Preclinical characterization of novel multi-client inhibitors of Sec61 with broad antitumor activity.

The Sec61 translocon mediates entry of most secreted and transmembrane proteins into the endoplasmic reticulum, providing a novel therapeutic target to block the expression of protumorigenic factors. Sec61 inhibitors with antitumor activity, mostly derived from natural products, have been reported. However, poor tolerability and suboptimal pharmaceutical properties have precluded their further development.

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.

Structural basis of mitochondrial protein import by the TIM23 complex.

Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope. Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing proteins (preproteins) into the mitochondrial matrix and inner membrane. Among about ten different subunits of the TIM23 complex, the essential multipass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel.

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.

P5-ATPases: Structure, substrate specificities, and transport mechanisms.

P5A- and P5B- ATPases, or collectively P5-ATPases, are eukaryotic-specific ATP-dependent transporters that are important for the function of the endoplasmic reticulum (ER) and endo-/lysosomes. However, their substrate specificities had remained enigmatic for many years. Recent cryo-electron microscopy (cryo-EM) and biochemical studies of P5-ATPases have revealed their substrate specificities and transport mechanisms, which were found to be markedly different from other members of the P-type ATPase superfamily.

Lysosomal GPCR-like protein LYCHOS signals cholesterol sufficiency to mTORC1.

Lysosomes coordinate cellular metabolism and growth upon sensing of essential nutrients, including cholesterol. Through bioinformatic analysis of lysosomal proteomes, we identified lysosomal cholesterol signaling (LYCHOS, previously annotated as G protein-coupled receptor 155), a multidomain transmembrane protein that enables cholesterol-dependent activation of the master growth regulator, the protein kinase mechanistic target of rapamycin complex 1 (mTORC1). Cholesterol bound to the amino-terminal permease-like region of LYCHOS, and mutating this site impaired mTORC1 activation.

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.

Structural basis of polyamine transport by human ATP13A2 (PARK9).

Polyamines are small, organic polycations that are ubiquitous and essential to all forms of life. Currently, how polyamines are transported across membranes is not understood. Recent studies have suggested that ATP13A2 and its close homologs, collectively known as P5B-ATPases, are polyamine transporters at endo-/lysosomes.

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.

The endoplasmic reticulum P5A-ATPase is a transmembrane helix dislocase.

Organelle identity depends on protein composition. How mistargeted proteins are selectively recognized and removed from organelles is incompletely understood. Here, we found that the orphan P5A-adenosine triphosphatase (ATPase) transporter ATP13A1 (Spf1 in yeast) directly interacted with the transmembrane segment (TM) of mitochondrial tail-anchored proteins.

Cryo-EM structure of the mitochondrial protein-import channel TOM complex at near-atomic resolution.

Nearly all mitochondrial proteins are encoded by the nuclear genome and imported into mitochondria after synthesis on cytosolic ribosomes. These precursor proteins are translocated into mitochondria by the TOM complex, a protein-conducting channel in the mitochondrial outer membrane. We have determined high-resolution cryo-EM structures of the core TOM complex from Saccharomyces cerevisiae in dimeric and tetrameric forms.

Structure of the substrate-engaged SecA-SecY protein translocation machine.

The Sec61/SecY channel allows the translocation of many proteins across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. In bacteria, most secretory proteins are transported post-translationally through the SecY channel by the SecA ATPase. How a polypeptide is moved through the SecA-SecY complex is poorly understood, as structural information is lacking.

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 by cryo-electron microscopy (cryo-EM).

Structure of the CLC-1 chloride channel from .

CLC channels mediate passive Cl conduction, while CLC transporters mediate active Cl transport coupled to H transport in the opposite direction. The distinction between CLC-0/1/2 channels and CLC transporters seems undetectable by amino acid sequence. To understand why they are different functionally we determined the structure of the human CLC-1 channel.

Structural and Mechanistic Insights into Protein Translocation.

Many proteins are translocated across the endoplasmic reticulum (ER) membrane in eukaryotes or the plasma membrane in prokaryotes. These proteins use hydrophobic signal sequences or transmembrane (TM) segments to trigger their translocation through the protein-conducting Sec61/SecY channel. Substrates are first directed to the channel by cytosolic targeting factors, which use hydrophobic pockets to bind diverse signal and TM sequences.

