Role of Hsl1p and Hsl7p catalytic activities in Swe1p regulation. (A)
Myc-tagged wild-type or K110R mutant Hsl1p proteins immunoprecipitated from
yeast lysates (strains JMY1500 and DLY4640) were incubated together with
[γ-32P]ATP to assess Hsl1p autophosphorylation (top) or
immunoblotted to assess protein abundance (bottom). (B) The
GAL1p-MIH1 strains JMY1340 (HSL1), JMY1289
(hsl1Δ), and DLY4994 (hsl1K110R) were grown
on galactose-containing medium and cells were streaked out onto
dextrose-containing medium to repress Mih1p expression. Microcolonies were
photographed after 2 d at 30°C. (C) GST-tagged wild-type or G386A mutant
Hsl7p proteins purified using glutathione-Sepharose were incubated together
with [3H]S-adenosylmethionine and histone H2A to assess
histone methylation (top) or stained with Coomassie Blue to assess protein
abundance (middle, histone H2A; bottom, GST-Hsl7p). (D) The
GAL1p-MIH1 strain JMY1290 (hsl7Δ) was transformed with
plasmids containing HSL7 (pDLB1545), empty vector (YCplac111), or
HSL7G386A (pDLB1608) and microcolonies were analyzed as
described above.