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

Abstract

Pancreatic β-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 β-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 β-cell death. Among the 4 components to maintain ER homeostasis in β-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 β-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 COPII-dependent ER exit by Sar1 mutants strongly induced ER morphology change, ER stress response, and β-cell apoptosis. These responses were mediated by the PKR (double-stranded RNA-dependent kinase)-like ER kinase (PERK)/eukaryotic translation initiation factor 2α (p-eIF2α) 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 β-cell survival.

The Noncanonical Role of ULK/ATG1 in ER-to-Golgi Trafficking Is Essential for Cellular Homeostasis

“…The COPII machinery distinguishes transmembrane and luminal secretory cargo from resident ER proteins and packages them into transport vesicles destined for Golgi. Defects in ER-to-Golgi trafficking can lead to the accumulation of protein in the ER and activation of the UPR (Fang et al, 2015; Preston et al, 2009); therefore, we further characterized the interaction between the ULKs and SEC16A.…”

Trk-fused gene (TFG) regulates pancreatic β cell mass and insulin secretory activity

“…Since TFG is suggested to play an important role in vesicle transport, loss of TFG might have resulted in increases in immature vesicles with reduced insulin content per vesicle. Impairment of proinsulin ER export and its conversion to mature insulin was reported in MIN6 cells and isolated islets overexpressing the defective mutant Sar1, the activation of which initiates COPII coat assembly and is crucial in COPII vesicle formation 28 . Sar1 mutant overexpressing β-cells showed drastically distended ER and upregulation of UPR markers including CHOP.…”

cTAGE5 deletion in pancreatic β cells impairs proinsulin trafficking and insulin biogenesis in mice

“…Proinsulin, the precursor of insulin, is rapidly translocated into ER, where it undergoes oxidative folding by forming three disulfide bonds ( Gupta et al, 2010 ; Liu et al, 2014 ). Only properly folded proinsulin can be assembled into COPII vesicles and transported from ER to Golgi ( Fang et al, 2015 ). Increases in misfolded proinsulin in the ER would perturb the ER environment and lead to ER stress ( Scheuner et al, 2005 ; Sun et al, 2015 ).…”