Figure 4. Dimerization of Cys-Y35A restores its ability to generate COPI-coated vesicles from Golgi membranes. (A) COPI-coated vesicles were reconstituted from Golgi membranes using Arf1-wt, cross-linked Cys-wt (cl-Cys-wt), or cross-linked Cys-Y35A (cl-Cys-Y35A) in the presence of GTPγS and purified coatomer. Vesicles were purified via sucrose density centrifugation. 1% of input (I) and 50% of vesicle (V) fractions were analyzed by SDS-PAGE and Western blotting against δ-COP and Arf1. Non-myrArf1, nonmyristoylated Arf1; myr-Arf1, myristoylated Arf1. (B) Purified vesicles were analyzed by negative staining EM. Bars, 250 nm. (C) Quantification of vesicle formation: 14 image sections of 3-µm2 size were randomly chosen, and the number of vesicles was counted. Error bars represent the standard deviation of the mean of three independent experiments. Molecular masses are given in kilodaltons.
Figure 4.

Dimerization of Cys-Y35A restores its ability to generate COPI-coated vesicles from Golgi membranes. (A) COPI-coated vesicles were reconstituted from Golgi membranes using Arf1-wt, cross-linked Cys-wt (cl-Cys-wt), or cross-linked Cys-Y35A (cl-Cys-Y35A) in the presence of GTPγS and purified coatomer. Vesicles were purified via sucrose density centrifugation. 1% of input (I) and 50% of vesicle (V) fractions were analyzed by SDS-PAGE and Western blotting against δ-COP and Arf1. Non-myrArf1, nonmyristoylated Arf1; myr-Arf1, myristoylated Arf1. (B) Purified vesicles were analyzed by negative staining EM. Bars, 250 nm. (C) Quantification of vesicle formation: 14 image sections of 3-µm2 size were randomly chosen, and the number of vesicles was counted. Error bars represent the standard deviation of the mean of three independent experiments. Molecular masses are given in kilodaltons.

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