Figure 5.
Biochemical characterization of EXO84 dominant mutants. (A) Immunoblot analysis of exocyst subunits in wild-type and EXO84 dominant mutant strains. C-terminally myc-tagged Sec8 (SEC8-myc) was introduced by transformation into strains containing wild-type or dominant mutants of EXO84 (on CEN plasmids) as the sole source of Exo84 in the cell. Cells were grown, lysed, and subjected to native immunoprecipitations with myc antibody to isolate and analyze the exocyst complex. Shown are immunoblots using antibodies against all subunits of the exocyst. (B and C) Vesicles labeled with the lipid dye FM4-64 were isolated from a sec6-4 mutant strain expressing Sec4-GFP and used in the in vitro vesicle-vesicle tethering assay with identical amounts of exocyst containing wild-type Exo84, Exo84-N12I, Exo84-L365W, Exo84-E626V, Exo70-I114F, or buffer only in the presence of Sro7 in B or in the absence of Sro7 in C. Scale bar, 5 μm. Vesicle-vesicle tethering was automatically detected and quantified as the percentage of fluorescence seen in clusters over the total fluorescence of the 60× magnification image. Error bar represents SD; P values were obtained using a two-tailed Student’s t test. *, P < 0.05; **, P < 0.01; *** P < 0.001; ns = no significant difference. (D) Purified wild-type exocyst and exocyst containing Exo84-N12I, Exo84-L365W, or Exo84-E626V were used in a GST pulldown assay with GST-Sec4-GTPγS immobilized on beads. Quantitation of the binding is shown to the right as well as a Coomassie stain of GST-fusion Sec4 proteins used. Error bars represent SD; P values were obtained using a two-tailed Student’s t test with significance annotated as above. (E and F) Purified wild-type exocyst, and exocyst containing Exo84-L365W or Exo84-E626V were bound to immobilized GST-Sec4-GTPγS or control GST-Sec4-GDP beads. Quantitation of binding was conducted as described above and is shown on the right. Error bars represent SD; P values were obtained using a two-tailed Student’s t test with significance annotated as above. Source data are available for this figure: SourceData F5.

Biochemical characterization of EXO84 dominant mutants. (A) Immunoblot analysis of exocyst subunits in wild-type and EXO84 dominant mutant strains. C-terminally myc-tagged Sec8 (SEC8-myc) was introduced by transformation into strains containing wild-type or dominant mutants of EXO84 (on CEN plasmids) as the sole source of Exo84 in the cell. Cells were grown, lysed, and subjected to native immunoprecipitations with myc antibody to isolate and analyze the exocyst complex. Shown are immunoblots using antibodies against all subunits of the exocyst. (B and C) Vesicles labeled with the lipid dye FM4-64 were isolated from a sec6-4 mutant strain expressing Sec4-GFP and used in the in vitro vesicle-vesicle tethering assay with identical amounts of exocyst containing wild-type Exo84, Exo84-N12I, Exo84-L365W, Exo84-E626V, Exo70-I114F, or buffer only in the presence of Sro7 in B or in the absence of Sro7 in C. Scale bar, 5 μm. Vesicle-vesicle tethering was automatically detected and quantified as the percentage of fluorescence seen in clusters over the total fluorescence of the 60× magnification image. Error bar represents SD; P values were obtained using a two-tailed Student’s t test. *, P < 0.05; **, P < 0.01; *** P < 0.001; ns = no significant difference. (D) Purified wild-type exocyst and exocyst containing Exo84-N12I, Exo84-L365W, or Exo84-E626V were used in a GST pulldown assay with GST-Sec4-GTPγS immobilized on beads. Quantitation of the binding is shown to the right as well as a Coomassie stain of GST-fusion Sec4 proteins used. Error bars represent SD; P values were obtained using a two-tailed Student’s t test with significance annotated as above. (E and F) Purified wild-type exocyst, and exocyst containing Exo84-L365W or Exo84-E626V were bound to immobilized GST-Sec4-GTPγS or control GST-Sec4-GDP beads. Quantitation of binding was conducted as described above and is shown on the right. Error bars represent SD; P values were obtained using a two-tailed Student’s t test with significance annotated as above. Source data are available for this figure: SourceData F5.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal