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Title: The Principles of Protein Targeting and Transport Across Cell Membranes

Yuanyuan Chen1, Sri Karthika Shanmugam1 and Ross E. Dalbey1* 1Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio, USA *Corresponding Author: dalbey.1@osu.edu, 614-292-2384

Abstract The past several decades have witnessed tremendous growth in the protein targeting, transport

and translocation field. Major advances were made during this time period. Now the molecular

details of the targeting factors, receptors and the membrane channels that were envisioned in

Blobel’s Signal Hypothesis in the 1970s have been revealed by powerful structural methods. It

is evident that there is a myriad of cytosolic and membrane associated systems that accurately

sort and target newly synthesized proteins to their correct membrane translocases for membrane

insertion or protein translocation. Here we will describe the common principles for protein

transport in prokaryotes and eukaryotes.

Keywords: Protein targeting, chaperones, translocase, insertases, energetics

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Introduction

One of the most significant events in the course of evolution is the development of cellular

compartmentalization [1-4]. It enables different physiological functions to be distributed among

different organelles. However, a great challenge arises at the same time; i.e. proteins synthesized

in the cytosol need to be sorted and transported to their destined locations.

In 1971, Blobel and Sabatini reported their signal hypothesis, which proposed that the

fundamental information for protein sorting and translocation is encoded in a signal sequence

found within the nascent chain, that can be recognized and shuttled to the endoplasmic reticulum

(ER) membrane by a “binding factor” [5-7]. Over the past half century, this hypothesis has been

confirmed and elaborated; the cellular targeting signals have been shown to direct proteins to the

mitochondria, chloroplast, peroxisome, and nucleus [8-13]. Playing center stage in protein

translocation among different organelles are the signal sequences of the respective substrates,

cytosolic chaperones, membrane receptors, membrane channels, and energy as reviewed in [14].

In this overview, we will revisit these principles, and update them with new findings from the

past two decades.

Protein translocation systems

In archaea and bacteria, proteins are synthesized by ribosomes in the cytoplasm. Over one-third

of these proteins need to be sorted, and either inserted into the plasma membrane, or exported

across one or two membranes to reach their destined location [15-17].

In contrast, this process is more sophisticated in eukaryotic cells [16], due to the variety of

compartments and endomembrane systems that they possess. The majority of proteins are

synthesized in the cytosol, and inserted into the ER, or imported into the mitochondria,

chloroplasts, peroxisomes or nucleus. Interestingly, some proteins imported into mitochondria

[18] and peroxisome [19] are done so by an ER-mediated process [18,20,21]. Lastly,

mitochondria [22,23] and chloroplasts [24] also have their own DNAs that encode a small group

of proteins that are synthesized by the organellar ribosomes, and inserted into or exported across

the inner membrane (or thylakoid membrane in chloroplasts).

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Interestingly, several translocon complexes have evolved in order to facilitate protein

translocation for a variety of substrates. Good examples are the Sec complex [25] and the Twin

arginine translocation (Tat) complex [26,27] for the export of bacterial proteins to the periplasm,

the Sec61 translocon [28-30] and the Get pathway [31,32] for membrane insertion of proteins

into the ER, or the different secretion systems in bacteria [33-36] for transporting proteins to the

extracellular space.

This overview will focus on the major players in protein targeting and translocation, as well as

the common principles that are shared among various systems. Briefly, membrane-targeted

proteins typically contain one or more N-terminal signal sequences that are recognized by

different cytosolic binding partners, which sort newly synthesized proteins to different transport

pathways or organelles. The binding partners include, in some cases, the signal recognition

particle (SRP) for co-translational translocation, or chaperones that help keep newly synthesized

proteins unfolded in the case of post-translational translocation. The binding partners then shuttle

the synthesized proteins to the surface of membranes via interaction with their membrane-

associated receptors, and then hand the proteins over to their respective membrane channels.

This process is most likely driven by a stepwise increment in the binding affinity. The

translocation through membrane channels is generally driven using ATP hydrolysis by motor

ATPases peripherally associated with the channels, or the membrane proton motif force. Finally,

during or after the translocation is complete, the precursor proteins containing cleavable signal

sequences are processed by signal peptidases, and subsequently fold into their mature forms.

Signal Sequence

It was exciting in the 70s and 80s to discover how precursor proteins are targeted to their correct

destinations in the cell and are able to discriminate among different translocation pathways.

