25Rosenblum J.S. Pemberton L.F. Blobel G. J. Cell Biol. 1997; 147: 1655-1661Crossref Scopus (72) Google Scholar) have been described previously. Apse1–1/KAP121 heterozygous strain was generated by mating PSY581 with pse1–1. After sporulation and tetrad dissection of pse1–1/KAP121, pse1–1 (mating type α) was obtained. A pse1–1/pse1–1 homozygous diploid strain was generated by mating pse1–1 with pse1–1(α). The entire coding region of SPO12 was PCR-amplified from S. cerevisiae genomic DNA and inserted into pYX242-GFP (26Rosenblum J.S. Pemberton L.F. Bonifaci N. Blobel G. J. Cell Biol. 1998; 143: 887-899Crossref PubMed Scopus (58) Google Scholar) using primer-encoded EcoRI andHindIII sites to generate an in-frame fusion with the green fluorescent protein (GFP), pSpo12-GFP. pSpo12-GFP derivatives pSpo121–143-GFP, pSpo121–90-GFP, pSpo1276–173-GFP, pSpo1291–173-GFP, pSpo12116–173-GFP, pSpo1276–143-GFP, pSpo1276–130-GFP, pSpo1276–115-GFP, and pSpo1291–130-GFP were generated similarly. A PCR fragment containing a deletion of amino acids 76–130 of Spo12p (Δ76–130) was created by the overlap extension method and cloned into pYX242-GFP as described above to generate pSpo12Δ76–130-GFP. Alanine-scanning mutants in the region 76–130 in pSpo12-GFP were generated by the same method. Oligonucleotides that sequentially introduced five alanine codons in place of wild-type sequences were used. Detailed information on plasmid construction is available upon request. Yeast strains were transformed with plasmids via the lithium acetate method or electroporation (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Spo12p fragments 1–173, 76–130, and Δ76–130 were PCR-amplified from pSpo12-GFP, pSpo1276–130-GFP, and pSpo12Δ76–130-GFP, and ligated into the bacterial expression vector pGEX4TTev (27Chook Y.M. Blobel G. Nature. 1999; 399: 230-237Crossref PubMed Scopus (293) Google Scholar) using primer-encodedEcoRI and SalI sites, resulting in N-terminal fusions with GST. All constructs were verified by sequencing analysis. hLa-GFP and Lhp1-GFP (26Rosenblum J.S. Pemberton L.F. Bonifaci N. Blobel G. J. Cell Biol. 1998; 143: 887-899Crossref PubMed Scopus (58) Google Scholar), GST-Kap114p (21Pemberton L.F. Rosenblum J.S. Blobel G. J. Cell Biol. 1999; 145: 1407-1417Crossref PubMed Scopus (99) Google Scholar), and pGEX-Kap121p (28Marelli M. Aitchison J.D. Wozniak R.W. J. Cell Biol. 1998; 143: 1813-1830Crossref PubMed Scopus (133) Google Scholar) were described previously. XL1-blue and BL21 gold (Stratagene, La Jolla, CA) were used for general cloning and expression purposes. Bacterial strains were transformed by heat shock. Post-nuclear, post-ribosomal cytosol was prepared from a strain expressing Spo12p-PrA grown to an A600 of 1.8 as described (23Aitchison J.D. Blobel G. Rout M.P. Science. 1996; 274: 624-627Crossref PubMed Scopus (270) Google Scholar). Spo12p-PrA-interacting proteins were immunoisolated by overnight incubation of cytosol with IgG-Sepharose at 4 °C. After washing with transport buffer (TB: 20 mm HEPES, pH 7.5, 110 mm KOAc, 2 mm MgCl2, 0.1% Tween 20), proteins were eluted with a step gradient of MgCl2 and concentrated using methanol-chloroform precipitation prior to SDS-PAGE analysis. Gels were stained with Coomassie Blue, and proteins of interest were excised, digested, and analyzed by MALDI-TOF mass spectrometry (MS) (29Gharahdaghi F. Kirchner M. Fernandez J. Mische S.M. Anal. Biochem. 1996; 233: PubMed Scopus Google Scholar). Gels were also to and Western procedures and were as described were grown at to an A600 of in containing and with mm for 2 were and in buffer mm mm 20 mm pH containing 20 and After was at for and the with for 20 at were with and proteins eluted with In the of Kap121p and were and in containing of or of overnight at 4 °C. was by addition of of All proteins were by the method. 