Figure 5.

Purified Sar1p shows enhanced recruitment and binds GalNAcT2. (A) Untreated or BFA-treated permeabilized NRK cells were incubated without or with Sar1p in absence or presence of GTPγS. Recovery of membrane bound Sar1p and the loading control GPP130 was determined by immunoblotting. The quantified result, showing enhanced Sar1p binding to cells with ER-localized Golgi enzymes, is the ratio of Sar1p/GPP130 at each condition (mean ± SD; n = 2). (B) Purified Sar1p was tested for binding to the cytoplasmic domain peptide of GalNAcT2 (wild type [wt] = MRRRSRC) or a version with alanine substituted for arginine (R→A = MAAASAC). After incubation in the presence or absence of GTPγS, recovery of Sar1p bound to unconjugated (Ø) or peptide-conjugated thiopropyl Sepharose 6B beads was determined by immunoblotting. Each reaction contained 0.5 μg Sar1p, and 10% of this was also analyzed (T). The quantified results showing the percentage bound indicate a specific interaction (mean ± SD; n = 2).