Blobel first proposed that proteins destined for export from the cytoplasm are synthesized with

an N-terminal signal sequence that contains the information needed for targeting [5]. This

hypothesis has been supported during the past ~50 years. Moreover, it is clear that different

signal peptides target proteins to different membrane systems in the cell (Figure 1).

Despite the identification of a great number of signal peptides of ER destined proteins in

eukaryotic cells and bacterial signal sequences, no significant sequence conservation could be

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found. In fact, artificial signal sequences [37,38] and randomly generated peptides [39] could

function as signal sequences, suggesting that it is the overall properties, rather than the precise

sequence, that play the role in targeting.

ER and bacterial plasma membrane targeting signal peptides contain a three-domain structure: an

N-terminal, 1-5 residues long, positively charged region (N-domain); a central, 7-15 residues

long, hydrophobic region (H-domain); and a C-terminal, 3-7 residues long, polar region (C-

domain) which contains small aliphatic residues at the -1 and -3 position allowing the secretory

protein to bind and be cleaved by signal peptidase [40-44]. It has been proposed that the

hydrophobicity of H-domain plays a role in discriminating between co-translational and post-

translational pathways [45-49]. A hydrophobic H-domain favors SRP mediated co-translational

translocation, while proteins with a less hydrophobic H-domain tend to be transported post-

translationally.

Distinct from ER and bacterial plasma membrane targeting sequences are the N-terminal

mitochondrial presequences [50-53] and chloroplast transit peptides [54,55] that target proteins

to mitochondria and chloroplasts, respectively. They are less hydrophobic than the signal

peptides mentioned earlier. The mitochondria presequence tend to form an amphiphilic D-helix,

while the chloroplast transit peptides are largely unstructured in aqueous solution, but become D-

helical upon insertion [56,57]. Mitochondrial presequences are typically rich in arginine [58,59],

while chloroplast transit peptides are often rich in hydroxylated amino acids [60]. Notably,

proteins synthesized with only a mitochondrial presequence or a chloroplast transit peptide are

destined to the mitochondrial matrix and chloroplasts stroma compartment, respectively. In the

matrix and stroma compartments, the presequence and transit peptide are removed by the

mitochondrial processing peptidase and stroma processing peptidase, respectively [61,62].

In addition, some mitochondrial and chloroplast proteins contain additional sequences to direct

the pre-proteins to other subcellular locations within the organelle. Chloroplast thylakoid

proteins are typically synthesized with a secondary thylakoid signal responsible for thylakoid

targeting, immediately following the transit peptide [63]. The thylakoid signal peptide is cleaved

following translocation into the thylakoid lumen by the thylakoidal processing peptidase [64].

Mitochondrial intermembrane space (IMS) proteins often contain an intermembrane IMS sorting

signal following the presequence. The IMS signal resembles bacterial and ER signal peptides.

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Alternatively, some IMS proteins have C-Xn-C motifs in the mature region of the mitochondrial

protein that form disulfide bonds and thereby function in retaining the proteins in the IMS [51].

Although protein import to peroxisome and nucleus seem to have less in common with other

systems, signal sequences are also required for these systems. The type 1 peroxisome targeting

signal (PTS1) is located at the extreme C-terminal, consisting of a conserved tripeptide (SKL).

PTS2 is a conserved nonapeptide (RL-X5-HL) embedded in an N-terminal sequence [65-67].

Nuclear localization signals (NLS) typically consist of one or two stretches of basic amino acids

[68,69].

Cytosolic Sorting and Membrane Receptors

One big challenge for precursor proteins is their tendency to aggregate in the cytosol prior to

translocation. Two major strategies have been developed to solve this problem: coupling

translocation with protein synthesis (co-translational translocation), or recruiting cytosolic

chaperones to prevent aggregation (post-translational translocation). The co-translational

pathway is commonly employed by bacterial inner membrane proteins and ER proteins in

eukaryotic cells that possess hydrophobic signal peptides. Examples of proteins that are co-

translationally imported have also been reported in mitochondria [70,71]. Post-translational

pathway can be found in all protein translocation systems.

