Báo cáo khoa học: Protein transport across the endoplasmic reticulum membrane pdf

In bacteria, the translocation of secretory and membrane proteins occurs through a homologous channel in the plasma membrane, employing signal and TM sequences that are similar to those

Protein transport across the endoplasmic reticulum

membrane

Delivered on 8 July 2007 at the 32nd FEBS Congress in Vienna,

Austria

Tom A Rapoport

Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, Boston, MA, USA

Introduction

Protein translocation across the eukaryotic

endopl-asmic reticulum (ER) membrane is a decisive step in

the biosynthesis of many proteins [1] These include

soluble proteins, such as those ultimately secreted from

the cell or localized to the ER lumen, and membrane

proteins, such as those in the plasma membrane or in

other organelles of the secretory pathway Soluble

proteins cross the membrane completely and usually

have cleavable N-terminal signal sequences, whose

major feature is a segment of approximately seven to

12 hydrophobic amino acids Integral membrane

proteins have one or more transmembrane (TM)

segments, each containing approximately 20 hydropho-bic amino acids, with intervening hydrophilic regions

on either side of the membrane Both types of proteins use the same machinery for transport across the membrane: a protein-conducting channel This channel allows polypeptides to cross the membrane and per-mits hydrophobic TM segments of membrane proteins

to exit laterally into the lipid phase In bacteria, the translocation of secretory and membrane proteins occurs through a homologous channel in the plasma membrane, employing signal and TM sequences that are similar to those in eukaryotes

The translocation channel is formed from an evolu-tionarily conserved heterotrimeric membrane protein

Keywords

endoplasmic reticulum; lipid; membrane

integration; protein-conducting channel;

protein translocation; ribosome; Sec

complex; Sec61; SecA; SecY

Correspondence

T A Rapoport, Howard Hughes Medical

Institute and Department of Cell Biology,

Harvard Medical School, 240 Longwood

Avenue, Boston, MA 02115, USA

Fax: +1 617 432 1190

Tel: +1 617 432 0676

E-mail: tom_rapoport@hms.harvard.edu

(Received 21 May 2008, accepted 18 June

2008)

doi:10.1111/j.1742-4658.2008.06588.x

A decisive step in the biosynthesis of many eukaryotic proteins is their par-tial or complete translocation across the endoplasmic reticulum membrane

A similar process occurs in prokaryotes, except that proteins are trans-ported across or are integrated into the plasma membrane In both cases, translocation occurs through a protein-conducting channel that is formed from a conserved, heterotrimeric membrane protein complex, the Sec61 or SecY complex Structural and biochemical data suggest mechanisms that enable the channel to function with different partners, to open across the membrane and to release laterally hydrophobic segments of membrane proteins into lipid

Abbreviations

EM, electron microscopy; ER, endoplasmic reticulum; SRP, signal recognition particle; TM, transmembrane.

complex, called the Sec61 complex in eukaryotes and

the SecY complex in bacteria and archaea [2] The

a-subunit forms the channel pore, as originally

demon-strated by systematic crosslinking experiments [3] In

addition, reconstitution experiments show that the

Sec61⁄ SecY complex is the only essential membrane

component for protein translocation in mammals and

bacteria [4–6] The channel has an aqueous interior,

as demonstrated by electrophysiology experiments

and measurements of the fluorescence life-time of

probes incorporated into a translocating polypeptide

chain [7–9]

Different modes of translocation

The channel must associate with partners that provide

the driving force for translocation Depending on the

channel partner, there are different known

transloca-tion modes [1] In co-translatransloca-tional translocatransloca-tion, the

major partner is the ribosome This translocation

mode is found in all cells of all species, and it is used

for the translocation of both secretory and membrane

proteins Co-translational translocation begins with a

targeting phase, during which a ribosome-nascent

chain complex is directed to the membrane by the signal recognition particle (SRP) The ribo-some⁄ SRP ⁄ nascent chain complex is bound to the membrane, first by an interaction between SRP and its membrane receptor (SR), and then by an interaction between the ribosome and the translocation channel (Fig 1) The elongating polypeptide chain sub-sequently moves directly from a tunnel inside the ribo-some into the associated membrane channel GTP hydrolysis during translation provides the energy for translocation

In most, if not all cells, some proteins are translocated after their completion This post-translational translo-cation occurs by different mechanisms in eukaryotes and bacteria In yeast (and probably in all eukaryotes), translocation occurs by a ratcheting mechanism and involves, as channel partners, the tetrameric Sec62⁄ 63 membrane protein complex and the ER luminal protein BiP, a member of the Hsp70 family of ATPases (Fig 2) [10] Following a transient interaction of BiP-ATP with the J-domain of Sec63p, BiP-ATP is hydrolyzed and the peptide-binding pocket of BiP closes around the translocation substrate BiP serves as a Brownian ratchet, preventing the bound polypeptide from sliding

SRP receptor

ribosome

SRP

Sec61/SecY complex

signal

sequence

cytosol

Fig 1 Model of co-translational trans-location The polypeptide chain moves from the tunnnel inside the ribosome into the membrane channel The energy for translocation is provided by GTP hydrolysis during translation Figure adapted from [1].

