Abstract
In interphase cells of fission yeast, the spindle pole body (SPB) is thought to be connected with chromosomal centromeres by an as yet unknown mechanism that spans the nuclear membrane. To elucidate this mechanism, we performed two-hybrid screens for proteins that interact with Kms1 and Sad1, which are constitutive membrane-bound components of the SPB that interact with each other. Seven and 26 genes were identified whose products potentially interact with Kms1 and Sad1, respectively. With the exception of Dlc1 (a homolog of the 14-kDa dynein light chain), all of the Kms1 interactors also interacted with Sad1. Among the genes identified were the previously known genes rhp9 +/ crb2 +, cut6 +, ags1 +/ mok1 +, gst3 +, kms2 +, and sid4 +. The products of kms2 + and sid4 + localize to the SPB. The novel genes were characterized by constructing disruption mutations and by localization of the gene products. Two of them, putative homologues of budding yeast UFE1 (which encodes a t-SNARE) and SFH1 (an essential component of a chromatin-remodeling complex), were essential for viability. Two further genes, which were only conditionally essential, genetically interact with sad1 +. One of these was named sif1 + (for Sad1-interacting factor) and is required for proper septum formation at high temperature. Cells in which this gene was overexpressed displayed a wee -like phenotype. The product of the other gene, apm1 +, is very similar to the medium chain of an adaptor protein complex in clathrin-coated vesicles. Apm1 appears to be required for SPB separation and spindle formation, and tended to accumulate at the SPB when it was overproduced. It was functionally distinct from its homologues Apm2 and Apm4. Other novel genes identified in this study included one for a nucleoporin and genes encoding novel membrane-bound proteins that were genetically related to Sad1. We found that none of the newly identified genes tested were necessary for centromere/telomere clustering.
Similar content being viewed by others
References
Adams IR, Kilmartin JV (2000) Spindle pole body duplication: a model for centrosome duplication? Trends Cell Biol 10:329–335
Alfa C, Fantes P, Hyams J, McLeod M, Warbrick E (1993) Experiments with fission yeast: a laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Al-Feel W, DeMar JC, Wakil SJ (2003) A Saccharomyces cerevisiae mutant strain defective in acetyl-CoA carboxylase arrests at the G2/M phase of the cell cycle. Proc Nat Acad Sci USA 100:3095–3100
Al-Khodairy F, Carr AM (1992) DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe. EMBO J 11:1343–1350
Bahler J, Wu J-Q, Longtine MS, Shah NG, McKenzie III A, Steever AB, Wach A, Philippsen P, Pringle JR (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14:943–951
Basi G, Schmid E, Maundrell K (1993) TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123:131–136
Boehm M, Bonifacino JS (2002) Genetic analyses of adaptin function from yeast to mammals. Gene 286:175–186
Bone N, Millar JB, Toda T, Armstrong J (1998) Regulated vacuole fusion and fission in Schizosaccharomyces pombe: an osmotic response dependent on MAP kinases. Curr Biol 8:135–144
Brands A, Ho TH (2002) Function of a plant stress-induced gene, HVA22. Synthetic enhancement screen with its yeast homolog reveals its role in vesicular traffic. Plant Physiol 130:1121–1131
Bridge AJ, Morphew M, Bartlett R, Hagan IM (1998) The fission yeast SPB component Cut12 links bipolar spindle formation to mitotic control. Genes Dev 12:927–942
Byers B (1981) Cytology of the yeast life cycle. In: Strathern JN, Jones EW, Broach JR (eds) Molecular biology of the yeast Saccharomyces. I. Life cycle and inheritance. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 59–96
Cao Y, Cairns BR, Kornberg RD, Laurent BC (1997) Sfh1p, a component of a novel chromatin-remodeling complex, is required for cell cycle progression. Mol Cell Biol 17:3323–3334