Co-translational translocation to the bacterial plasma membrane or the ER requires the initial

recognition of the nascent polypeptide containing a hydrophobic signal peptide or TM segment

by SRP. In E. coli, SRP consists of a stem-loop shaped 4.5S RNA and a single polypeptide Ffh

with three conserved domains: an N-terminal N domain; a Ras-like G domain with GTPase

activity; and a C-terminal methionine-rich M domain, which possesses a hydrophobic groove for

signal peptide binding [72,46]. After substrate recognition, SRP shuttles the ribosome-nascent

chain complex (RNC) to its membrane associated receptor (SR, FtsY in E. coli). FtsY is also

composed of three domains: The N and G domains, similar to the NG domains of SRP; and an

acidic A domain involved in membrane binding [73]. The GTP-loaded NG domains of SRP and

FtsY interact with each other to form a hetero dimer, bringing the RNC to the vicinity of the Sec

translocon. The dimer formation also triggers the hydrolysis of GTPs, leading to the hand-over of

the RNC to the translocon and the release of SRP [74,75]. A snapshot of the molecular details of

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these interactions and how the hand-over takes place have been provided by a cryo-electron

microscopy study where a complex of the ribosome nascent chain, SRP, SR and the SecYEG

was formed [75] (Figure 2).

Post-translational translocation is arguably less understood but typically occurs when the signal

peptide is less hydrophobic. Most precursor proteins that are exported post-translationally require

cytosolic chaperones to keep them in an unfolded state. Trigger factor or SecB binds to the

preproteins that are not recognized by SRP in the cytosol, and deliver them to a SecY-associated

SecA [76-81]. In bacteria, the proteins that go post-translational are secreted proteins while most

membrane proteins go co-translational. In ER targeting, Hsp70 [82-84] or calmodulin [85] have

been reported to function as cytosolic binding factors, and Sec72 has been suggested to play the

role of a membrane receptor for Hsp70 in yeast [86]. In mitochondrial import, the presequences

are recognized by membrane receptors Tom20/Tom22 or Tom70, with the assistance of cytosolic

heat shock proteins Hsp70 and Hsp90 [87]. In the case of chloroplast import, transit peptides are

recognized by Hsp70/14-3-3 or Hsp90 in the cytosol, and the precursor is delivered to the surface

of the outer membrane via the receptors Toc34/Toc159 or Toc64, respectively [88-90].

Preproteins imported into peroxisome or nucleus also require cytosolic binding factors for

targeting, although their subsequent import mechanisms are different from other systems.

Peroxisome destined preproteins with PTS1 signals are recognized by peroxin 5 (Pex5), while

those with PTS2 signals bind to Pex7 [65,66,91-95]. In the case of Pex5, in addition to

recognizing peroxisome proteins, it also forms a part of the translocation channel for the

substrate [96]. Nuclear targeted proteins with NLS are recognized by importin-D (ImpD) and are

transported through nuclear pore complexes (NPC) with the help of carrier proteins like

importin-E (ImpE1) or E-Kap [68,69].

The common features that are shared by cytosolic binding factors and membrane receptors

among different protein translocation systems are: 1) cytosolic binding factors contain binding

sites that recognize the translocation signal of exported proteins. For example, cytosolic factors

that bind preproteins with bacterial or ER signal peptides contain hydrophobic pockets

surrounded by charged and polar amino acids, to accommodate for the H-region and positively

charged N-region of signal sequences, respectively; 2) receptor binding step is generally

accomplished by the formation of heterogenous oligomers between the chaperone and receptor; 3)

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Nucleotide triphosphate/nucleotide diphosphate exchange is often involved in receptor binding

and dissociation, but the energy from the hydrolysis at this step is not necessarily essential for

driving protein translocation; 4) the unidirectional hand-over of cargoes from the chaperone to

the receptor, and subsequently to the membrane channel is mostly driven by an increased affinity

chain [45,56].

Membrane channels

Once the preproteins are targeted to the cytosolic surface of the membrane, most are either

inserted into or membrane translocated through hetero-oligomeric translocases with channel-like

structures. A small group of these channels are wide enough for large, folded substrates to pass

through, like the Tat complex found in the plasma membrane in prokaryotes or the thylakoid

membrane of chloroplast in which the TatA component has been suggested to form channels of

various sizes under various conditions [26,97]. However, it is still under debate whether these

TatA-derived channels, which have been observed in detergent, are indeed formed in biological

membranes and function in protein translocation. However, the majority of membrane channels

possess highly regulated pores with relatively small diameters (ranging from 1.4 nm to 2.6 nm),

which allow the translocation of unfolded polypeptides without jeopardizing the permeability

barrier of the membrane [29,98-100].