Sec61

complex ADP ADP

ADP ADP ADP

ADP ATP

ATP ATP cytosol

ATP

Sec62/63 complex

signal

sequence

BiP J-domain

Fig 2 Model of post-translational transloca-tion in eukaryotes A Brownian ratcheting mechanism is responsible for moving a poly-peptide chain through the membrane Trans-location might be mediated by oligomers of the Sec61p complex, as in the other modes

of translocation Figure adapted from [1].

back into the cytosol, but allowing polypeptide

move-ment in the forward direction When moved

suffi-ciently, the next BiP molecule binds, and this process

is repeated until the entire polypeptide chain is

translo-cated Finally, nucleotide exchange of ADP for ATP

opens the peptide binding pocket and releases BiP

from the polypeptide

In eubacterial post-translational translocation,

poly-peptides are ‘pushed’ through the channel by its

part-ner, the cytosolic ATPase SecA (Fig 3) [11] SecA has

two nucleotide binding domains (NBD1 and NBD2),

which bind the nucleotide between them and move

relative to one another during the ATP hydrolysis

cycle Exactly how these movements are used to ‘push’

a polypeptide chain through the channel remains

unknown The size of SecA makes it unlikely that it

inserts deeply into the SecY channel, as proposed

pre-viously [11] Bacterial translocation in vivo requires an

electrochemical gradient across the membrane, but the

mechanism by which the gradient is utilized is unclear

Archaea probably have both co- and

post-transla-tional translocation Although co-translapost-transla-tional

trans-location is likely to be similar to that in eukaryotes

and eubacteria, it is not known how post-translational

translocation occurs because archaea lack SecA, the

Sec62⁄ 63 complex and BiP

Structure and function of the

translocation channel

The crystal structure of an archaeal SecY complex

pro-vides much insight into channel function [2] The

struc-ture is likely representative of all species, as indicated

by sequence conservation and by the similarity to a

lower resolution structure of the Escherichia coli SecY

complex determined by electron microscopy (EM)

from 2D crystals [12] The a-subunit consists of two

halves, TMs 1–5 and TMs 6–10, which form a lateral

gate at the front and are clamped together at the back

by the c-subunit (Fig 4A) The 10 helices of the

a-sub-unit form an hourglass-shaped pore that consists of

cytoplasmic and external funnels whose tips meet approximately half way across the membrane (Fig 4B) The cytoplasmic funnel is empty, whereas the external funnel is filled by a short helix, the ‘plug’ The constriction of the hourglass is formed by a ‘pore

signal sequence

NBF1

SecY complex

SecA

cytosol

Fig 3 Model of post-translational

transloca-tion in bacteria The ATPase SecA uses the

energy of ATP hydrolysis to push a

poly-peptide through the channel Figure adapted

from [1].

β

γ

pore ring

pore ring

A

B cytosol

hinge

plug

plug

Fig 4 Structure of the translocation channel (A) View from the cytosol of the X-ray structure of the SecY complex from Methano-coccus jannaschii The two halves of SecY are colored blue (TM 1–5) and red (TM 6–10) The plug is shown in yellow and pore ring residues are shown in green The purple arrow indicates how the lateral gate opens The black arrow indicates how the plug moves

to open the channel across the membrane (B) Cross-section of the channel from the side Figure adapted from [1].

ring’ of hydrophobic amino acid residues that project

their side chains radially inward The crystal structure

represents a closed state of the channel, but

biochemi-cal data indicate how it can open to translocate

proteins (see below)