Chang L, Gould KL (2000) Sid4p is required to localize components of the septation initiation pathway to the spindle pole body in fission yeast. Proc Natl Acad Sci USA 97:5249–5254
Chikashige Y, Ding D-Q, Imai Y, Yamamoto M, Haraguchi T, Hiraoka Y (1997) Meiotic nuclear reorganization: switching the position of centromeres and telomeres in the fission yeast Schizosaccharomyces pombe. EMBO J 16:193–202
Craven RA, Griffiths DJF, Sheldrick KS, RAndall RE, Hagan IM, Carr AM (1998) Vectors for the expression of tagged proteins in Schizosaccharomyces pombe. Gene 221:59–68
Decottignies A, Zarzov P, Nurse P (2001) In vivo localisation of fission yeast cyclin-dependent kinase cdc2p and cyclin B cdc13p during mitosis and meiosis. J Cell Sci 114:2627–2640
Ding R, West RR, Morphew DM, Oakley BR, McIntosh JR (1997) The spindle pole body of Schizosaccharomyces pombe enters and leaves the nuclear envelope as the cell cycle proceeds. Mol Biol Cell 8:1461–1479
Ding DQ, Chikashige Y, Haraguchi T, Hiraoka Y (1998) Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules, as revealed by continuous observation of chromosomes and microtubules in living cells. J Cell Sci 111:701–712
Ding DQ, Tomita Y, Yamamoto A, Chikashige Y, Haraguchi T, Hiraoka Y (2000) Large-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA library. Genes Cells 5:169–190
Dreger M, Bengtsson L, Schoneberg T, Otto H, Hucho F (2001) Nuclear envelope proteomics: novel integral membrane proteins of the inner nuclear membrane. Proc Natl. Acad Sci USA 98:11943–11948
Esashi F, Yanagida M (1999) Cdc2 phosphorylation of Crb2 is required for reestablishing cell cycle progression after the damage checkpoint. Mol Cell 4:167–174
Flory MR, Morphew M, Joseph JD, Means AR, Davis TN (2002) Pcp1p, an Spc110p-related calmodulin target at the centrosome of the fission yeast Schizosaccharomyces pombe. Cell Growth Differ 13:47–58
Forsberg SL (1993) Comparison of Schizosaccharomyces pombe expression systems. Nucleic Acids Res 21:2955–2956
Funabiki H, Hagan I, Uzawa S, Yanagida M (1993) Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast. J Cell Biol 121:961–976
Galway ME, Heckman JW Jr, Schiefelbein JW (1997) Growth and ultrastructure of the Arabidopsis root hair: the rhd3 mutation alters vacuole enlargement and tip growth. Planta 201:209–218
Goto B, Okazaki K, Niwa O (2001) Cytoplasmic microtubular system implicated in de novo formation of a Rabl-like orientation of chromosomes in fission yeast. J Cell Sci 114:2427–2435
Guertin DA, Venkatram S, Gould KL, McCollum D (2002) Dma1 prevents mitotic exit and cytokinesis by inhibiting the septation initiation network (SIN) Dev Cell 3:779–790
Hagan IM, Petersen J (2000) The microtubule organizing centers of Schizosaccharomyces pombe. Curr Topics Dev Biol 49:133–159
Hagan I, Yanagida M (1995) The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability. J Cell Biol 129:1033–1047
Hayashi N, Murakami S (2002) STM1, a gene which encodes a guanine quadruplex binding protein, interacts with CDC13 in Saccharomyces cerevisiae. Mol Genet Genomics 267:806–813
Hirata A, Shimoda C (1994) Structural modification of spindle pole bodies during meiosis II is essential for the normal formation of ascospores in Schizosaccharomyces pombe: ultrastructural analysis of spo mutants. Yeast 10:173–183
Hirata A, Tanaka K (1982) Nuclear behavior during conjugation and meiosis in the fission yeast Schizosaccharomyces pombe. J Gen Appl Microbiol 28:263–274
Hochstenbach F, Klis FM, van den Ende H, van Donselaar E, Peters PJ, Klausner RD (1998) Identification of a putative alpha-glucan synthase essential for cell wall construction and morphogenesis in fission yeast. Proc Natl Aca Sci USA 95:9161–9166
Hoyt MA (2000) Exit from mitosis: spindle pole power. Cell 102:267–270
Ikemoto S, Nakamura T, Kubo M, Shimoda C (2000) S. pombe sporulation-specific coiled-coil protein Spo15p is localized to the spindle pole body and essential for its modification. J Cell Sci 113:545–554
