Fig. 4. Interaction of EMF2 and CLF in yeast and in vitro. Two-hybrid assays were performed in yeast strain HF7c. For nutritional assays, three independent transformants were stamped onto –LW and –LWH media. Growth on –LW selects for markers carried on the bait and prey plasmids, whereas growth on –LWH also indicates activity of the HIS3 reporter gene. ß-Galactosidase activity was quantified using the assay and units of Miller (Miller, 1972; Miller, 1992). Each value is an average from assays of three independent transformants; the standard error of the mean is also indicated. (A) The EMF2 protein is shown schematically, with the zinc finger motif indicated by the black box and the conserved VEFS domain by the blue box. The uppermost row is a control to show that the CLF bait does not have transcriptional activation activity by itself and cannot interact with an `empty' GAL4-TA prey. The smallest region of EMF2 that was sufficient for interaction with CLF comprised residues 510-631. (B) The CLF protein is shown schematically, with the C5 domain indicated in orange and the CXC region, which precedes the SET domain, shown in turquoise. All prey fusions that contained an intact C5 domain were able to interact with the EMF2-VEFS domain (427-631). The shortest region of CLF sufficient for interaction comprised residues 257-331. (C) Split ubiquitin assay using the system of Kim et al. (Kim et al., 2002). CLF protein, lacking the C-terminal SET domain, was fused to the C-terminal half of ubiquitin (CUB) and the EMF2 VEFS domain was fused to an N-terminal portion of a modified ubiquitin (NUB). The NUB and CUB peptides are unable to interact on their own. Interaction of NUB and CUB fusions reconstitutes ubiquitin activity and results in proteolysis of a URA3 reporter. This allows growth on media containing FOA. Growth on –HW medium selects for the markers on the CUB and NUB constructs. (D) (lane A) In-vitro binding of CLF C5 domain and EMF2 VEFS domain. Bacterial extract containing His₆–EMF2 VEFS protein was tested for binding to GST–CLF C5 (lane B) or GST (lane C). Proteins that bound to GST or GST-CLF C5 were separated by SDS–PAGE, transferred to PVDF membrane, and incubated with anti-His₆ antibodies. The input lane (lane A) contains 1.5% of the volume of bacterial extract used in the binding assay. The lower band in the input lane corresponds to a His₆–EMF2 VEFS degradation product. Note that this is not bound by GST-CLF C5.