The most well studied membrane channel is SecYEG in bacteria (Sec61 in eukaryotic cells)

[101,102]. The central component, SecY (Sec61D), is composed of 10 D-helical transmembrane

segments arranged in an hour-glass shape, with the pore ring blocked by a plug domain in the

resting state (Figure 3). The SecY (Sec61D) channel is typically stabilized and regulated by

auxiliary proteins such as SecE, SecG, SecDF, YajC, or SecA in bacteria (Sec61E, Sec61J,

Sec62/63, BiP in the ER) [103,104]. The SecY (Sec61D) channel is capable of both vertical and

lateral opening, and the binding of the signal peptide or stop transfer sequence of the inserting

protein plays an important role in its gate regulation [105,29,106-110]. Details of the lateral

opening and the plug displacement have been elucidated from structures of the Sec channel

actively involved in membrane insertion of a substrate [110].

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At the outer membrane of mitochondria and chloroplast, Tom40 and Toc75 form translocation

channels for protein import into mitochondria and chloroplasts [56,99,111]. Interestingly, these

E-barrel channels are capable of forming oligomers. The Tom40 complex, along with the

auxiliary proteins Tom20/Tom22, can contain two to three pore forming units [112,113], while

the Toc75 complex with the auxiliary Toc159/Toc34 components can contain three to four pore

forming channels [114-116]. The exact gating mechanisms of these channels are not clear, but it

has been suggested that the mitochondrial Tom22 and the chloroplast Toc159 proteins might be

involved in regulating their pore opening [117-119]. Once the preproteins are translocated across

the outer membrane, they can either be reinserted into the outer membrane, remain in the inter-

membrane space, or be further imported across the inner membrane to the matrix of

mitochondria or the stroma of chloroplasts. This secondary import step across the inner

membrane requires the membrane channels formed by Tim23 or Tic110, respectively [120,121].

The core architecture of TIM complex is composed of Tim23, which forms the D-helical channel;

Tim50 with a large intermembrane space domain, which functions to recruit the matrix targeting

signal of the imported protein at Tom40 channel and brings it to Tim23 [122] and Tim17 which

coordinates the pmf-dependent channel opening [123]. The TIC channel is mainly composed of

Tic110, and the IMS chaperone, Tic22, which plays a similar shuttle role as Tim50 [124,125].

Tic20 has also been reported to form channels at the inner envelop [126].

The membrane channel system is unique in peroxisome compared to other organelles. It has been

reported that after recognizing preproteins with PTS1, the import receptor Pex5, at least a

fraction of it, is integrated into the membrane, forming part of the translocation channel in

collaboration with Pex14 [96]. Following the completion of translocation, by a mechanism that is

not yet understood, Pex5 is mono- or poly-ubiquitylated, and released back to the cytosol for

recycling [127,128].

In contrast, the Oxa1/Alb3/YidC family members found in mitochondria, chloroplast and

bacteria, respectively, function as insertases without forming channels [131-133]. They contain

a 5-transmembrane-helix core domain that folds in such a way to form a hydrophilic groove

within the inner leaflet of membrane bilayer (Fig. 3). In addition to the polar groove, another

important structural element is the greasy slide where the TM segments of YidC substrates make

contact during insertion. The protein substrates of YidC family insertases are typically

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membrane proteins that have small hydrophilic domains that need to be translocated [134-

144,133].

Another unique channel-forming insertase is the BAM family of proteins that insert E-barrel

proteins into the outer membrane of gram-negative bacteria, mitochondria and chloroplasts [145].

The BAM machinery contains a E-barrel domain that catalyzes insertion, and several

polypeptide-transport-associated (POTRA) domains that bind the proteins destined to insert into

the outer membrane (Fig. 3). Two mechanisms have been proposed for the E. coli BAM to insert

and fold E-barrel proteins into the outer membrane. The E-augmentation mechanism proposes

that the substrate initiates E-barrel formation, possibly using the exposed BamA E1 strand as a

template [146]. The protein would then insert into the membrane, possibly using pairs of E-

strands, to form a substrate-BamA super-barrel complex, where the existing E-strand would

function as a template for the newly forming strand. The building of the E-barrel of the substrate

most likely occurs at the exposed edge of the BamA barrel after a lateral opening between the

unstable E1 and E16 strands. When the substrate is still bound to BamA and the E-barrel is

almost completely formed, the complex collapses and the substrate barrel buds off and moves

away from BamA. The second mechanism proposes that BamA functions to thin and destabilize

the membrane region adjacent to the BamA barrel [147,148]. With these perturbations in the

membrane, the substrate is proposed to insert and fold into a E-barrel structure. It should be

noted that the chloroplast Toc75 protein is a member of the BamA family of proteins (Fig. 3e).

