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. 2014 Nov;34(21):3955–3967. doi: 10.1128/MCB.00077-14

FIG 3.

FIG 3

Interactions with CPF involve more than one region of Mpe1. (A) Pulldown assays with extracts prepared from cells expressing Myc-His6-tagged forms of mpe1, mpe1-ΔRING, or mpe1-ΔUBL. Cells were grown at 30°C. Pulldown assays were performed using nickel affinity beads, and the eluates were analyzed by Western blotting with antibodies against CF IA and CPF subunits. Mpe1 was detected with the anti-Myc antibody. The control lane shows a pulldown with an extract from cells expressing only untagged Mpe1. The asterisk indicates a nonspecific band detected with the anti-Myc antibody. The amount of protein pulled down in the mutant relative to wild-type Mpe1 is given under each lane. (B) Yeast two-hybrid analysis reveals an interaction between GBD-Mpe1 and GAD-Cft1. A positive two-hybrid interaction was detected by transcription activation of the HIS3 reporter gene, which allowed for growth on a medium lacking histidine. Pta1 and Ssu72, which have been shown previously to interact with each other (47), were used as a positive control. The pairings of two-hybrid constructs are as follows: lane 1, pGBD-PTA1 plus pGAD-SSU72; lane 2, pGBD-MPE1 plus pGAD; lane 3, pGBD plus pGAD-CFT1; lane 4, pGBD-MPE1 plus pGAD-CFT1. (C) Schematic representation of the different Mpe1 regions encoded by pGBD plasmids. Numbers indicate the amino acid boundaries of each Mpe1 fragment. UBL, ubiquitin-like; ZK, zinc knuckle. (D) The region containing the zinc knuckle domain of Mpe1 and the second linker mediates the two-hybrid interaction with Cft1. Yeast two-hybrid analyses were performed as described for panel B. The pairings of two-hybrid constructs are as follows: lane 1, pGBD-PTA1 plus pGAD-SSU72; lane 2, pGBD-ZK plus pGAD; lane 3, pGBD plus pGAD-CFT1; lane 4, pGBD-UBL plus pGAD-CFT1; lane 5, pGBD-ZK plus pGAD-CFT1; lane 6, pGBD-RING plus pGAD-CFT1. (E) Mpe1 interacts with Cft1 in vitro. GST pulldown assays were performed with purified recombinant GST or GST-Mpe1 and 35S-labeled in vitro-translated Cft1. The input lane represents 1/5 of the in vitro-translated Cft1 used for binding.