Download Protein Synthesis and Quality Control at the Endoplasmic Reticulum

Membrane Group Colloquium Edited by N. Bulleid (Manchester) and S. High (Manchester). Sponsored by AstraZeneca and Wolf Laboratories. 679th Meeting
of the Biochemical Society held at the University of Essex, Colchester, on July 2–4 2003.
Maintaining the permeability barrier during
protein trafficking at the endoplasmic
reticulum membrane
A.E. Johnson1
Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, TX 77843-1114, U.S.A., Department
of Chemistry, Texas A&M University, College Station, TX 77843, U.S.A., and Department of Biochemistry and Biophysics, Texas A&M University, College
Station, TX 77843, U.S.A.
Abstract
Many proteins are translocated across or integrated into a cellular membrane without disrupting its integrity,
although it is difficult to imagine how such macromolecular transmembrane movement can occur without
simultaneously allowing significant small-molecule and ion diffusion across the bilayer. Recent studies
have identified some molecular mechanisms that are involved in maintaining the permeability barrier of
the endoplasmic reticulum membrane during co-translational protein translocation and integration. These
mechanisms are both simple and direct in concept, but are operationally complex and require the coordinated and regulated interaction of several multicomponent complexes.
The apparent contradiction:
macromolecular transport without
coincident small-molecule transport
In every cell, a significant fraction of the proteins synthesized
by ribosomes in the cytoplasm must be transported through
a cellular membrane to reach the external or organellar destination in which they normally function. In addition, every
cell synthesizes a large number of proteins that are inserted
into a cellular membrane. Every cell has therefore evolved the
molecular machinery and mechanisms necessary to identify
those proteins destined to leave the cytoplasm and to facilitate their movement through or into a phospholipid bilayer.
In eukaryotic cells, secretory proteins and membrane proteins are typically first translocated across or integrated into
the membrane of the ER (endoplasmic reticulum) at sites termed translocons [1]. Protein translocation and integration at
the ER membrane are mostly co-translational in mammalian
cells, which means that the mammalian secretory or memKey words: endoplasmic reticulum membrane (ER membrane), fluorescence spectroscopy,
permeability barrier, protein integration, protein translocation, translocon.
Abbreviations used: ER, endoplasmic reticulum; SRP, signal-recognition particle; RNC, ribosomenascent chain; BiP, Ig heavy-chain-binding protein; TM, transmembrane.
1
Address correspondence to College of Medicine, 116 Reynolds Medical Building, TAMUS
HSC, 1114 TAMU, College Station, TX 77843-1114, U.S.A. (e-mail [email protected]).
brane proteins are being translated by the ribosome at the
same time that they are being translocated or integrated at
the translocon. Hence, ribosomes synthesizing secretory and
membrane proteins are usually not found in the cytosol, but
instead are bound to translocons at the ER membrane. Such
ribosomes are identified when a signal sequence in the nascent
chain emerges from the ribosome and binds very tightly
to the SRP (signal-recognition particle) [2,3]. The resulting
RNC (ribosome-nascent chain) complex with SRP is then
targeted to the translocon via a GTP-dependent process that
involves the SRP receptor [1,2]. Not surprisingly, the processes that accomplish protein trafficking at membranes are
extraordinarily complex, and we have only begun to appreciate the subtleties and sophistication of various mechanistic aspects of protein targeting to, translocation across
and integration into different cellular membranes. Most of
the mechanistic details are still unknown.
One of the most important and fundamental issues that
has intrigued scientists from the beginning is the mechanism
by which the cell is able to maintain the permeability barrier
of the membrane while proteins are being translocated across
or integrated into that membrane. After all, the purpose of
a membrane is to separate two aqueous compartments or
milieux. Yet protein trafficking requires the movement of a
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Figure 1 Three models for co-translational protein translocation
(A) The nascent protein chain moves through an aqueous pore formed by ER membrane proteins that comprise the translocon
[1]. Ion movement through the pore is prevented by a tight junction between the ribosome and the translocon. (B) The
nascent chain moves directly through the non-polar core of the bilayer without the assistance of any ER membrane proteins.
(C) Translocation occurs through an aqueous pore, but ion movement through the pore is prevented by a structural constriction
in the translocon that allows only the nascent chain to pass through the pore.
protein macromolecule, either wholly or in part, through a
membrane. How can this occur without the simultaneous
movement of many small ions and molecules across the
membrane?
There are many examples of the need to maintain a
membrane’s permeability barrier during protein trafficking.