The Toc75 POTRA domain has been found to possess chaperone activity and function to

promote import of proteins into chloroplast [149].

Energetics of Protein Translocation

It is clear that energy is required to transport proteins across the hydrophobic barrier of

membranes. For co-translational translocation, GTP hydrolysis occurring during translation

provides the energy source for the coupled translocation process. For post-translational

translocation, a combination of membrane potential and ATP hydrolysis powers the translocation

of proteins across bacterial plasma membrane, ER and inner membrane of mitochondria and the

thylakoid membrane of chloroplasts [150-158].

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In several translocation systems, specific subunits function to couple membrane potential (or pmf)

with protein translocation. SecDF possesses a proton tunnel that utilizes the influx of protons to

fuel protein translocation through SecYEG [159]. In mitochondria, Tim17 has been reported to

coordinate membrane potential dependent pore opening of Tim23 [123]. Similar functioning

subunits are yet to be identified for other systems.

ATPase motors are broadly required for protein translocation in different organelles: SecA in

bacterial cytoplasm [160], BiP in ER lumen [161,105], mtHsp70 in the mitochondria matrix

[51,162,50], and cpHsp70 in the stroma of chloroplasts [163].

While SecA, the motor ATPase, performs its function on the cis-side of the membrane, other

ATPases function on the trans-side. For example, figure 4 shows a model of BiP in the ER

lumen binding to Sec63 within the Sec61-Sec63-Sec71-Sec72 complex [104]. Itskanov and Park

[104] used cryo-electron microscopy to solve the structure of the post-translational protein

translocation machinery in yeast, which determined the positioning of the Sec63, Sec71 and

Sec72 on Sec61. Due to flexibility, the Sec62 and the J domain of Sec63 could not be fitted to

the electron density. Sec63 has 3 N-terminal TM segments and the J domain that interacts with

BiP. The structure revealed that it is mainly the 3 N-terminal TM segments and a cytosolic

region of Sec63 (of the Sec71-Sec72 complex), which makes the connections with Sec61. They

showed that the structure had a wide-open lateral gate and an open pore as a result of the Sec63

interactions that would allow it to translocate bigger peptide chains compared to a partially

opened channel with SecA or ribosome bound. They used a homology model based on a

structure of the bacterial J domain bound to Hsp70 complex to provide insight into how the J

domain might bind and position BiP. Intriguingly, the homology model showed that the peptide-

binding cleft of BiP was right below the translocation pore, in an ideal location to latch onto the

polypeptide chain in the ER lumen and promote translocation. It should be noted that insight

into how the Sec61 channel is activated for post-translational translocation was also provided by

Wu et al. where they solved the post-translational Sec complex in yeast as well [164].

One popular model proposed for the ATPase motors is the power stroke mechanism. A recent

study on SecA [165] showed that the two-helix finger of SecA pushes the polypeptide to be

translocated into the SecYEG channel. During ATP hydrolysis, the finger retracts and the clamp

region of SecA binds the preprotein preventing backsliding. Another model argues that the

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ATPase motors are likely functioning via a Brownian Ratchet mechanism where the polypeptide

moves through the channel by Brownian diffusion, and the ATPase motors drive translocation

through the channel by repetitive binding, which prevents backsliding [166-168]. In addition, a

recent model called “entropic pulling” has also been proposed [169], suggesting that the

translocation is driven by the change of conformational freedom of both motors and incoming

preprotein.

Peroxisomal import also requires ATP as the energy source, but in a different manner [170].

Although no ATPase activity has been shown to be involved in the import process, the release of

receptor Pex5 requires AAA ATPase Pex1 and Pex6 [171,172]. Since the import of preprotein

and export of Pex5 are closely coupled, this phenomenon is called “export-driven protein import”

[173].

Finally, GTP hydrolysis is involved in nuclear trafficking by creating a RanGTP gradient across

the nuclear envelope, which leads to the unidirectional import of most nuclear proteins [68,174].

