Substrate recognition mechanism of the endoplasmic reticulum-associated ubiquitin ligase Doa10.

Doa10 (MARCHF6 in metazoans) is a large polytopic membrane-embedded E3 ubiquitin ligase in the endoplasmic reticulum (ER) that plays an important role in quality control of cytosolic and ER proteins. Although Doa10 is highly conserved across eukaryotes, it is not understood how Doa10 recognizes its substrates. Here, we define the substrate recognition mechanism of Doa10 by structural and functional analyses on Saccharomyces cerevisiae Doa10 and its model substrates. Cryo-EM analysis shows that Doa10 has unusual architecture with a large lipid-filled central cavity, and its conserved middle domain forms an additional water-filled lateral tunnel open to the cytosol. Our biochemical data and molecular dynamics simulations suggest that the entrance of the substrate's degron peptide into the lateral tunnel is required for efficient polyubiquitination. The N- and C-terminal membrane domains of Doa10 seem to form fence-like features to restrict polyubiquitination to those proteins that can access the central cavity and lateral tunnel. Our study reveals how extended hydrophobic sequences at the termini of substrate proteins are recognized by Doa10 as a signal for quality control.

Substrate recognition mechanism of the endoplasmic reticulum-associated ubiquitin ligase Doa10.

Doa10 (MARCH6 in metazoans) is a large polytopic membrane-embedded E3 ubiquitin ligase in the endoplasmic reticulum (ER) that plays an important role in quality control of cytosolic and ER proteins. Although Doa10 is highly conserved across eukaryotes, it is not understood how Doa10 recognizes its substrates. Here, we defined the substrate recognition mechanism of Doa10 by structural and functional analyses on Saccharomyces cerevisiae Doa10 and its well-defined degron Deg1. Cryo-EM analysis shows that Doa10 has unusual architecture with a large lipid-filled central cavity, and its conserved middle domain forms an additional water-filled lateral tunnel open to the cytosol. Our biochemical data and molecular dynamics simulations suggest that the entrance of the substrate's degron peptide into the lateral tunnel is required for efficient polyubiquitination. The N- and C-terminal membrane domains of Doa10 seem to form fence-like features to restrict polyubiquitination to those proteins that can access the central cavity and lateral tunnel.

A common mechanism of Sec61 translocon inhibition by small molecules.

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit Sec61, generating cellular effects that are useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of human Sec61 inhibited by a comprehensive panel of structurally distinct small molecules-cotransin, decatransin, apratoxin, ipomoeassin, mycolactone, cyclotriazadisulfonamide and eeyarestatin. All inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of Sec61. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain in a closed state, thereby preventing the protein-translocation pore from opening. Our study provides the atomic details of Sec61-inhibitor interactions and the structural framework for further pharmacological studies and drug design.

Mechanism of Protein Translocation by the Sec61 Translocon Complex.

The endoplasmic reticulum (ER) is a major site for protein synthesis, folding, and maturation in eukaryotic cells, responsible for production of secretory proteins and most integral membrane proteins. The universally conserved protein-conducting channel Sec61 complex mediates core steps in these processes by translocating hydrophilic polypeptide segments of client proteins across the ER membrane and integrating hydrophobic transmembrane segments into the membrane. The Sec61 complex associates with several other molecular machines and enzymes to enable substrate engagement with the channel and coordination of protein translocation with translation, protein folding, and/or post-translational modifications. Recent cryo-electron microscopy and functional studies of these translocon complexes have greatly advanced our mechanistic understanding of Sec61-dependent protein biogenesis at the ER. Here, we will review the current models for how the Sec61 channel performs its functions in coordination with partner complexes.

Stepwise gating of the Sec61 protein-conducting channel by Sec63 and Sec62.

Many proteins are transported into the endoplasmic reticulum by the universally conserved Sec61 channel. Post-translational transport requires two additional proteins, Sec62 and Sec63, but their functions are poorly defined. In the present study, we determined cryo-electron microscopy (cryo-EM) structures of several variants of Sec61-Sec62-Sec63 complexes from Saccharomyces cerevisiae and Thermomyces lanuginosus and show that Sec62 and Sec63 induce opening of the Sec61 channel. Without Sec62, the translocation pore of Sec61 remains closed by the plug domain, rendering the channel inactive. We further show that the lateral gate of Sec61 must first be partially opened by interactions between Sec61 and Sec63 in cytosolic and luminal domains, a simultaneous disruption of which completely closes the channel. The structures and molecular dynamics simulations suggest that Sec62 may also prevent lipids from invading the channel through the open lateral gate. Our study shows how Sec63 and Sec62 work together in a hierarchical manner to activate Sec61 for post-translational protein translocation.

Structure of the posttranslational Sec protein-translocation channel complex from yeast.

The Sec61 protein-conducting channel mediates transport of many proteins, such as secretory proteins, across the endoplasmic reticulum (ER) membrane during or after translation. Posttranslational transport is enabled by two additional membrane proteins associated with the channel, Sec63 and Sec62, but its mechanism is poorly understood. We determined a structure of the Sec complex (Sec61-Sec63-Sec71-Sec72) from Saccharomyces cerevisiae by cryo-electron microscopy (cryo-EM). The structure shows that Sec63 tightly associates with Sec61 through interactions in cytosolic, transmembrane, and ER-luminal domains, prying open Sec61's lateral gate and translocation pore and thus activating the channel for substrate engagement. Furthermore, Sec63 optimally positions binding sites for cytosolic and luminal chaperones in the complex to enable efficient polypeptide translocation. Our study provides mechanistic insights into eukaryotic posttranslational protein translocation.

The mycotoxin phomoxanthone A disturbs the form and function of the inner mitochondrial membrane.

Mitochondria are cellular organelles with crucial functions in the generation and distribution of ATP, the buffering of cytosolic Ca2+ and the initiation of apoptosis. Compounds that interfere with these functions are termed mitochondrial toxins, many of which are derived from microbes, such as antimycin A, oligomycin A, and ionomycin. Here, we identify the mycotoxin phomoxanthone A (PXA), derived from the endophytic fungus Phomopsis longicolla, as a mitochondrial toxin. We show that PXA elicits a strong release of Ca2+ from the mitochondria but not from the ER. In addition, PXA depolarises the mitochondria similarly to protonophoric uncouplers such as CCCP, yet unlike these, it does not increase but rather inhibits cellular respiration and electron transport chain activity. The respiration-dependent mitochondrial network structure rapidly collapses into fragments upon PXA treatment. Surprisingly, this fragmentation is independent from the canonical mitochondrial fission and fusion mediators DRP1 and OPA1, and exclusively affects the inner mitochondrial membrane, leading to cristae disruption, release of pro-apoptotic proteins, and apoptosis. Taken together, our results suggest that PXA is a mitochondrial toxin with a novel mode of action that might prove a useful tool for the study of mitochondrial ion homoeostasis and membrane dynamics.