Figure 2.
Sro7-NT peptide but not the mutant Sro7-NT-A5,A6 peptide stimulates exocyst-mediated in vitro vesicle tethering. (A) Vesicles labeled with the lipid dye FM4-64 were isolated from a sec6-4 mutant strain expressing GFP-Sec4 and used in an in vitro vesicle-vesicle tethering assay with suboptimal concentrations of purified Sro7, identical amounts of wild-type exocyst complex, and purified Sro7-NT (aa 1–90) or Sro7-NT-5A,6A peptides. Vesicle-vesicle tethered clusters were automatically detected (see Materials and methods for details) and quantified as the percentage of fluorescence seen in clusters over the total fluorescence of the image using the TRITC channel. Error bars represent SD obtained from counting images at 60× magnification. 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. Scale bar, 5 μm. (B) Labeled vesicles were incubated with purified exocyst and Sro7 or exocyst and wild-type Sro7-NT peptide or mutant Sro7-NT-5A,6A peptide as described above. Clusters were automatically detected and quantified as a percentage of total fluorescence with significance annotated as above. Scale bar, 5 μm. (C) Labeled vesicles were incubated with exocyst and either wild-type or mutant Sro7-NT peptides [Sro7-NT; Sro7-NT-A5,A6; Sro7-NT-A7,A8; Sro7-NT-A9,A10 or Sro7-NT-A(4-8)]. Clusters were automatically detected and quantified as a percentage of total fluorescence with significance annotated as above. Scale bar, 5 μm. Coomassie of the peptides used is shown to the right. (D) Wild-type SRO7 and the Sro7 charge-to-alanine mutants SRO7-A5,A6, SRO7-A7,A8, SRO7-A9,A10, and SRO7-A(4-8), and vector only (CEN) were transformed into a sec15-1 strain. The growth of three independent transformants is shown for each sro7 mutant, under permissive and restrictive conditions. (E) Wild-type SRO7 and the Sro7 charge-to-alanine mutants SRO7-A5,A6, SRO7-A7,A8, SRO7-A9,A10, and SRO7-A(4-8), and vector only (CEN) were transformed into an sro7Δ;sro77Δ strain. The growth of three independent transformants is shown for each sro7 mutant, under permissive and restrictive conditions. Source data are available for this figure: SourceData F2.

Sro7-NT peptide but not the mutant Sro7-NT-A5,A6 peptide stimulates exocyst-mediated in vitro vesicle tethering. (A) Vesicles labeled with the lipid dye FM4-64 were isolated from a sec6-4 mutant strain expressing GFP-Sec4 and used in an in vitro vesicle-vesicle tethering assay with suboptimal concentrations of purified Sro7, identical amounts of wild-type exocyst complex, and purified Sro7-NT (aa 1–90) or Sro7-NT-5A,6A peptides. Vesicle-vesicle tethered clusters were automatically detected (see Materials and methods for details) and quantified as the percentage of fluorescence seen in clusters over the total fluorescence of the image using the TRITC channel. Error bars represent SD obtained from counting images at 60× magnification. 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. Scale bar, 5 μm. (B) Labeled vesicles were incubated with purified exocyst and Sro7 or exocyst and wild-type Sro7-NT peptide or mutant Sro7-NT-5A,6A peptide as described above. Clusters were automatically detected and quantified as a percentage of total fluorescence with significance annotated as above. Scale bar, 5 μm. (C) Labeled vesicles were incubated with exocyst and either wild-type or mutant Sro7-NT peptides [Sro7-NT; Sro7-NT-A5,A6; Sro7-NT-A7,A8; Sro7-NT-A9,A10 or Sro7-NT-A(4-8)]. Clusters were automatically detected and quantified as a percentage of total fluorescence with significance annotated as above. Scale bar, 5 μm. Coomassie of the peptides used is shown to the right. (D) Wild-type SRO7 and the Sro7 charge-to-alanine mutants SRO7-A5,A6, SRO7-A7,A8, SRO7-A9,A10, and SRO7-A(4-8), and vector only (CEN) were transformed into a sec15-1 strain. The growth of three independent transformants is shown for each sro7 mutant, under permissive and restrictive conditions. (E) Wild-type SRO7 and the Sro7 charge-to-alanine mutants SRO7-A5,A6, SRO7-A7,A8, SRO7-A9,A10, and SRO7-A(4-8), and vector only (CEN) were transformed into an sro7Δ;sro77Δ strain. The growth of three independent transformants is shown for each sro7 mutant, under permissive and restrictive conditions. Source data are available for this figure: SourceData F2.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal