Download Supplementary Figure 1 - Word file (395 KB )

Supplementary Figure 1
Supplementary Figure 1. In vivo analysis of full length wild-type GFP-Yap1
and Phe302A, Met306A and Val309A mutant derivatives. Full length GFP-Yap1 was
expressed using the YAP1 promoter from a yeast CEN plasmid 6. Point
mutations were made using standard oligonucleotide PCR-based mutagenesis
procedures. GFP-Yap1 plasmids were transformed into an isogenic
yap1::KanMX strain derived from the S. cerevisiae YPH499 parental strain.
Cells were grown to mid-log phase at 30 °C in minimal media containing 0.67%
(w/v) yeast nitrogen bases, 2% (w/v) glucose and amino acid dropout mix
supplemented with adenine and treated with 0.5 mM H2O2 for 5 min. a, Confocal
fluorescence microscopy of wild-type GFP-Yap1 and Phe302A, Met306A and Val309A
mutants before and after treatment with H2O2. Images were taken with a LSM
510 model confocal microscope (Carl Zeiss MicroImaging, Inc.) using 100x planneoflaur objective, 1.3 na lens, 488 nm excitation and a 505-530 nm long pass
filter. Wild-type GFP-Yap1 accumulates in the nucleus upon treatment with
H2O2. The Phe302A and Met306A mutations impaired the ability of Yap1 to
accumulate in the nucleus in response to H2O2. The Val309A mutant behaved
similar to wild-type GFP-Yap1. b, Oxidized and reduced wild-type GFP-Yap1
and Phe302A, Met306A and Val309A mutants extracted from cells before and after
H2O2 treatment. Cell extracts were prepared as described in the materials and
methods section, run on 8% acrylamide SDS-PAGE gels, transferred to
nitrocellulose and probed with anti-GFP monoclonal antibodies. Under nonreducing conditions, wild-type GFP-Yap1 was observed as a faster-migrating
species in the H2O2-treated sample consistent with an oxidized form of the
protein. When the same sample was run under reducing conditions, wild-type
GFP-Yap1 reverted back to a slower-migrating, reduced species. The oxidized
form of Phe302A GFP-Yap1 was not observed for the H2O2-treated sample.
Although a slight amount of oxidized Met306A GFP-Yap1 was observed upon H2O2
treatment, there was considerably less than for the wild-type protein. Val309A
GFP-Yap1 behaved in a manner similar to wild-type GFP-Yap1. Under reducing
conditions, wild-type and Val309A GFP-Yap1 H2O2-treated samples appeared to
be partially phosphorylated. This observation is consistent with previous results
where Myc-tagged Yap1 was observed to be slightly phosphorylated after 5 min
treatment with 0.3 mM H2O2 8.