ATF6 transport from the endoplasmic reticulum to the Golgi during the unfolded protein response

The secretory pathway processes proteins for secretion or for localization in the plasma membrane and intracellular organelles. Quality control is an important aspect of the secretory pathway, as improperly folded proteins are degraded at the endoplasmic reticulum (ER). Under some conditions, quality control in the ER is insufficient to prevent an accumulation of misfolded proteins. This condition is termed ER stress, and if unchecked can lead to apoptosis. To respond to stress, the ER activates the unfolded protein response (UPR), which slows protein synthesis and upregulates chaperones that assist in folding and degradation. The three sensors of the UPR are ER transmembrane proteins: Ire1 and PERK are activated by dimerization and signal via cytoplasmic proteins, while ATF6 is packaged into COPII vesicles for transport to the Golgi. In the Golgi, ATF6 is cleaved by proteases to release a soluble transcription factor that upregulates ER chaperones. The precise mechanisms by which ATF6 senses stress and becomes competent for transport are unclear. ER stress is a lumenal event, whereas the COPII proteins that mediate ER to Golgi transport are cytoplasmic. To study ATF6 trafficking, I examined mutant and chimeric forms of the protein. I also developed an in vitro assay that recapitulated the ER-stress induced transport of ATF6. When mammalian cells were permeabilized and treated with the ER-stress inducer dithiothreitol, ATF6 was packaged into COPII vesicles. The in vitro reaction was analogous to the in vivo response. ATF6 budding by individual COPII isoforms was examined in the in vitro assay and in vivo by RNAi. Although these altered ATF6 transport, there was not a single COPII protein or isoform that affected ATF6 uniquely. A second series of experiments was conducted to isolate proteins that interact with ATF6. There is evidence that ER proteins may form complexes with ATF6 to both retain it in the ER during unstressed conditions and allow it to transport during stress. These experiments identified a previously uncharacterized interacting protein, ATAD3. The biological significance of this interaction is under investigation.