Abstract
CFTR is a transmembrane protein that reaches the cell surface via the conventional Golgi mediated secretion pathway. Interestingly, ER-to-Golgi blockade or ER stress induces alternative GRASP-mediated, Golgi-bypassing unconventional trafficking of wild-type CFTR and the disease-causing ΔF508-CFTR, which has folding and trafficking defects. Here, we show that Sec16A, the key regulator of conventional ER-to-Golgi transport, plays a critical role in the ER exit of protein cargos during unconventional secretion. In an initial gene silencing screen, Sec16A knockdown abolished the unconventional secretion of wild-type and ΔF508-CFTR induced by ER-to-Golgi blockade, whereas the knockdown of other COPII-related components did not. Notably, during unconventional secretion, Sec16A was redistributed to cell periphery and associated with GRASP55 in mammalian cells. Molecular and morphological analyses revealed that IRE1α-mediated signaling is an upstream regulator of Sec16A during ER-to-Golgi blockade or ER stress associated unconventional secretion. These findings highlight a novel function of Sec16A as an essential mediator of ER stress-associated unconventional secretion.
Highlights
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is an N-glycosylated transmembrane protein with anion channel activity that permeates chloride and bicarbonate at the apical surface of secretory epithelia of the airways, intestine, pancreas, and exocrine glands[1,2]
In an initial RNA interference (RNAi) screen of COPII-associated components, we found that Sec16A knockdown abolished the unconventional secretion of wild-type and ΔF508 CFTR induced by endoplasmic reticulum (ER)-to-Golgi blockade, whereas the knockdown of other COPII-related components did not
We examined the role of Sec16A in the unconventional secretion pathway and found that Sec16A is a critical component in the ER stress-associated, Golgi reassembly stacking protein (GRASP)-mediated unconventional secretion of core-glycosylated CFTR
Summary
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is an N-glycosylated transmembrane protein with anion channel activity that permeates chloride and bicarbonate at the apical surface of secretory epithelia of the airways, intestine, pancreas, and exocrine glands[1,2]. Under ER-to-Golgi blockade or ER-stress conditions, core-glycosylated wild-type and ΔF508 CFTR in the ER can travel to the cell surface through an unconventional Golgi reassembly stacking protein (GRASP)-dependent pathway that bypasses the Golgi[7]. Augmentation of this unconventional secretion pathway via GRASP55 overexpression has been shown to rescue the defects caused by ΔF508-CFTR in a murine CF model[7]. The blockade of ER-to-Golgi transport, either direct via the inhibition of COPII-mediated vesicular transport (e.g., transfection with the dominant-negative form of Sar1), or indirect via the inhibition of COPI-mediated transport (e.g., transfection with the dominant-negative form of Arf1), triggers the activation of ER stress[18] and evokes the unconventional secretion of core-glycosylated CFTR via the GRASP-dependent mechanism in mammalian cells[7]
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