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. 2014 Mar 27;3(4):297–306. doi: 10.1242/bio.20146866

Fig. 3. Limited trypsin digestion time course for co-expressed wild-type and T7-Ste2-F204S mutant receptors.

Fig. 3.

Purified plasma membranes were incubated with trypsin in the absence (−) or presence (+) of 10−8 M α-factor (αF). Resulting cleavage products were analyzed by SDS-PAGE and western blotting. Digestion reaction I contained membranes from cells expressing T7-Ste2-F204S and no wild-type Ste2 (strain DJ213-7-3 containing plasmid pDJ655). Digestion reaction II contained membranes from cells expressing T7-Ste2-F204S and over-produced wild-type Ste2 (strain DJ484-1 containing plasmid pDJ655). Membranes from cells expressing HA-Ste2 (strain DJ213-6-3::pDJ281) were added to both digestion reactions as an internal control. (A) Digestion reaction I probed with anti-T7 antiserum. (B) Digestion reaction II probed with anti-T7 antiserum. (C) Digestion reaction I probed with anti-HA antiserum. (D) Digestion reaction II probed with anti-HA antiserum. (E) Control experiment showing that Ste2-F204S receptors (T7-Ste2-F204S,N25Q,N32Q) undergo qualitatively similar ligand-induced changes when the sites are partially occupied at 5×10−6 M α-factor (strain DJ213-7-1 containing plasmid pDJ657). (F) Binding proficient receptors (T7-Ste2-N25Q,N32Q) with 5×10−6 M α-factor (strain DJ213-7-1 containing plasmid pDJ656).