Opening the channel across the

membrane

The crystal structure indicates that a single copy of the

Sec61⁄ SecY complex forms the pore [2] The

transloca-tion of a secretory protein begins with insertransloca-tion of a

loop into the channel, such that the signal sequence

is intercalated into the walls of the channel and the

segment distal to it is inserted into the pore proper

(Figs 1–3) In a first step, the binding of a channel

partner (i.e the ribosome, the Sec62⁄ 63p complex, or

SecA) likely weakens interactions that keep the plug in

the center of the Sec61⁄ SecY molecule, as indicated by

an increased ion conductance when nontranslating

ribosomes are bound to the channel [7] Next, the

hydrophobic segment of a signal sequence intercalates

into the lateral gate, between TM2b and TM7, as

indi-cated by photo-crosslinking experiments [13] This

fur-ther destabilizes plug interactions, causing the plug to

be displaced from the center of Sec61⁄ SecY, as shown

by disulfide bridge crosslinking [14,15] During

sub-sequent translocation, the signal sequence remains

stationary, whereas the rest of the polypeptide moves

through the pore from the cytoplasmic funnel through

the pore ring into the extracellular funnel (Figs 1–3),

as indicated by systematic disulfide crosslinking

experi-ments [16] The aqueous interior of the channel and its

shape help to minimize the energy required for the

translocation of a polypeptide through the membrane

The plug can only return to the center of Sec61⁄ SecY

when the polypeptide chain has left the pore

The diameter of the pore ring, as observed in the

crystal structure, has to increase during translocation,

probably by movements of the helices to which the

pore ring residues are attached The pore ring is indeed

flexible, as shown by molecular dynamics simulations

and electrophysiology experiments [17,18] The

maxi-mum size of the pore could be 15· 20 A˚, which is

much smaller than the pore size estimated from

fluo-rescence quenching experiments (40–60 A˚) [19] These

data could be reconciled with the crystal structure if

two or more Sec61⁄ SecY complexes associated at their

front surfaces, opened their lateral gates, and fused

their pores to form a larger channel However,

disul-fide bridge crosslinking experiments argue against

fusion of different pores because they show that,

during SecA-mediated translocation, both the signal

sequence and the mature region of a polypeptide chain are located in the same SecY molecule [20] In addi-tion, a detergent-solubilized translocation intermediate also contains just one copy of SecY associated with one SecA and one translocation substrate molecule [21] Two SecY molecules in a nearly front-to-front orientation were proposed to be associated with a translating E coli ribosome [22] However, this conclu-sion was based on a low-resolution ( 15 A˚) EM structure, and the docking of the crystal structure required its drastic modification Furthermore, the position and orientation of both SecY molecules are different from that of the single SecY molecule observed in recent EM structures of nontranslating ribosome-SecY complexes [23] (Fig 5) It should be noted that, in the fluorescence quenching experiments, the fluorescent probes were located deep inside the ribosome, and therefore the same large diameter (40–60 A˚) must be assumed for the ribosome tunnel, a size that does not agree with that seen in ribosome structures (< 20 A˚) determined by crystallography

or cryo-EM [24] Taken together, it is likely that the translocation pore is formed by just one copy of the Sec61⁄ SecY complex

Oligomeric translocation channels Although the pore is formed by only one Sec61⁄ SecY molecule, translocation of a polypeptide chain appears

to be mediated by oligomers This conclusion is based

on the observation that a SecY molecule defective in SecA-mediated translocation can be rescued by linking

it covalently with a wild-type SecY copy [20] Disulfide bridge crosslinking showed that SecA interacts through its NBD1 with a nontranslocating SecY copy and moves the polypeptide chain through a neighboring SecY copy The Sec61⁄ SecY complex probably forms oligomers during co-translational translocation as well When a ribosome⁄ nascent chain ⁄ SRP complex binds

to the SRP receptor, a domain of SRP undergoes a conformational change, exposing a site on the ribo-some to which a single Sec61⁄ SecY molecule could bind [25] This is likely to be the molecule seen in recent EM structures of complexes of nontranslating ribosomes with either the SecY or the Sec61 complex [23,23a] The bound SecY⁄ Sec61 molecule is close to the point where a polypeptide exits the ribosome and could thus become the translocating copy (Fig 5)

At a later stage of translocation, SRP completely detaches from the ribosome, and an additional copy

of the Sec61⁄ SecY complex may associate (Fig 1),

as suggested by crosslinking and freeze-fracture EM experiments [26,27] These copies could stabilize the

ribosome–channel junction and possibly recruit other

components, such as signal peptidase and

oligosaccha-ryl transferase, or the translocon-associated protein

complex Upon termination of translocation,

disso-ciation of the Sec61⁄ SecY oligomers could facilitate

the release of the ribosome from the membrane

Dissociable oligomers may also allow the Sec61⁄ SecY

complex to change channel partners and modes of

translocation

The EM structures of detergent-solubilized

ribo-some–channel complexes suggested the presence of

three or four Sec61 molecules [28,29] However, it now

appears that only one Sec61 molecule is present and

that the additional density can be attributed to lipid

and⁄ or detergent

Membrane protein integration

During the synthesis of a membrane protein,

hydro-phobic TM segments move from the aqueous interior

of the channel through the lateral gate into the lipid

phase The lateral gate may continuously open and

close, exposing polypeptide segments located in the

aqueous channel to the surrounding hydrophobic lipid

phase Alternatively, there may be a ‘window’ in the

lateral gate that would allow the hydrocarbon chains

of lipids to make contact with a translocating polypep-tide at the same time as preventing charged head groups from entering the channel Polypeptide seg-ments inside the channel would partition between the aqueous and hydrophobic environments This model is supported by photo-crosslinking experiments [30] and

by the close correlation between a hydrophobicity scale and the tendency of a peptide to span the membrane [31] Hydrophilic segments between the TMs would alternately move from the ribosome through the aque-ous channel to the external side of the membrane, or emerge into the cytosol between the ribosome and channel through a ‘gap’ that can be visualized in EM structures [28,29]

