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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.
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