Figure 1
Direct specific interaction between Cos2 and Fu. (A). GST pull down assay: In vitro translated, 35S-methionine-labeled Fu, Fu1-305, Fu306-805 or Su(fu) before (Input, Lanes 1, 4, 7 and 10) or after incubation with equal amount of, respectively, GST (used as a control; Lanes 2, 5, 8 and 11) or GST-Cos2 (Lanes 3, 6, 9 and 12). Input equals one third of the amount used in the pull down assay. (B). Yeast two hybrid assay: Left: β-galactosidase assay, Right: leucine assay. Each dot corresponds to a yeast diploid coexpressing, respectively, various regions of Cos2 (as shown below) or Rab3 fused to the DNA binding domain of LexA (DBD LexA) (rows I to V) and respectively Fu306-805, Fu437-805, GGTIIβ fused to the transactivation domain B42 (columns 1 to 3). Both assays were performed using RFY231-pSH18-34 (Rows I to IV) or EGY189-pSH18-34 (Row V). The negative control assays with Rab3 give the same result in both strains (Row I and data not shown). In combination with Fureg, Cos538-751 leads to even higher reporter activation than does Cos538-1201. This could be due to numerous causes as differences in stability of the protein fusion, in their nuclear targeting, and/or affinity for Fureg. Cos2: heptad repeats domain (AA643-990), in black Cos2 motor domain (AA172-357) in grey.