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. 2013 Nov 11;288(52):37057–37070. doi: 10.1074/jbc.M113.518340

FIGURE 4.

FIGURE 4.

Identification of Rrt6 as a p24-binding protein. A, coprecipitation of Rrt6 with an Erp3/5-containing p24 complex. The ER-enriched P13 (P13) and the Golgi-containing P100 (P100) fractions were isolated from cells expressing either 3myc-Erp5 (Erp5) or 3myc-Rrt6 (Rrt6). From these fractions (P13–3myc-Erp5, P13–3myc-Rrt6, P100–3myc-Erp5, and P100–3myc-Rrt6), the tagged proteins were immunoprecipitated as in Fig. 3A, and proteins in the precipitated fractions were resolved by SDS-PAGE and detected by silver staining. Parental wild-type cells were processed in the same way as control (P13-(−), P100-(−)). Positions of Rrt6 and p24 proteins are shown. B, coprecipitation of Rrt6 with Erp3 and Erp5. Cells expressing one of the 3myc-tagged p24α/γ (lanes 1–6, numbers correspond to the ERP gene numbers), 3myc-tagged Rrt6 (lane R), and control wild-type cells (lane 0) were grown in glycerol medium, and tagged proteins were immunoprecipitated as in Fig. 3A. Proteins in the solubilized fractions (input) and immunoprecipitated fractions (IP and Co-IP) were resolved by SDS-PAGE, and the myc-tagged proteins (anti-myc), Rrt6 (anti-Rrt6), Emp24 (anti-Emp24), and Erv25 (anti-Erv25) were detected by Western blot analysis. Despite that 3myc-Erp6 is functional and has shown to be assembled with Emp24 and Erv25 (Fig. 2A and Table 4), these p24 isoforms were not detected in the 3myc-Erp6 immunoprecipitates in this experiment. We speculate that this is due to the low expression level of 3myc-Erp6. C, steady-state levels of Rrt6 in wild-type (wt), erp1Δ to erp6Δ (Δ1 to Δ6), emp24Δ (Δ24), and erv25Δ (Δ25) cells. Steady-state levels of Rrt6 were determined as in Fig. 1C. D, partial restoration of erv25Δ by overexpression of RRT6. Kar2 secretion was assayed as in Fig. 1A. The graph represents the relative amount of secreted Kar2. Column 1 (spot 1), erv25Δ cells carrying the empty vector. Columns 2–4, erv25Δ cells overexpressing 3myc-RRT6 (column 2), ERP3 and ERP5 (column 3), or 3myc-RRT6, ERP3, and ERP5 (column 4). Column 5, erv25Δ cells expressing 3myc-ERV25. 3myc-Rrt6 used in this experiment has ERV25 promoter and Erv25 signal peptide. Error bars represent S.E. (n = 6). Asterisks indicate p < 0.05.