Atg39 selectively captures inner nuclear membrane into lumenal vesicles for delivery to the autophagosome

Mechanisms that turn over components of the nucleus and inner nuclear membrane (INM) remain to be fully defined. We explore how components of the INM are selected by a cytosolic autophagy apparatus through a transmembrane nuclear envelope–localized cargo adaptor, Atg39. A split-GFP reporter showed that Atg39 localizes to the outer nuclear membrane (ONM) and thus targets the INM across the nuclear envelope lumen. Consistent with this, sequence elements that confer both nuclear envelope localization and a membrane remodeling activity are mapped to the Atg39 lumenal domain; these lumenal motifs are required for the autophagy-mediated degradation of integral INM proteins. Interestingly, correlative light and electron microscopy shows that the overexpression of Atg39 leads to the expansion of the ONM and the enclosure of a network of INM-derived vesicles in the nuclear envelope lumen. Thus, we propose an outside–in model of nucleophagy where INM is delivered into vesicles in the nuclear envelope lumen, which can be targeted by the autophagosome.

The stress-sensing domain of activated IRE1α forms helical filaments in narrow ER membrane tubes

The signaling network of the unfolded protein response (UPR) adjusts the protein folding capacity of the endoplasmic reticulum (ER) according to need. The most conserved UPR sensor, IRE1α, spans the ER membrane and activates through oligomerization. IRE1α oligomers accumulate in dynamic foci. We determined the in-situ structure of IRE1α foci by cryogenic correlated light and electron microscopy (cryo-CLEM), combined with electron cryo-tomography (cryo-ET) and complementary immuno-electron microscopy. IRE1α oligomers localize to a network of narrow anastomosing ER tubes (diameter ~28 nm) with complex branching. The lumen of the tubes contains protein filaments, likely composed of linear arrays of IRE1α lumenal domain dimers, arranged in two intertwined, left-handed helices. Our findings define a previously unrecognized ER subdomain and suggest positive feedback in IRE1 signaling.

Atg39 selectively captures inner nuclear membrane into lumenal vesicles for delivery to the autophagosome

Mechanisms that turnover components of the nucleus and inner nuclear membrane (INM) remain to be fully defined. We explore how components of the INM are selected by a cytosolic autophagy apparatus through a transmembrane nuclear envelope-localized cargo adaptor, Atg39. A split-GFP reporter shows that Atg39 localizes to the outer nuclear membrane (ONM) and thus targets the INM across the nuclear envelope lumen. Consistent with this, sequence elements that confer both nuclear envelope localization and a membrane remodeling activity are mapped to the Atg39 lumenal domain; these lumenal motifs are required for the autophagy-mediated degradation of an integral INM protein. Interestingly, correlative light and electron tomography shows that the overexpression of Atg39 leads to the expansion of the ONM and the enclosure of a network of INM-derived vesicles in the nuclear envelope lumen. Thus, we propose an outside-in model of nucleophagy where INM is delivered into vesicles in the nuclear envelope lumen, which can be targeted by the autophagosome.

Activation of the IRE1 RNase through remodeling of the kinase front pocket by ATP-competitive ligands

Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response. IRE1 spans the endoplasmic reticulum membrane, comprising a sensory lumenal domain, and tandem kinase and endoribonuclease (RNase) cytoplasmic domains. Excess unfolded proteins in the ER lumen induce dimerization and oligomerization of IRE1, triggering kinase trans-autophosphorylation and RNase activation. Known ATP-competitive small-molecule IRE1 kinase inhibitors either allosterically disrupt or stabilize the active dimeric unit, accordingly inhibiting or stimulating RNase activity. Previous allosteric RNase activators display poor selectivity and/or weak cellular activity. In this study, we describe a class of ATP-competitive RNase activators possessing high selectivity and strong cellular activity. This class of activators binds IRE1 in the kinase front pocket, leading to a distinct conformation of the activation loop. Our findings reveal exquisitely precise interdomain regulation within IRE1, advancing the mechanistic understanding of this important enzyme and its investigation as a potential small-molecule therapeutic target.

Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients

Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies distinguish between two separable EMC activities, as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC’s multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes.

Structural modelling of the lumenal domain of human GPAA1, the metallo-peptide synthetase subunit of the transamidase complex, reveals zinc-binding mode and two flaps surrounding the active site