Structure of a CLC chloride ion channel by cryo-electron microscopy.

CLC proteins transport chloride (Cl) ions across cellular membranes to regulate muscle excitability, electrolyte movement across epithelia, and acidification of intracellular organelles. Some CLC proteins are channels that conduct Cl ions passively, whereas others are secondary active transporters that exchange two Cl ions for one H. The structural basis underlying these distinctive transport mechanisms is puzzling because CLC channels and transporters are expected to share the same architecture on the basis of sequence homology.

Crystal structure of a substrate-engaged SecY protein-translocation channel.

Hydrophobic signal sequences target secretory polypeptides to a protein-conducting channel formed by a heterotrimeric membrane protein complex, the prokaryotic SecY or eukaryotic Sec61 complex. How signal sequences are recognized is poorly understood, particularly because they are diverse in sequence and length. Structures of the inactive channel show that the largest subunit, SecY or Sec61α, consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces lipid.

Structure of the SecY channel during initiation of protein translocation.

Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY or eukaryotic Sec61 complexes, and are translocated across the membrane during their synthesis. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug.

Structural alteration in the pore motif of the bacterial 20S proteasome homolog HslV leads to uncontrolled protein degradation.

In all cells, ATP-dependent proteases play central roles in the controlled degradation of short-lived regulatory or misfolded proteins. A hallmark of these enzymes is that proteolytic active sites are sequestered within a compartmentalized space, which is accessible to substrates only when they are fed into the cavity by protein-unfolding ATPases. HslVU is a prototype of such enzymes, consisting of the hexameric HslU ATPase and the dodecameric HslV protease.

Bacterial protein translocation requires only one copy of the SecY complex in vivo.

The transport of proteins across the plasma membrane in bacteria requires a channel formed from the SecY complex, which cooperates with either a translating ribosome in cotranslational translocation or the SecA ATPase in post-translational translocation. Whether translocation requires oligomers of the SecY complex is an important but controversial issue: it determines channel size, how the permeation of small molecules is prevented, and how the channel interacts with the ribosome and SecA. Here, we probe in vivo the oligomeric state of SecY by cross-linking, using defined co- and post-translational translocation intermediates in intact Escherichia coli cells.

Mechanisms of Sec61/SecY-mediated protein translocation across membranes.

The Sec61 or SecY channel, a universally conserved protein-conducting channel, translocates proteins across and integrates proteins into the eukaryotic endoplasmic reticulum (ER) membrane and the prokaryotic plasma membrane. Depending on channel-binding partners, polypeptides are moved by different mechanisms. In cotranslational translocation, the ribosome feeds the polypeptide chain directly into the channel.

Preserving the membrane barrier for small molecules during bacterial protein translocation.

Many proteins are translocated through the SecY channel in bacteria and archaea and through the related Sec61 channel in eukaryotes. The channel has an hourglass shape with a narrow constriction approximately halfway across the membrane, formed by a pore ring of amino acids. While the cytoplasmic cavity of the channel is empty, the extracellular cavity is filled with a short helix called the plug, which moves out of the way during protein translocation.

HslVU ATP-dependent protease utilizes maximally six among twelve threonine active sites during proteolysis.

HslVU is a bacterial ATP-dependent protease distantly related to eukaryotic proteasomes consisting of hexameric HslU ATPase and dodecameric HslV protease. As a homolog of the 20 S proteasome beta-subunits, HslV also uses the N-terminal threonine as the active site residue. However, unlike the proteasome that has only 6 active sites among the 14 beta-subunits, HslV has 12 active sites that could potentially contribute to proteolytic activity.

Binding of MG132 or deletion of the Thr active sites in HslV subunits increases the affinity of HslV protease for HslU ATPase and makes this interaction nucleotide-independent.

HslVU is an ATP-dependent protease in bacteria consisting of HslV dodecamer and HslU hexamer. Upon ATP binding, HslU ATPase allosterically activates the catalytic function of HslV protease by 1-2 orders of magnitude. However, relatively little is known about the role of HslV in the control of HslU function.