Eunyong Park

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.

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.

Single Copies of Sec61 and TRAP Associate with a Nontranslating Mammalian Ribosome

During cotranslational protein translocation, the ribosome associates with a membrane channel, formed by the Sec61 complex, and recruits the translocon-associated protein complex (TRAP). Here we report the structure of a ribosome-channel complex from mammalian endoplasmic reticulum in which the channel has been visualized at 11 A resolution. In this complex, single copies of Sec61 and TRAP associate with a nontranslating ribosome and this stoichiometry was verified by quantitative mass spectrometry. A bilayer-like density surrounds the channel and can be attributed to lipid and detergent. The crystal structure of an archaeal homolog of the Sec61 complex was then docked into the map. In this model, two cytoplasmic loops of Sec61 may interact with RNA helices H6, H7, and H50, while the central pore is located below the ribosome tunnel exit. Hence, this copy of Sec61 is positioned to capture and translocate the nascent chain. Finally, we show that mammalian and bacterial ribosome-channel complexes have similar architectures.

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 GYVG93 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.

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.

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.

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

In vivo probing of the oligomeric state of SecY during co- and post-translational translocation reveals that oligomerization is not required for this process.

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. Here we determined the structure of a bovine CLC channel (CLC-K) using cryo-electron microscopy. A conserved loop in the Cl− transport pathway shows a structure markedly different from that of CLC transporters. Consequently, the cytosolic constriction for Cl− passage is widened in CLC-K such that the kinetic barrier previously postulated for Cl−/H+ transporter function would be reduced. Thus, reduction of a kinetic barrier in CLC channels enables fast flow of Cl− down its electrochemical gradient.

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.

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.