Fig. 1. Sly1^ts protein has a lower binding affinity for Sed5 than Sly1 protein. (A) Recombinant genes encoding myc-tagged Sly1, Sly1-20 and Sly1^ts on CEN plasmids under its native promoter were expressed in the sly1^ts strain. Lysates of the sly1^ts strains containing vector, Sly1-6myc, Sly1-20-6myc or Sly1^ts-6myc were prepared with glass beads and 1% Triton X-100, and immunoprecipitation was performed using an anti-myc monoclonal IgG and equal starting amounts of lysate. The Sly1-6myc/Sly1-20-6myc/Sly1^ts-6myc protein and Sed5 protein immunoprecipitated by anti-myc antibody were detected by western blotting. The bands of Sly1-6myc/Sly1-20-6myc/Sly1^ts-6myc and Sed5 are indicated. (B) MBP-fusion proteins of Sed5TMD and its coiled-coil regions with a C-terminal myc tag were produced in E. coli and purified by amylose resin. GST-fusion Sly1/Sly1^ts proteins were also produced and purified by glutathione-Sepharose 4B. Proteins were collected by anti-myc IgG and Protein A-Sepharose beads from the mixture of MBP-Sed5-myc (3 µg) and GST-Sly1 (6 µg, lane 1); MBP-Sed5-myc and GST-Sly1^ts (6 µg, lane 2); MBP-Sed5H1-myc (3 µg) and GST-Sly1 (lane 3); MBP-Sed5H1-myc and GST-Sly1^ts (lane 4); MBP-Sed5H3-myc (3 µg) and GST-Sly1 (lane 5); MBP-Sed5H3-myc and GST-Sly1^ts (lane 6). Note that equal amounts of GST-Sly1 and GST-Sly1^ts proteins were used for the binding assay (6 µg). The bands of MBP-Sed5-myc, MBP-Sed5H1-myc, MBP-Sed5H3-myc and GST-Sly1/Sly1^ts are indicated by arrowheads on the right. Positions of molecular mass markers are shown on the left in kDa.

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