Reinvestigation of Disulfide-bonded Oligomeric Forms of the Unfolded Protein Response Transducer ATF6

ATF6 alpha is an endoplasmic reticulum (ER)-embedded transcription factor which is rapidly activated by ER stress, and a major regulator of ER chaperone levels in vertebrates. We previously suggested that ATF6 alpha occurs as a monomer, dimer and oligomer in the unstressed ER of Chinese hamster ovary cells due to the presence of two evolutionarily conserved cysteine residues in its luminal region (C467 and C618), and showed that ATF6 alpha is reduced upon ER stress, such that only reduced monomer ATF6 alpha is translocated to the Golgi apparatus for activation by proteolysis. However, mutagenesis analysis (C467A and C618A) revealed that the C618A mutant behaves in an unexpected manner (monomer and oligomer) during non-reducing SDS-PAGE, for reasons which remained unclear. Here, we used human colorectal carcinoma-derived HCT116 cells deficient in ATF6 alpha and its relevant ATF6 beta, and found that ATF6 alpha dimer and oligomer are both dimers, which we designated C618-dimer and C467-dimer, respectively. We demonstrated that C467-dimer (previously considered an oligomer) behaved bigger than C618-dimer (previously considered a dimer) during non-reducing SDS-PAGE, based on their disulfide-bonded structures. Furthermore, ATF6 alpha monomer physically associates with another ATF6 alpha monomer in the absence of disulfide bonding, which renders two C467 residues in close proximity so that formation of C467-dimer is much easier than that of C618-dimer. In contrast, C618-dimer is more easily reduced upon ER stress. Thus, our analysis revealed that all forms of ATF6 alpha, namely monomer, C618-dimer and C467dimer, are activated by single reduction of a disulfide bond in response to ER stress, ensuring the rapidity of ATF6 alpha activation.