Belyy, V. | Search bioRxiv

bioRxiv2021

Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remained unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1s lumenal domain. Phosphorylation of IRE1s kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans- autophosphorylation, which in turn drives IRE1s RNase activity.

biophysics↗

bioRxiv2021

Conserved structural elements specialize ATAD1 as a membrane protein extraction machine

The mitochondrial AAA protein ATAD1 (in humans; Msp1 in yeast) removes mislocalized membrane proteins, as well as stuck import substrates from the mitochondrial outer membrane, facilitating their re-insertion into their cognate organelles and maintaining mitochondrias protein import capacity. In doing so, it helps to maintain proteostasis in mitochondria. How ATAD1 tackles the energetic challenge to extract hydrophobic membrane proteins from the lipid bilayer and what structural features adapt ATAD1 for its particular function has remained a mystery. Previously, we determined the structure of Msp1 in complex with a peptide substrate (Wang et al., 2020). The structure showed that Msp1s mechanism follows the general principle established for AAA proteins while adopting several structural features that specialize it for its function. We used the yeast growth assay to test the function of key amino acids within these structural elements. In this work, we determined the cryo-EM structures of the human ATAD1 in complex with a peptide substrate at near atomic resolution. The structures show that phylogenetically conserved structural elements adapt ATAD1 for its function while generally adopting a conserved mechanism shared by many AAA proteins. ATAD1s distinguishing features include an abundance of aromatic amino acids that line the central pore proposed to give ATAD1 a firm grip on its substrate and a short -helix at the C-terminus that strongly facilitates ATAD1s oligomerization. We also developed a microscopy-based assay reporting on protein mislocalization, with which we directly assessed ATAD1s activity in live cells.

biochemistry↗