The SPF27 Homologue Num1 Connects Splicing and Kinesin 1-Dependent Cytoplasmic Trafficking in Ustilago maydis
(A) Schematic representation of the protein-protein interactions (blue arrows) within the human hPrp19/CDC5L-complex . Proteins depicted in blue represent the core complex, additional components (yellow) are associated with individual proteins. Green arrows indicate U. maydis protein interactions identified in this study. (B) The Prp19 N-terminus and the Cef1 C-terminus interact with the full length Num1 protein in a yeast two-hybrid analysis. p53/SV40 T-antigen and Lamin/SV40 T-antigen (Clontech) serve as positive and negative control, respectively. (C) In vivo co-immunoprecipitation of HA-tagged Prp19 and Cef1 and 3eGFP-tagged Num1. α-HA coupled agarose beads were used to precipitate Prp19 and Cef1, respectively. The Num1 protein was detected on a Western blot using an α-GFP antibody. The negative control shows no unspecific binding of the Num1 protein to α-HA agarose. (D) Subcellular localization of the Num1:3eGFP fusion protein. In addition to the nuclear localization of Num1 (enclosed by dashed circle), a cytoplasmic distribution to distinct foci can be observed (arrowhead). Scale bar: 5 µm. (E, F) Num1:3eGFP as well as Cef1:RFP and Prp19:RFP were co-expressed in AB31 under control of their native promoters. Num1 co-localizes with both Cef1 and Prp19 in the nuclei. Additional cytoplasmic Num1:3eGFP foci are indicated by arrowheads. Scale bars: 10 µm.