title: Purification and characterization of the fission yeast telomere clustering factors, Bqt1 and Bqt2.
abstract: During meiosis, chromosomes adopt a bouquet arrangement, which is widely conserved among eukaryotes. This arrangement is assumed to play an important role in the normal progression of meiosis, by mediating the proper pairing of homologous chromosomes. In Schizosaccharomyces pombe, the complex of Bqt1 and Bqt2 plays a key role in telomere clustering and the subsequent bouquet arrangement of chromosomes during early meiotic prophase. Bqt1 and Bqt2 are part of a multi-protein complex that mediates the attachment of the telomere to the nuclear membrane. However, the structural details of the complex are needed to clarify the mechanism of telomere clustering. To enable biophysical studies of Bqt1 and Bqt2, we established a purification procedure for the Schizosaccharomyces japonicus Bqt1-Bqt2 complex, which is closely related to the S. pombe Bqt1-Bqt2 complex. A co-expression vector, in which one of the expressed subunits is fused to a removable SUMO tag, yielded high amounts of the proteins in the soluble fraction. The solubility of the Bqt1-Bqt2 complex after the removal of the SUMO tag was maintained by including CHAPS, a nondenaturing, zwitterionic detergent, in the purification buffers. These procedures enabled us to rapidly purify the stable Bqt1-Bqt2 complex. The co-purified Bqt1 and Bqt2 proteins formed a stable heterodimer, consistent with results from in vivo studies showing the requirement of both proteins for the bouquet arrangement. The expression and purification procedures established here will facilitate further biophysical studies of the Bqt1-Bqt2 complex.
authors: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162 8480, Japan.
authors: Yuichi
authors: Y
authors: Ichikawa
authors: Wataru
authors: W
authors: Kagawa
authors: Kengo
authors: K
authors: Saito
authors: Yuji
authors: Y
authors: Chikashige
authors: Tokuko
authors: T
authors: Haraguchi
authors: Yasushi
authors: Y
authors: Hiraoka
authors: Hitoshi
authors: H
authors: Kurumizaka
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012568:Schizosaccharomyces / Q000737:chemistry / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D034501:Telomere-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: C510171:Bqt1 protein, S pombe; C510172:Bqt2 protein, S pombe; D011993:Recombinant Fusion Proteins; C099362:SMT3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D034501:Telomere-Binding Proteins