More than 90% of mitochondrial proteins are synthesized by
cytoplasmic ribosomes and then imported into mitochondria
[4,5]. Since mitochondrial production of ATP requires that an
electrical potential difference is maintained across the inner
mitochondrial membrane, the transport of large numbers of
proteins into the mitochondrial matrix or inner membrane
must occur without compromising its permeability barrier
and allowing significant numbers of ions to travel through
the bilayer and dissipate that potential difference. Similarly,
ion gradients are maintained in bacteria across the plasma
membrane while proteins are being exported from the cell.
Thus bacteria have also evolved mechanisms that successfully
export proteins without simultaneously allowing significant
ion passage through the membrane. As a final example,
calcium ions are stored in the ER in eukaryotic cells. Since
these ions function as very potent second messengers when
released from the ER into the cytoplasm, the cells must
prevent unregulated calcium ion release from the ER during
protein translocation and integration to avoid disrupting
cell metabolism. These three examples demonstrate that
all cells have successfully evolved mechanisms for eliminating
or reducing small-molecule and ion leakage to acceptable
levels during protein trafficking, and hence for maintaining
a membrane’s permeability barrier. Yet few studies have
examined the molecular details of this aspect of protein
trafficking.
Secretory proteins are translocated though
aqueous holes in the ER membrane
Blobel and Dobberstein [6] proposed many years ago that
proteins in the ER membrane form an aqueous pore through
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which secretory proteins are transported to the ER lumen
(Figure 1A). However, experimental confirmation of this
hypothesis proved very difficult to obtain, and alternative
models involving direct movement of the nascent chain
through the non-polar core of the ER membrane (Figure 1B)
were also proposed (e.g. [7]). These competing models were
debated vigorously, largely because the lack of direct experimental evidence for the presence or absence of a translocon
and an aqueous pore did not allow a resolution of the issue.
Simon and Blobel [8] then reported conductivity measurements that were consistent with the formation of an aqueous
channel in the ER membrane. This channel was observed only
after puromycin treatment of membrane-bound ribosomes,
presumably because puromycin released the nascent chain
from the ribosome and translocon. Although these experiments did not directly address nascent chain surroundings,
the data strongly supported the aqueous pore model (Figure 1A).
The most direct approach to determine which of the two
models shown in Figures 1(A) and 1(B) is correct is to identify
the environment of a nascent secretory protein as it is being
translocated across the ER membrane. To accomplish this experimentally, Crowley et al. [9] incorporated water-sensitive
fluorescent dyes into the nascent chain during in vitro translation using a modified aminoacyl-tRNA analogue [10–12].
The fluorescent probes in the nascent chains were then positioned at specific locations within the ER membrane using
truncated mRNAs that yielded a homogeneous sample
of translocation intermediates with the same length of nascent
chain and the same probe position(s) upon translation. Since
these RNCs are formed in situ in the presence of SRP and
ER microsomes, the resulting translocation intermediates are
intact, fully assembled and contain only functional components. The fluorescence lifetimes of the probes positioned at
all locations within the ER membrane then provided a direct
measure of the extent of nascent chain exposure to water,
and these data revealed that a nascent secretory protein was
surrounded by water throughout its passage through the ER
Protein Synthesis and Quality Control at the Endoplasmic Reticulum
Figure 2 The translocon functional cycle during translocation
(A) A ribosome-free translocon is sealed at its luminal end by the action of BiP. (B) Shortly after SRP-dependent targeting
of an RNC to the translocon, the translocon pore is sealed on both ends. The ribosome closes the cytoplasmic end, while
BiP continues to seal the luminal end. (C) Once the nascent chain reaches a length of about 70 amino acids, BiP is released
and the pore is sealed solely by the binding of the ribosome to the translocon. The nascent chain is then translocated through
the large (40–60 Å diameter) pore into the ER lumen. (D) After termination of translation, BiP effects closure of the luminal
end of the pore, the translocon contracts to form a 9–15 Å-diameter pore, and the ribosome is released. The relative timing
of these last three events is unknown.
membrane [9]. This work therefore demonstrated unambiguously that the ER translocon forms an aqueous pore that
spans the ER membrane and is occupied by the nascent
secretory protein.
The aqueous hole in the translocon is
sealed at one end or the other to prevent
ion movement through the ER membrane
The existence of aqueous pores in ER translocons means that
eukaryotic cells have evolved a mechanism for preventing
Ca2+ and other ion movement from one side of the membrane
to the other through the pore. The two most reasonable
alternatives for the molecular origin of the permeability
barrier during translocation involve closure of the pore either
by the translocon itself or by interaction with other molecular
species. Specifically, one possibility is that the ribosome binds
tightly to the cytoplasmic end of the translocon and seals
off the pore from the cytosol, thereby blocking any ion
movement (Figure 1A). The other likely possibility is that
the translocon proteins form a constriction in the pore so
that it is only large enough to pass an extended polypeptide chain and cannot pass any ions in addition to the nascent
chain (Figure 1C).