Jin QW, Fuchs J, Loidl J (2000) Centromere clustering is a major determinant of yeast interphase nuclear organization. J Cell Sci 113:1903–1912
Katayama S, Hirata D, Arellano M, Perez P, Toda T (1999) Fission yeast alpha-glucan synthase Mok1 requires the actin cytoskeleton to localize the sites of growth and plays an essential role in cell morphogenesis downstream of protein kinase C function. J Cell Biol 144:1173–1186
Kniola B, O’Toole E, McIntosh JR, Bellone B, Allshire R, Mengarelli S, Hultenby K, Ekwall K (2001) The domain structure of centromeres is conserved from fission yeast to humans. Mol Biol Cell 12:2764–2775
Knop M, Strasser K (2000) Role of the spindle pole body of yeast in mediating assembly of the prospore membrane during meiosis. EMBO J 19:3657–3667
Lewis MJ, Pelham HR (1996) SNARE-mediated retrograde traffic from the Golgi complex to the endoplasmic reticulum. Cell 85:205–215
MacNeill SA, Moreno S, Reynolds N, Nurse P, Fantes PA (1996) The fission yeast Cdc1 protein, a homologue of the small subunit of DNA polymerase delta, binds to Pol3 and Cdc27. EMBO J 15:4613–4628
Malone CJ, Fixsen WD, Horvitz HR, Han M (1999) UNC-84 localizes to the nuclear envelope and is required for nuclear migration and anchoring during C. elegans development. Development 126:3171–3181
Marelli M, Aitchison JD, Wozniak RW (1998) Specific binding of the karyopherin Kap121p to a subunit of the nuclear pore complex containing Nup53p, Nup59p, and Nup170p. J Cell Biol 143:1813–1830
Maundrell K (1993) Thiamine-repressible expression vectors prep and pRIP for fission yeast. Gene 123:127–130
Miki F, Okazaki K, Shimanuki M, Yamamoto A, Hiraoka Y, Niwa O (2002) The 14-kDa dynein light chain-family protein Dlc1 is required for regular oscillatory nuclear movement and efficient recombination during meiotic prophase in fission yeast. Mol Biol Cell 13:930–946
Miyata M, Doi H, Miyata H, Johnson BF (1997) Sexual coflocculation by heterothallic cells of the fission yeast Schizosaccharomyces pombe modulated by medium constituents. Antonie Leeuwenhoek 71:207–215
Moreno S, Klar A, Nurse P (1991) Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol 194:795–823
Moser MJ, Flory MR, Davis TN (1997) Calmodulin localizes to the spindle pole body of Schizosaccharomyces pombe and performs an essential function in chromosome segregation. J Cell Sci 110:1805–1812
Niwa O, Shimanuki M, Miki F (2000) Telomere-led bouquet formation facilitates homologous chromosome pairing and restricts ectopic interaction in fission yeast meiosis. EMBO J 19:3831–3840
Okamoto CT, McKinney J, Jeng YY (2000) Clathrin in mitotic spindles. Am J Physiol Cell Physiol 279: C369–374
Okazaki K, Okayama H, Niwa O (2000) The polyubiquitin gene is essential for meiosis in fission yeast. Exp Cell Res 254:143–152
Raff JW (1999) Nuclear migration: the missing (L)UNC? Curr Biol 9: R708–R710
Rowley R, Subramani S, Young PG (1992) Checkpoint controls in Schizosaccharomyces pombe: rad1. EMBO J 11:1335–1342
Russell P, Nurse P (1987) Negative regulation of mitosis by wee1 +, a gene encoding a protein kinase homolog. Cell 49:559–567
Saitoh S, Takahashi K, Nabeshima K, Yamashita Y, Nakaseko Y, Hirata A, Yanagida M (1996) Aberrant mitosis in fission yeast mutants defective in fatty acid synthetase and acetyl CoA corboxylase. J Cell Biol 134:949–961
Saka Y, Esashi F, Matsusaka T, Mochida S, Yanagida M (1997) Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with a BRCT motif, with Cut5 and Chk1. Genes Dev 11:3387–3400
Shimanuki M, Miki F, Ding D-Q, Chikashige Y, Hiraoka Y, Horio T, Niwa O (1997) A novel fission yeast gene kms1 +, is required for the formation of meiotic prophase-specific nuclear architecture. Mol Gen Genet 254:238–249
Sohrmann M, Schmidt S, Hagan I, Simanis V (1998) Asymmetric segregation on spindle poles of the Schizosaccharomyces pombe septum-inducing protein kinase Cdc7p. Genes Dev 12:84–94
