Abstract
The Golgi apparatus is important for the transport of secretory cargo. Glycosylation is a major post-translational event. Recognition of O-glycans on proteins is necessary for glycoprotein trafficking. In this study, specific inhibition of O-glycosylation (Golgi stress) induced the expression of endoplasmic reticulum (ER)-resident heat shock protein (HSP) 47 in NIH3T3 cells, although cell death was not induced by Golgi stress alone. When HSP47 expression was downregulated by siRNA, inhibition of O-glycosylation caused cell death. Three days after the induction of Golgi stress, the Golgi apparatus was disassembled, many vacuoles appeared near the Golgi apparatus and extended into the cytoplasm, the nuclei had split, and cell death assay-positive cells appeared. Six hours after the induction of Golgi stress, HSP47-knockdown cells exhibited increased cleavage of Golgi-resident caspase-2. Furthermore, activation of mitochondrial caspase-9 and ER-resident unfolded protein response (UPR)-related molecules and efflux of cytochrome c from the mitochondria to the cytoplasm was observed in HSP47-knockdown cells 24 h after the induction of Golgi stress. These findings indicate that (i) the ER-resident chaperon HSP47 protected cells from Golgi stress, and (ii) Golgi stress-induced cell death caused by the inhibition of HSP47 expression resulted from caspase-2 activation in the Golgi apparatus, extending to the ER and mitochondria.
Highlights
The cellular locations of major cytoplasmic organelles, such as the nucleus, endoplasmic reticulum (ER), Golgi apparatus, and mitochondria, are unique, and the organelles have specific functions based on their unique location
We further investigated the expression or activation of unfolded protein response (UPR)-related molecules, i.e., ATF6, a sensor-transcription factor embedded in the ER, PKR-like ER kinase (PERK), a sensor-kinase located in the ER membrane, and IRE1α, a sensor RNase located in the ER membrane [51]
The present study demonstrated that Golgi stress caused by the inhibition of O-glycosylation induced the expression of heat shock protein 47 (HSP47) in the ER of NIH3T3 cells and that downregulation of HSP47 caused Golgi dysfunction, leading to cell death (Figure S6)
Summary
The cellular locations of major cytoplasmic organelles, such as the nucleus, endoplasmic reticulum (ER), Golgi apparatus, and mitochondria, are unique, and the organelles have specific functions based on their unique location. ER dysfunctioninduced cell death was reported, in neurodegeneration [8,9,10,11,12,13,14,15,16,17,18] This organelle exhibits specific mechanisms for counteracting stress stimuli [19,20,21,22,23]. Glycosylation is a major post-translational event, and the recognition of O-glycans on proteins is required for glycoprotein trafficking [24,25] Such an event occurs through sorting in the trans-Golgi network, and glycoprotein trafficking is disturbed when O-glycosylation is inhibited [27,28,29,30]. Benzyl 2-acetamido-2-deoxy-α-dgalactopyranoside (GalNAc-bn) was used as an O-
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