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 envelope1-5. 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 channel6-11. However, the mechanism by which these subunits form a translocation path in the membrane and enable the import process remains unclear due to a lack of structural information. Here we determined the cryo-electron microscopy structure of the core TIM23 complex (heterotrimeric Tim17-Tim23-Tim44) from Saccharomyces cerevisiae. Contrary to the prevailing model, Tim23 and Tim17 themselves do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Our structural and biochemical analyses show that the cavity of Tim17, but not Tim23, forms the protein translocation path, whereas Tim23 probably has a structural role. The results further suggest that, during translocation of substrate polypeptides, the nonessential subunit Mgr2 seals the lateral opening of the Tim17 cavity to facilitate the translocation process. We propose a new model for the TIM23-mediated protein import and sorting mechanism, a central pathway in mitochondrial biogenesis.

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. The P5A-ATPase extracts mistargeted or mis-inserted transmembrane helices from the ER membrane for protein quality control, while the P5B-ATPases mediate export of polyamines from late endo-/lysosomes into the cytosol. In this review, we discuss the mechanisms of their substrate recognition and transport based on the cryo-EM structures of the yeast and human P5-ATPases. We highlight how structural diversification of the transmembrane domain has enabled the P5-ATPase subfamily to adapt for transport of atypical substrates.

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.

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. Loss-of-function mutations of ATP13A2 in humans cause hereditary early-onset Parkinson's disease. To understand the polyamine transport mechanism of ATP13A2, we determined high-resolution cryoelectron microscopy (cryo-EM) structures of human ATP13A2 in five distinct conformational intermediates, which together, represent a near-complete transport cycle of ATP13A2. The structural basis of the polyamine specificity was revealed by an endogenous polyamine molecule bound to a narrow, elongated cavity within the transmembrane domain. The structures show an atypical transport path for a water-soluble substrate, in which polyamines may exit within the cytosolic leaflet of the membrane. Our study provides important mechanistic insights into polyamine transport and a framework to understand the functions and mechanisms of P5B-ATPases.

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. P5A-ATPase activity mediated the extraction of mistargeted proteins from the endoplasmic reticulum (ER). Cryo-electron microscopy structures of Saccharomyces cerevisiae Spf1 revealed a large, membrane-accessible substrate-binding pocket that alternately faced the ER lumen and cytosol and an endogenous substrate resembling an α-helical TM. Our results indicate that the P5A-ATPase could dislocate misinserted hydrophobic helices flanked by short basic segments from the ER. TM dislocation by the P5A-ATPase establishes an additional class of P-type ATPase substrates and may correct mistakes in protein targeting or topogenesis.

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. Here, we report an electron cryo-microscopy (cryo-EM) structure of a translocating SecA-SecY complex in a lipid environment. The translocating polypeptide chain can be traced through both SecA and SecY. In the captured transition state of ATP hydrolysis, SecA's two-helix finger is close to the polypeptide, while SecA's clamp interacts with the polypeptide in a sequence-independent manner by inducing a short ß-strand. Taking into account previous biochemical and biophysical data, our structure is consistent with a model in which the two-helix finger and clamp cooperate during the ATPase cycle to move a polypeptide through the channel.

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.

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. Subsequently, these hydrophobic sequences insert into the channel, docking into a groove on the outside of the lateral gate of the channel, where they also interact with lipids. Structural data and biochemical experiments have elucidated how channel partners, the ribosome in cotranslational translocation, and the eukaryotic ER chaperone BiP or the prokaryotic cytosolic SecA ATPase in posttranslational translocation move polypeptides unidirectionally across the membrane. Structures of auxiliary components of the bacterial translocon, YidC and SecD/F, provide additional insight. Taken together, these recent advances result in mechanistic models of protein translocation.

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. The cytoplasmic funnel is empty, while the extracellular funnel is filled with a plug domain. In bacteria, the SecY channel associates with the translating ribosome in co-translational translocation, and with the SecA ATPase in post-translational translocation. How a translocating polypeptide inserts into the channel is uncertain, as cryo-electron microscopy structures of the active channel have a relatively low resolution (~10 Å) or are of insufficient quality. Here we report a crystal structure of the active channel, assembled from SecY complex, the SecA ATPase, and a segment of a secretory protein fused into SecA. The translocating protein segment inserts into the channel as a loop, displacing the plug domain. The hydrophobic core of the signal sequence forms a helix that sits in a groove outside the lateral gate, while the following polypeptide segment intercalates into the gate. The carboxy (C)-terminal section of the polypeptide loop is located in the channel, surrounded by residues of the pore ring. Thus, during translocation, the hydrophobic segments of signal sequences, and probably bilayer-spanning domains of nascent membrane proteins, exit the lateral gate and dock at a specific site that faces the lipid phase.

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. During initiation of translocation, a ribosome-nascent chain complex binds to the SecY (or Sec61) complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here we have addressed this question by determining structures of inactive and active ribosome-channel complexes with cryo-electron microscopy. Non-translating ribosome-SecY channel complexes derived from Methanocaldococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome-channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the amino- and carboxy-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than entering the channel directly.

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. We show that nontranslocating SecY associated transiently through different interaction surfaces with other SecY molecules inside the membrane. These interactions were significantly reduced when a translocating polypeptide inserted into the SecY channel co- or post-translationally. Mutations that abolish the interaction between SecY molecules still supported viability of E. coli. These results show that a single SecY molecule is sufficient for protein translocation.

