REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity
Figure 8
SGMF analysis of REEP membrane localization.
To differentiate plasma and ER membrane localization of REEPs, sucrose gradient membrane fractionation (SGMF) analysis was performed with HEK293A cells transfected with HA-α2C ARs, and Flag-REEP1, -REEP2, or -REEP6. Forty-eight hrs post-transfection, total cell membranes were isolated and separated by layering upon a discontinuous sucrose gradient. Eleven fractions were collected and analyzed by SDS-PAGE and immunoblotting techniques. Top. Plasma and ER membrane fractions were detected by probing with antibodies against Na/K ATPase (A) or calnexin (B) respectively. Fractions containing lighter plasma membranes (#1-4) and heavier ER membranes (#6-11) are demarcated with bars above the fraction number. Molecular weight markers (M) and input loading control lanes (I) were also included. Note progression of α2C ARs (C) from ER to plasma membrane fractions, demonstrating increasing mature glycosylation in plasma membrane and decreasing immature glycosylation in ER membrane fractions (see Figure 10). Bottom. Similar SGMF analysis of REEP1 (D), REEP2 (E), and REEP6 (F) demonstrating that all three REEPs tested were only found in heavier ER, but not plasma, membrane fractions. Mr standards (kDa) are indicated. Representative of three separate transfections.