Outlook

Considerable progress has been achieved in the protein transport field in the past 40 to 45 years

since Blobel proposed the Signal Hypothesis [5-7]. The signal peptide address codes for

subcellular targeting have been elucidated. Advanced algorithms have been developed to predict

the subcellular localization of a protein based on the signal peptides [175]. We now understand a

great deal about the molecular mechanism of protein targeting. Structures of targeting factors,

chaperones, receptors, and membrane channels have now been solved at atomic resolution

[106,72,108-110,107,29,104,102,145,75]. We can now visualize the ribosome-Sec complex in

the process of inserting a substrate in its membrane environment. We are gaining understanding

about how the transmembrane segments are recognized at the molecular level by the Sec

translocon [75,110,104].

Nonetheless, there are still many intriguing questions yet to be answered. For instance, what are

the structures of the mitochondrial (TIM) and chloroplasts (TOC/TIC) transport channels? What

are the non-traditional protein translocation mechanisms used to import proteins into the

peroxisome and to translocate folded proteins by the Tat pathway? Can single molecule

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fluorescence techniques shed light on how proteins pass across the translocation channels in real

time? Obviously, there is much to be done in the field of protein transport and new works will

ultimately lead to deeper understanding of how cellular compartmentalization is achieved.

Acknowledgements

This work was partially supported by National Science Foundation grant MCB-1814936 (R.E.D).

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Figure 1 Targeting signals. Targeting signals for proteins destined to the ER, mitochondria, chloroplasts, peroxisome and nucleus are shown in red and blue. Blank boxes represent hydrophobic helices; boxes with stripes represent amphiphilic helices; chloroplast transit peptides tend to be unstructured in solution, as shown by red line; black arrows represent signal peptidase (processing peptidase) cleavage sites. Figure 2 Structure of the ribosome-SRP-SR-SecYEG quaternary complex. One example of cytosolic sorting and the stepwise delivery of the ribosome-bound SRP protein to the membrane receptor/translocation machinery is shown in the cryo-EM structure of the ribosome-SRP-SR-SecYEG quaternary complex (pdb: 5NCO) (Jomaa et al. [75]). The ribosome had synthesized the FtsQ nascent chain containing the first transmembrane segment. Each of the components are color-coded as follows: ribosome in grey; SRP RNA in orange; SRP M domain in cyan; SRP NG domain in blue; SR NG domain in green; SR A domain (which is not resolved) in light green; SecYEG in red; signal sequence in magenta. Figure 3 Structures of common translocation devices. A. X-ray structure of SecYEG from Thermus thermophiles (pdb: 5AWW). SecY, SecE and SecG subunits are shown in light blue, yellow and magenta, respectively. The lateral gate is shown in red and dark blue. The plug domain is shown in green. B. X-ray structure of YidC from Escherichia coli (pdb: 3WVF). The greasy slide comprised of TM3 and TM5 is shown in dark blue and red, respectively. The conserved positively charged residue in the hydrophilic groove is shown in green. C. Structure of BamABCDE complex. It was modeled by combining X-ray structure of BamACDE (pdb: 5EKQ) and X-ray structure of BamB fused to BamA POTRA domain (pdb: 4PK1) from Escherichia coli. BamA in light blue; BamB in orange; BamC in magenta; BamD in cyan; BamE in green. D. Dimer of Tom40. The model is based on cryo-EM structure from Neurospora crassa (pdb: 5O8O). E. Model of full-length Toc75 from Arabidopsis thaliana adapted from O’Neil et al [149]. X-ray structures of FhaC (pdb: 2QDZ) [176] and Toc75 POTRA1-3 (pdb: 5UAY) are superpositioned by aligning POTRA2 of FhaC (not shown) and POTRA3 (yellow) of Toc75. The barrel domain (blue) is from FhaC, and POTRA domains 1, 2 and 3 (green, purple and yellow respectively) are from Toc75. POTRA2 contains an extra D-helix (P2h), as shown in cyan. Figure 4 Model of the post-translational Sec61 translocase pathway with the BiP ATPase

promoting translocation. The model is constructed by combining the cryo-EM structure of the yeast Sec post-translational complex (pdb: 6N3Q) and the bacterial J domain bound to DnaK Hsp70 (pdb: 5NRO) as a model for J-domain-BiP (see Itskanov et al. [104]). Each of the components are color-coded as follows: Sec61 channel (Sec61D) in light blue; SSS1 (Sec61J) in cyan; SBH1 (Sec61E) in magenta; Sec63 in orange; Sec66 in yellow; Sec72 in green; the homology model of the J-domain of Sec63 in red; the hypothetical model of BiP ATPase in pink. The blue line represents a model of the substrate.

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Figure 4