2 of recombinant and were on of a of in by incubation for 30 at After was with 2 of Kap121p or for 30 in containing mm and The was with of and proteins were eluted with buffer and analyzed by SDS-PAGE and Coomassie using and were similarly. was overnight of yeast strains in or on for the of or one in of was on a All were with a on a were with a using and to Spo12p was previously to the nucleus (13Grether M.E. Herskowitz I. Mol. Biol. Cell. 1999; 10: 3689-3703Crossref PubMed Scopus (24) Google Scholar). proteins involved in import with their substrates in the prior to their import into the the specific factors the leading to the of the cargo and the of the kap to the cytosol for of import. To determine which karyopherins a role in Spo12p we isolated proteins with the of To do we first the Spo12p terminus in with the IgG binding of S. aureus This in the expression of Spo12p-PrA from the SPO12 We prepared a post-ribosomal of this After incubation with proteins were eluted with a step gradient of The eluted were analyzed by SDS-PAGE and Coomassie The of a are shown in A. by of the of to is to only with a of the of is the by an that eluted only in the A eluted at A protein with an mass of eluted in the the of MgCl2 to the complex The was excised, with and resulting to MALDI-TOF analysis. of a of were with the mass of resulting from the of Kap121p. The of isolated Kap121p to be in of this is a we on the complex proteins have in of MgCl2, which in a of between the of isolated proteins and the on the We also sought to determine of Kap121p was with After of Spo12p-PrA in the described were on an SDS-PAGE gel and to Western blot with a Kap121p that the of Kap121p in the cytosol remains in the and is with Spo12p Since the complex was isolated from it is that this interaction is by we whether the interaction between Spo12p and Kap121p is direct. Spo12p-PrA from yeast cytosol was with recombinant and proteins eluted with After SDS-PAGE and Coomassie we binding of in addition to the Kap121p not We and on and with recombinant Kap121p or which was used as a The was proteins were eluted with buffer and analyzed by SDS-PAGE and Coomassie in we found that Kap121p to We not kap to only a of to relative to Kap121p. We that Spo12p and Kap121p To determine the role of Kap121p in Spo12p nuclear we the localization of Spo12p to in In an the fusion was the control of the from a low number but was not We a plasmid to Spo12p to from a number which was found to be nuclear in wild-type cells as and In significant mislocalization to the cytoplasm was inpse1–1, a temperature-sensitive strain a mutation in Kap121p, at the permissive La cerevisiae (also known as which are imported via the and (26Rosenblum J.S. Pemberton L.F. Bonifaci N. Blobel G. J. Cell Biol. 1998; 143: 887-899Crossref PubMed Scopus (58) Google Scholar), were to the nucleus in pse1–1, that import pathways were not in this strain not We whether Kap121p is also responsible for Spo12p import into the nucleus of diploid cells. We found that Spo12p in the nucleus of a wild-type diploid strain and was mislocalized in heterozygous strain significant mislocalization to the cytoplasm was in a strain suggesting the import pathway is the same in both haploid and diploid cells. We whether pse1–1/pse1–1 a sporulation similar to that ofspo12Δ/spo12Δ. We found the cells that the number of containing two spores was at in pse1–1/pse1–1 in wild-type cells of with two in each of four were for each The were and the in a in each of four were for each The were and the In order to in Spo12p that are for we deletion mutants of Spo12p in with and analyzed their localization in vivo We found the region containing amino acids 76–130 to be both and for import into the nucleus we some mislocalization of to the This was or constructs were used suggesting that the kap/substrate leading to transport The fusion is mislocalized to the cytosol in pse1–1 indicating that it is to the nucleus in a To further this we whether this region was also and for interaction with Kap121p. recombinant and were on and with recombinant Kap121p. After proteins were eluted with buffer and analyzed by SDS-PAGE and Coomassie was as a The was in of the by Western blot not suggesting that this protein a that there was binding of Kap121p to this fusion and not to there was more or In order to more which residues of to NLS we by sequentially stretches of residues in this region with mutants were in with and their localization assessed in cells shown in we found that mutation of residues to alanine to significant mislocalization to the the localization of the mutants was from that of the wild-type protein We that Kap121p is the kap used for import of Kap121p was first described as a karyopherin whose overexpression was to for in the import of the ribosomal protein NLS (14Rout M.P. Blobel G. Aitchison J.D. Cell. 1997; 89: 715-725Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). also suggesting an pathway with mRNA export was shown to be in but or pse1–1 (15Seedorf M. Silver P.A Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8590-8595Crossref PubMed Scopus (116) Google Scholar). it be that the defect be It be that import of a protein involved in mRNA export in the More recently, Kap121p was shown to import transcription Pho4p This role was not on Pho4p not with was it mislocalized in kap123Δ (16Kaffman A. Rank N.M. O'Shea E. Genes Dev. 1998; 12: 2673-2683Crossref PubMed Scopus (205) Google Scholar). This was the first Kap121p and were this report was Kap121p was shown to be responsible for import of transcription Pdr1p A. Pandjaitan R. Corral-Debrinski M. Jacq C. Mol. Microbiol. 