Sparks CA, Morphew M, McCollum D (1999) Sid2p, a spindle pole body kinase that regulates the onset of cytokinesis. J Cell Biol 146:777–790
Starr DA, Hermann GJ, Malone CJ, Fixsen W, Priess JR, Horvitz HR, Han M (2001) unc-83 encodes a novel component of the nuclear envelope and is essential for proper nuclear migration. Development 128:5039–5050
Stearns T (1997) Motoring to the finish: kinesin and dynein work together to orient the yeast mitotic spindle. J Cell Biol 138:957–960
Stepp JD, Pellicena-Palle A, Hamilton S, Kirchhausen T, Lemmon SK (1995) A late Golgi sorting function for Saccharomyces cerevisiae Apm1p, but not for Apm2p, a second yeast clathrin AP medium chain-related protein. Mol Biol Cell 6:41–58
Tanaka H, Tanaka K, Murakami H, Okayama H (1999) Fission yeast cdc24 is a replication factor C- and proliferating cell nuclear antigen-interacting factor essential for S-phase completion. Mol Cell Biol 19:1038–1048
Tanaka K, Yonekawa T, Kawasaki Y, Kai M, Furuya K, Iwasaki M, Murakami H, Yanagida M, Okayama H (2000) Fission yeast Eso1p is required for establishing sister chromatid cohesion during S phase. Mol Cell Biol 20:3459–3469
Tange Y, Niwa O (1995) A selection system for diploid and against haploid cells in Schizosaccharomyces pombe. Mol Gen Genet 248:644–648
Tange Y, Horio T, Shimanuki M, Ding D-Q, Hiraoka Y, Niwa O (1998) A novel fission yeast gene, tht1 +, is required for the fusion of nuclear envelopes during karyogamy. J Cell Biol 140:247–258
Tange Y, Hirata A, Niwa O (2002) An evolutionarily conserved fission yeast protein, Ned1, implicated in normal nuclear morphology and chromosome stability, interacts with Dis3, Pim1/RCC1 and an essential nucleoporin. J Cell Sci 115:4375–4385
Tomlin GC, Morrell JL, Gould KL (2002) The spindle pole body protein Cdc11p links Sid4p to the fission yeast septation initiation network. Mol Biol Cell 13:1203–1214
Tran PT, Marsh L, Doye V, Inoue S, Chang F (2001) A mechanism for nuclear positioning in fission yeast based on microtubule pushing. J Cell Biol 153:397–411
Trelles-Sticken E, Loidl J, Scherthan H (1999) Bouquet formation in budding yeast: initiation of recombination is not required for meiotic telomere clustering. J Cell Sci 112:651–658
Veal EA, Toone WM, Jones N, Morgan BA (2002) Distinct roles for glutathione S-transferases in the oxidative stress response in Schizosaccharomyces pombe. J Biol Chem 277:35523–35531
Wang H, Lockwood SK, Hoeltzel MF, Schiefelbein JW (1997) The ROOT HAIR DEFECTIVE3 gene encodes an evolutionarily conserved protein with GTP-binding motifs and is required for regulated cell enlargement in Arabidopsis. Genes Dev 11:799–811
West RR, Vaisberg EV, Ding R, Nurse P, McIntosh JR (1998) cut11 +: a gene required for cell cycle-dependent spindle pole body anchoring in the nuclear envelope and bipolar spindle formation in Schizosaccharomyces pombe. Mol Biol Cell 9:2839–2855
Willson J, Wilson S, Warr N, Watts FZ (1997) Isolation and characterization of the Schizosaccharomyces pombe rhp9 gene: a gene required for the DNA damage checkpoint but not the replication checkpoint. Nucleic Acids Res 25:2138–2146
Woods A, Sherwin T, Sasse R, MacRae TH, Baines AJ, Gull K (1989) Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. J Cell Sci 93:491–50
Acknowledgements
This work was supported by the Kazusa DNA Research Institute Foundation.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by C. P. Hollenberg
The first three authors contributed equally to this work
Rights and permissions
About this article
Cite this article
Miki, F., Kurabayashi, A., Tange, Y. et al. Two-hybrid search for proteins that interact with Sad1 and Kms1, two membrane-bound components of the spindle pole body in fission yeast. Mol Genet Genomics 270, 449–461 (2004). https://doi.org/10.1007/s00438-003-0938-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00438-003-0938-8