Background The transamidase complex is a molecular machine in the endoplasmic reticulum of eukaryotes that attaches a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins after cleaving a C-terminal propeptide with a defined sequence signal. Its five subunits are very hydrophobic; thus, solubility, heterologous expression and complex reconstruction are difficult. Therefore, theoretical approaches are currently the main source of insight into details of 3D structure and of the catalytic process. Results In this work, we generated model 3D structures of the lumenal domain of human GPAA1, the M28-type metallo-peptide-synthetase subunit of the transamidase, including zinc ion and model substrate positions. In comparative molecular dynamics (MD) simulations of M28-type structures and our GPAA1 models, we estimated the metal ion binding energies with evolutionary conserved amino acid residues in the catalytic cleft. We find that canonical zinc binding sites 2 and 3 are strongest binders for Zn1 and, where a second zinc is available, sites 2 and 4 for Zn2. Zinc interaction of site 5 with Zn1 enhances upon substrate binding in structures with only one zinc. Whereas a previously studied glutaminyl cyclase structure, the best known homologue to GPAA1, binds only one zinc ion at the catalytic site, GPAA1 can sterically accommodate two. The M28-type metallopeptidases segregate into two independent branches with regard to one/two zinc ion binding modality in a phylogenetic tree where the GPAA1 family is closer to the joint origin of both groups. For GPAA1 models, MD studies revealed two large loops (flaps) surrounding the active site being involved in an anti-correlated, breathing-like dynamics. Conclusions In the light of combined sequence-analytic and phylogenetic arguments as well as 3D structural modelling results, GPAA1 is most likely a single zinc ion metallopeptidase. Two large flaps environ the catalytic site restricting access to large substrates. Reviewers This article was reviewed by Thomas Dandekar (MD) and Michael Gromiha.

Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients

Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies revealed at least two separable EMC activities, as an insertase regulating tail-anchored protein levels and as a polytopic membrane protein holdase chaperone. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a novel, non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC’s multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes.

Signature of N-terminal domain (NTD) structural re-orientation in NPC1 for proper alignment of cholesterol transport: Molecular dynamics study with mutation

The lysosomal membrane protein NPC1 (Niemann-Pick type C1) and NPC2 (Niemann-Pick type C2) are main players of cholesterol control in lysosome and it is known that mutation on these proteins leads to cholesterol trafficking related disease, called Niemann-Pick disease type C (NPC) disease. The mutation R518W or R518Q on NPC1 is one of such disease-related mutations, causing reduced cholesterol transport by half, resulting in accumulation of cholesterol and lipids in late endosomal/lysosomal region of the cell. Even though there has been significant progress in understanding cholesterol transport by NPC1 in combination with NPC2, especially after the structural determination of full length NPC1 in 2016, many details such as interaction of full length NPC1 with NPC2, molecular motions responsible for cholesterol transport during and after this interaction, and structure and function relations of many mutations are still not well understood.We report the extensive molecular dynamics simulations to gain insight into the structure and motions of NPC1 lumenal domain for cholesterol transport and disease behind the mutation (R518W). It is found that the mutation induces structural shift of NTD (N-terminal domain), toward the loop region in MLD (middle lumenal domain), which is believed to play central role in interaction with NPC2 protein, such that the interaction with NPC2 protein might be less favorable compare to wild NPC1. Also, the simulation indicates the possible re-orientation of the NTD, aligning to form an internal tunnel, after receiving the cholesterol from NPC2 with wild NPC1 unlike the mutated one, a possible pose for further action in cholesterol trafficking. We believe the current study can provide better understanding on the cholesterol transport by NPC1, especially the role of NTD of NPC1, in combination with NPC2 interaction.Synopsismodeling study of cholesterol binding protein NPC1

Structural Modelling of the Lumenal Domain of Human GPAA1, the Metallo-Peptide Synthetase Subunit of the Transamidase Complex, Reveals Zinc-Binding Mode and Two Flaps Surrounding the Active Site

Background The transamidase complex is a molecular machine in the endoplasmic reticulum of eukaryotes that attaches a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins after cleaving a C-terminal propeptide with a defined sequence signal. Its five subunits are very hydrophobic; thus, solubility, heterologous expression and complex reconstruction are difficult. Therefore, theoretical approaches are currently the main source of insight into details of 3D structure and of the catalytic process. Results In this work, we generated model 3D structures of the lumenal domain of human GPAA1, the M28-type metallo-peptide-synthetase subunit of the transamidase, including zinc ion and model substrate positions. In comparative molecular dynamics (MD) simulations of M28-type structures and our GPAA1 models, we estimated the metal ion binding energies with evolutionary conserved amino acid residues in the catalytic cleft. We find that canonical zinc binding sites 2 and 3 are strongest binders for Zn1 and, where a second zinc is available, sites 2 and 4 for Zn2. Zinc interaction of site 5 with Zn1 enhances upon substrate binding in structures with only one zinc. Whereas a previously studied glutaminyl cyclase structure, the best known homologue to GPAA1, binds only one zinc ion at the catalytic site, GPAA1 can sterically accommodate two. The M28-type metallopeptidases segregate into two independent branches with regard to one/two zinc ion binding modality in a phylogenetic tree where the GPAA1 family is closer to the joint origin of both groups. For GPAA1 models, MD studies revealed two large loops (flaps) surrounding the active site being involved in an anti-correlated, breathing-like dynamics. Conclusions In the light of combined sequence-analytic and phylogenetic arguments as well as 3D structural modelling results, GPAA1 is most likely a single zinc ion metallopeptidase. Two large flaps environ the catalytic site restricting access to large substrates.