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. In posttranslational translocation, a ratcheting mechanism is used by the ER-lumenal chaperone BiP in eukaryotes, and a pushing mechanism is utilized by the SecA ATPase in bacteria. In prokaryotes, posttranslational translocation is facilitated through the function of the SecD/F protein. Recent structural and biochemical data show how the channel opens during translocation, translocates soluble proteins, releases hydrophobic segments of membrane proteins into the lipid phase, and maintains the barrier for small molecules.

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. The mechanism by which the channel transports large polypeptides and yet prevents the passage of small molecules, such as ions or metabolites, has been controversial. Here, we have addressed this issue in intact Escherichia coli cells by testing the permeation of small molecules through wild-type and mutant SecY channels, which are either in the resting state or contain a defined translocating polypeptide chain. We show that in the resting state, the channel is sealed by both the pore ring and the plug domain. During translocation, the pore ring forms a 'gasket-like' seal around the polypeptide chain, preventing the permeation of small molecules. The structural conservation of the channel in all organisms indicates that this may be a universal mechanism by which the membrane barrier is maintained 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. Here, by using a series of HslV dodecamers containing different numbers of active sites, we demonstrate that like the proteasome, HslV with only approximately 6 active sites is sufficient to support full catalytic activity. However, a further reduction of the number of active sites leads to a proportional decrease in activity. Using proteasome inhibitors, we also demonstrate that substrate-mediated stabilization of the HslV-HslU interaction remains unchanged until the number of the active sites is decreased to approximately 6 but is gradually compromised upon further reduction. These results with a mathematical model suggest HslVU utilizes no more than 6 active sites at any given time, presumably because of the action of HslU. These results also suggest that each ATP-bound HslU subunit activates one HslV subunit and that substrate bound to the HslV active site stimulates the HslU ATPase activity by stabilizing the HslV-HslU interaction. We propose this mechanism plays an important role in supporting complete degradation of substrates while preventing wasteful ATP hydrolysis in the resting state by controlling the interaction between HslV and HslU through the catalytic engagement of the proteolytic active sites.

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. Here we describe the involvement of the N-terminal Thr active sites (Thr-1) of HslV in the communication between HslV and HslU. Binding of proteasome inhibitors to Thr-1 led to a dramatic increase in the interaction between HslV and HslU with a marked increase in ATP hydrolysis by HslU. Moreover, carbobenzoxy-leucyl-leucyl-leucinal (MG132) could bind to Thr-1 of free HslV, and this binding induced a tight interaction between HslV and HslU with the activation of HslU ATPase, suggesting that substrate-bound HslV can allosterically regulate HslU function. Unexpectedly, the deletion of Thr-1 also caused a dramatic increase in the affinity between HslV and HslU even in the absence of ATP. Furthermore, the increase in the number of the Thr-1 deletion mutant subunit in place of HslV subunit in a dodecamer led to a proportional increase in the affinity between HslV and HslU with gradual activation of HslU ATPase. Although the molecular mechanism elucidating how the Thr-1 deletion influences the interaction between HslV and HslU remains unknown, these results suggest an additional allosteric mechanism for the control of HslU function by HslV. Taken together, our findings indicate a critical involvement of Thr-1 of HslV in the reciprocal control of HslU function and, thus, for their communication.

Nucleotide triphosphates inhibit the degradation of unfolded proteins by HslV peptidase.

Escherichia coli HslVU is an ATP-dependent protease consisting of two heat shock proteins, the HslU ATPase and HslV peptidase. In the reconstituted enzyme, HslU stimulates the proteolytic activity of HslV by one to two orders of magnitude, while HslV increases the rate of ATP hydrolysis by HslU several-fold. Here we show that HslV alone can efficiently degrade certain unfolded proteins, such as unfolded lactalbumin and lysozyme prepared by complete reduction of disulfide bonds, but not their native forms. Furthermore, HslV alone cleaved a lactalbumin fragment sandwiched by two thioredoxin molecules, indicating that it can hydrolyze the internal peptide bonds of lactalbumin. Surprisingly, ATP inhibited the degradation of unfolded proteins by HslV. This inhibitory effect of ATP was markedly diminished by substitution of the Arg86 residue located in the apical pore of HslV with Gly, suggesting that interaction of ATP with the Arg residue blocks access of unfolded proteins to the proteolytic chamber of HslV. These results suggest that uncomplexed HslV is inactive under normal conditions, but may can degrade unfolded proteins when the ATP level is low, as it is during carbon starvation.

Role of the GYVG pore motif of HslU ATPase in protein unfolding and translocation for degradation by HslV peptidase.

HslVU is an ATP-dependent protease consisting of HslU ATPase and HslV peptidase. In an HslVU complex, the central pores of HslU hexamer and HslV dodecamer are aligned and the proteolytic active sites are sequestered in the inner chamber of HslV. Thus, the degradation of natively folded proteins requires unfolding and translocation processes for their access into the proteolytic chamber of HslV. A highly conserved GYVG(93) sequence constitutes the central pore of HslU ATPase. To determine the role of the pore motif on protein unfolding and translocation, we generated various mutations in the motif and examined their effects on the ability of HslU in supporting the proteolytic activity of HslV against three different substrates: SulA as a natively folded protein, casein as an unfolded polypeptide, and a small peptide. Flexibility provided by Gly residues and aromatic ring structures of the 91st amino acid were essential for degradation of SulA. The same structural features of the GYVG motif were highly preferred, although not essential, for degradation of casein. In contrast, none of the features were required for peptide hydrolysis. Mutations in the GYVG motif of HslU also showed marked influence on its ATPase activity, affinity to ADP, and interaction with HslV. These results suggest that the GYVG motif of HslU plays important roles in unfolding of natively folded proteins as well as in translocation of unfolded proteins for degradation by HslV. These results also implicate a role of the pore motif in ATP cleavage and in the assembly of HslVU complex.