COPII-Dependent ER Export: A Critical Component of Insulin Biogenesis and β-Cell ER Homeostasis

Pancreatic beta-cells possess a highly active protein synthetic and export machinery in the endoplasmic reticulum (ER) to accommodate the massive production of proinsulin. ER homeostasis is vital for beta-cell functions and is maintained by the delicate balance between protein synthesis, folding, export, and degradation. Disruption of ER homeostasis by diabetes-causing factors leads to beta-cell death. Among the 4 components to maintain ER homeostasis in beta-cells, the role of ER export in insulin biogenesis is the least understood. To address this knowledge gap, the present study investigated the molecular mechanism of proinsulin ER export in MIN6 cells and primary islets. Two inhibitory mutants of the secretion-associated RAS-related protein (Sar) 1 small GTPase, known to specifically block coat protein complex II (COPII)-dependent ER export, were overexpressed in beta-cells using recombinant adenoviruses. Results from this approach, as well as small interfering RNA-mediated Sar1 knockdown, demonstrated that defective Sar1 function blocked proinsulin ER export and abolished its conversion to mature insulin in MIN6 cells, isolated mouse, and human islets. It is further revealed, using an in vitro vesicle formation assay, that proinsulin was packaged into COPII vesicles in a GTP-and Sar1-dependent manner. Blockage of COPIIdependent ER exit by Sar1 mutants strongly induced ER morphology change, ER stress response, and beta-cell apoptosis. These responses were mediated by the PKR (double-stranded RNA-dependent kinase)-like ER kinase (PERK)/ eukaryotic translation initiation factor 2 alpha (p-eIF2 alpha) and inositol- requiring protein 1 (IRE1)/ x-box binding protein 1 (Xbp1) pathways but not via activating transcription factor 6 (ATF6). Collectively, results from the study demonstrate that COPII-dependent ER export plays a vital role in insulin biogenesis, ER homeostasis, and beta-cell survival.