The first TM segment of a membrane protein can have its N-terminus on either side of the membrane, depending on the amino acid sequence of the protein, which often determines the orientation of subsequent TMs If the first TM is long and the preceding sequence not retained in the cytosol by positive charges or by its folding, the N-terminus can flip across the channel and subsequently exit laterally into the lipid phase When the N-terminus is retained in the cytosol and the polypeptide chain is further elongated,

SecY

Fig 5 Structure of the E coli ribosome-associated SecY channel (A) Bottom view showing the single copy of the SecY complex that is bound to a nontranslating ribosome The docked X-ray structure is indicated by the ribbons The electron density of the channel is shown in transparent pink (B) Comparison of the single-SecY model (left) with a model [22] in which two SecY copies are bound in a near front-to-front orientation to a translating ribosome (right) The lateral gate of the SecY channel is indicated by an arrow, and the tunnel exit by a star The smaller picture above shows the orientation of the ribosome.

the C-terminus can translocate across the channel,

inserting the polypeptide as a loop, as in the case of a

secretory protein

Maintaining the permeability barrier

The channel must prevent the free movement of small

molecules, such as ions or metabolites The crystal

structure suggests a simple model for the maintenance

of the membrane barrier The resting channel would

be closed, which is consistent with electrophysiology

experiments showing that, in the absence of other

com-ponents, the SecY channel is impermeable to ions and

water [18] In the active channel, the pore ring would

fit around the translocating polypeptide chain like a

gasket to restrict the passage of small molecules The

seal would not be expected to be perfect, but leakage

could be compensated for by powerful ion pumps

During the synthesis of a multi-spanning membrane

protein, the seal would be provided in an alternating

manner by either the nascent chain in the pore or,

once the chain has left the pore, by the plug returning

to the center of Sec61⁄ SecY Although this model

needs further experimental verification, it would

explain how the membrane barrier can be maintained

in both co- and post-translational translocation

Surprisingly, plug deletion mutants are viable in

Saccharomyces cerevisiae and E coli and have only

moderate translocation defects [32–34] However, the

crystal structures of these mutants show that new plugs

are formed from neighboring polypeptide segments

[34] The new plugs still seal the closed channel, but

they have lost many interactions that normally keep

the plug in the center of SecY This results in

continu-ous channel opening and closing, and permits

polypep-tides with defective or even missing signal sequences to

be translocated The plug sequences are only poorly

conserved among Sec61⁄ SecY channels, supporting the

idea that promiscuous segments can seal the channel

and lock it in its closed state

Perspective

We are beginning to understand protein translocation

across the eukaryotic ER and bacterial plasma

mem-branes at the molecular level In particular, progress

during the recent years has led to important insights

into the function of the Sec61⁄ SecY channel

Neverthe-less, there are major questions in the field that remain

controversial and unresolved, and further progress will

require a combination of approaches

Electrophysi-ology experiments are needed to complement the

fluo-rescence quenching method, particularly because the

results obtained from the latter are difficult to recon-cile with structural data Important questions with respect to co-translational translocation include how the SRP receptor and channel collaborate, how many Sec61⁄ SecY complexes participate in translocation, and how the ribosome ultimately dissociates from the channel Both the precise role of the Sec62⁄ 63 compo-nents in post-translational translocation and the mech-anism by which SecA moves polypeptides need to be clarified Membrane protein integration is still particu-larly poorly understood, and new methods are required

to follow the membrane integration of TMs Several other translocation components have been identified, such as the TRAM protein and the translocon-associ-ated protein complex in mammalian cells, or the YidC and SecDF proteins in prokaryotes These components may be required as chaperones for the folding of TM segments, or to increase the efficiency of translocation

of some substrates, but their precise functions remain

to be clarified Much of the progress in the field will hinge on structural data, with the ‘holy grail’ being

a picture of an active translocon, where a channel associated with both a partner and a translocating polypeptide chain is visualized at the atomic level The results obtained will likely serve as a paradigm for other protein translocation systems, such as those in mitochondria, chloroplasts and peroxisomes

Acknowledgements Work in the author’s laboratory was supported by grants from the National Institute of Health The author is a Howard Hughes Medical Institute Investi-gator Briana Burton and Sol Schulman are thanked for critically reading the manuscript

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