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. 2005 Nov 17;24(23):4133–4143. doi: 10.1038/sj.emboj.7600873

Figure 1.

Figure 1

BARS is a peripheral Golgi membrane protein critical for reconstituting COPI vesicles. (A) GAP is no longer sufficient to complete the second stage of the vesicle reconstitution system when Golgi membrane is washed with 3 M KCl. CHO Golgi membrane was subjected to salt washes as indicated and then used for the two-stage incubation system, followed by immunoblotting of pellet (P) and supernatant (S) for β-COP after the second-stage incubation. (B) The critical activity lost upon washing Golgi membrane with 3 M KCl contains BARS. The 3 M KCl wash fraction was analyzed by gel filtration with eluted fractions monitored for total proteins (top panel), assayed for activity in restoring COPI vesicle formation (middle panel), and immunoblotted for BARS (bottom panel). (C) Endogenous BARS on Golgi membrane is markedly reduced by 3 M KCl wash. CHO Golgi membrane is subjected to salt washes as indicated, followed by immunoblotting for proteins as indicated. The level of the transmembrane KDELR is used to assess the levels of Golgi membrane in each condition. (D) The addition of BARS restores the ability of GAP to complete the second-stage incubation using Golgi membrane washed with 3 M KCl. CHO Golgi membrane washed with 3 M KCl was used for the two-stage incubation system, followed by immunoblotting of pellet (P) and supernatant (S) for β-COP after the second-stage incubation. (E) Antibody against endogenous BARS on Golgi membrane inhibits the reconstitution of COPI vesicles. CHO Golgi membrane washed with 0.5 M KCl was incubated with an anti-BARS antibody or an anti-mannosidase I antibody (as control), and then subjected to the two-stage incubation system, followed by immunoblotting of pellet (P) and supernatant (S) for β-COP after the second-stage incubation.