Figure 1.

Interactions of UP9C and LSU proteins. (A) The pJK1 and pJK2 plasmids contain UP9C in the BD- and AD-vectors, respectively. The yeast strains co-transformed with indicated BD- and AD-plasmids were grown on selective media without leucine and tryptophan (SD-LT), leucine, tryptophan and adenine (SD-LTA), leucine, tryptophane and histidine (SD-LTH) or were screened for β-galactosidase expression (+X-gal). (B) The protein gel blot shown on the left-hand side verifies expression of the GST-fusion proteins in the extracts from bacteria producing GST-ZZ (2b), GsT-Joka8 (8) and GST-Joka20 (20); the GST-PB1 protein was not detected. The protein gel blot shown on the righthand side shows results of the “pull-down” assay performed as described in Materials and Methods. The results confirm interaction of UP9c with Joka8, Joka20 and the ZZ domain of Joka2; the extract containing His-tagged UP9C protein (C+) was loaded as a positive control and the arrows indicate the positions of the proteins corresponding to the expected sizes of the recombinant proteins: GST-ZZ (54.5 kDa), GST-Joka8 (66.5 kDa), GST-Joka20 (42 kDa) and His-UP9C (17.2 kDa). (C) The scheme of the protein (truncated NpJoka2) encoded by the insert present in pJoka2. The position of EcoRI used for the subcloning is shown and the domains PB1 and ZZ are indicated. (D) The pJK11 plasmid contains the insert as pJoka2 but in pGAD424. Plasmids pLUs1–4 contain the corresponding orfs (LSU1–4 from Arabidopsis) cloned into pGBT9. The remaining explanations as in (A).