Abstract
Expression of genes in the endoplasmic reticulum (ER) beyond its protein folding capacity activates signaling pathways that are collectively referred to as the Unfolded Protein Response (UPR). A major branch of the UPR pathway is mediated by IRE1, an ER-tethered endonuclease. Upon ER stress-induced activation, IRE1 splices the mRNA of XBP1, thereby generating an active isoform of this transcription factor. During normal Drosophila development, tissues with high protein secretory load show signs of IRE1/XBP1 activity indicative of inherent ER stress associated with those cell types. Here, we report that the XBP1 promoter activity itself is enhanced in secretory tissues of Drosophila, and it can be induced by excessive ER stress. Specifically, we developed a Drosophila XBP1 transcription reporter by placing dsRed under the control of the XBP1 intergenic sequence. DsRed expression in these xbp1p>dsRed transgenic flies showed patterns similar to that of xbp1 transcript distribution. In healthy developing flies, the reporter expression was highest in salivary glands and the intestine. In the adult, the male reproductive organs showed high levels of dsRed. These tissues are known to have high protein secretory load. Consistently, the xbp1p>dsRed reporter was induced by excessive ER stress caused by mutant Rhodopsin-1 overexpression. These results suggest that secretory cells suffer from inherent ER stress, and the xbp1p>dsRed flies provide a useful tool in studying the function and homeostasis of those cells.
Highlights
Eukaryotic cells express most of their membrane and secretory proteins in the endoplasmic reticulum (ER), where those proteins undergo folding before being trafficked to their ultimate destination
A Unfolded Protein Response (UPR) pathway that is conserved between yeast, C. elegans, Drosophila and mammals is mediated by IRE1 and XBP1
We report the development of an xbp1 transcription reporter, which drives dsRed under the control of the xbp1 promoter. We find that this reporter is expressed in cells with high protein secretory load and is further induced by experimentally imposed ER stress. These results suggest that secretory cells suffer from inherent ER stress, and cells respond by inducing the transcription of xbp1, in addition to the IRE1-mediated splicing of these mRNAs
Summary
Eukaryotic cells express most of their membrane and secretory proteins in the endoplasmic reticulum (ER), where those proteins undergo folding before being trafficked to their ultimate destination. Most eukaryotic cells are equipped with signaling pathways that can induce the expression of ER quality control genes, which are widely referred to as the Unfolded Protein Response (UPR) [1]. IRE1 forms oligomers on the ER membrane and activates its cytoplasmic RNAse domain [10,11]. IRE1 mediated cleavage, and a subsequent ligation reaction results in an unconventional mRNA splicing event that occurs in the cytoplasm, and results in the shift of the XBP1 reading frame, thereby generating an isoform of XBP1 that acts as an active transcription factor. XBP1 induces the expression of a number of ER quality control genes, including chaperones that fold misfolded proteins, and those involved in the degradation of misfolded ER proteins [16,17]
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