2001; 39: 304-313Crossref PubMed Scopus (39) Google Scholar). We have shown that Spo12p is imported by Kap121p. We have the two proteins from yeast cytosol and reconstituted this complex with recombinant proteins, which this interaction is direct. Spo12p is mislocalized in pse1–1, but not in of the kap strains a role for Kap121p in Spo12p import. The that some nuclear of Spo12p was in pse1–1 remains to be is that of Kap121p remains in this strain at the permissive (15Seedorf M. Silver P.A Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8590-8595Crossref PubMed Scopus (116) Google Scholar), the Kap121p is for import of some of the In we Spo12p localization in diploid a of the fusion in the cytosol of was there was in wild-type cells and and haploid pse1–1. This to a between the of Kap121p and the of Spo12p nuclear with this There is also the that karyopherin Spo12p in this It has been suggested that some kaps for the absence of suggesting function in pathways and that family have (14Rout M.P. Blobel G. Aitchison J.D. Cell. 1997; 89: 715-725Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, L.F. Rosenblum J.S. Blobel G. J. Cell Biol. 1999; 145: 1407-1417Crossref PubMed Scopus (99) Google Scholar). Since Kap121p is a complex of Spo12p with kap in the absence of Kap121p not be identified in Spo12p absence in sporulating diploid cells in the formation of We found that of Kap121p in sporulating diploid cells to an number of containing two This is with import of Spo12p in we it is Kap121p with a large number of proteins in the cytosol not proteins constitute substrates. of substrates provide more into and it is essential for Kap121p be responsible for import of essential substrates or constitute a pathway in the import of a large number of proteins, and be in for the or of kaps L.F. Rosenblum J.S. Blobel G. J. Cell Biol. 1999; 145: 1407-1417Crossref PubMed Scopus (99) Google Scholar). We have characterized the Spo12p NLS and that the region containing residues 76–130 is both and for import and for binding to Kap121p. we to determine which residues were important for NLS function by alanine we only identified of of five to mislocalization of Spo12p to the cytoplasm for this is that residues the entire NLS with Kap121p. Since the mapped NLS is it be that mutation of five residues to alanine was not in order to a the the that we not a deletion with nuclear that a large of the protein is in order to provide the for interaction with Kap121p. This the one NLS analysis. The for a large to nuclear has been previously. the region containing residues of import. some signal is in contrast with the protein (26Rosenblum J.S. Pemberton L.F. Bonifaci N. Blobel G. J. Cell Biol. 1998; 143: 887-899Crossref PubMed Scopus (58) Google Scholar). residues of are and for import and binding to its import more in the cytoplasm is in with the protein B. G. Bischoff F.R. A. M. E. J. 1998; PubMed Scopus Google Scholar). The Spo12p NLS is between residues and but only a will which residues are required for interaction with Kap121p and constitute the It that it is from the It is and there are some stretches of basic residues in the mapped are not for nuclear mutation of residues to a large number of proteins have it not be that will be imported via the The mapped Spo12p NLS and the Pho4p NLS do not so a of substrate recognition by Kap121p not be The and the Pdr1p NLSs were not shown to bind Kap121p also do not sequence NLS of substrates a of recognition by Kap121p. is the for Kap121p, kaps recognize It that only of a large number of kap/substrate are known we to to this recognition We the for the and and Silver for plasmids and We also Wozniak for the Kap121p We the by We Rosenblum and of the Blobel as as Pemberton and for We are also to Rosenblum and for of this