To address these two possibilities experimentally, Crowley
et al. [9] employed collisional quenching techniques. Fluorescent probes were incorporated into nascent chains undergoing translocation, and the probes in the resulting
translocation intermediates were examined to determine
whether the fluorescence intensity of the sample was reduced
(quenched) by the addition of quenching agents (certain
molecules and ions) to the medium. Since quenching occurs
only when these molecules or ions (quenchers) are able to
collide with the fluorescent probes, and since nascent chain
fluorescence in membrane-bound RNCs was not quenched
by cytoplasmic iodide ions (or larger ions, either negatively
or positively charged), the aqueous pore in the translocon and
the nascent chain tunnel inside the ribosome were inaccessible
to cytoplasmic ions [1,9,13]. The nascent chain was therefore
sealed off from the cytoplasm by the binding of the ribosome
to the cytoplasmic end of the translocon.
Could a constriction in the translocon also be involved
in maintaining the permeability barrier of the ER membrane
during translocation? Cryo-electron microscopy images of
ribosomes associated with detergent-purified translocons
revealed a ‘gap’ between the ribosome and the translocon that
appears to be incompatible with the ribosome binding tightly
to the translocon [14,15]. These authors therefore suggested
that ion movement through the pore is prevented during
translocation not by a ribosome–translocon seal, but rather
by the structure of the translocon itself. Specifically, they
proposed that the translocon pore was only large enough to
accommodate an unfolded nascent polypeptide and that ion
movment was prevented for steric reasons (Figure 1C).
Yet fluorescent probes located in the nascent chain
inside the ribosome were quenched by collisional quenchers
from the luminal side of the membrane when the nascent
chains were longer than about 70 residues [9,13]. In fact,
quenchers as large as NAD+ were able to move through the
translocon aqueous pore and collide with a probe inside
the ribosome when the nascent chain occupied the translocon
pore and spanned the membrane [9,13]. Thus the translocon does not contain a constriction that limits ion movement during translocation as depicted in Figure 1(C). Instead,
the ribosome–translocon junction prevents ion diffusion
from one side of the ER membrane to the other (Figure 1A).
The discrepancy between the fluorescence and cryo-electron
microscopy studies presumably results from the fact that the
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fluorescence experiments used intact, fully assembled and
functional membrane-bound translocation intermediates,
while the cryo-electron microscopy experiments examined
detergent-extracted translocons or translocation intermediates that lacked some translocon proteins and the lipid
components of the ER membrane.
If the ribosome is responsible for maintaining the permeability barrier during translocation, what happens when
translation terminates and the ribosome is released from the
translocon? Hamman et al. [13,16] showed that the translocon
did not disassemble, but did undergo a major structural
change that reduced the size of the aqueous pore from 40–
60 Å during co-translational translocation to 9–15 Å when
no ribosome was bound to the translocon. In the latter case,
closure of the aqueous pore was mediated by the binding of
BiP (Ig heavy-chain-binding protein) to the luminal side
of the ER membrane [16] (Figure 2A). Although Figure 2
depicts BiP itself closing the luminal end of the aqueous
translocon pore, it has not yet been determined whether
BiP effects pore closure directly or indirectly by binding to
another protein. However, the important mechanistic point is
that a ribosome-free translocon is sealed on the luminal side
of the ER membrane.
Figure 3 Translocon pore closure during integration
(A) When the TM sequence (indicated by the black rectangle in
the nascent chain) of a signal-cleaved membrane protein is being
synthesized by a ribosome, the aqueous ribosome tunnel and translocon
pore are contiguous and open to the ER lumen. (B) Shortly after the
TM sequence has been synthesized, the luminal end of the pore is
also sealed by the direct or indirect action of BiP. This requires a long
signal-transduction pathway that extends from the detection of the TM
sequence far inside the ribosome to the binding of BiP to the other side
of the membrane [17]. (C) After the nascent chain has been elongated
by a few more residues, the ribosome–translocon seal is broken to
allow the cytoplasmic domain of the membrane protein to enter the
cytosol. This arrangement (open cytoplasmic end and sealed luminal
end) is maintained until translation of the single-spanning membrane
protein is terminated and the ribosome dissociates from the translocon. BiP seals the luminal end of the pore even though the nascent
chain spans the translocon and extends into the ER lumen [17,18], and
(D) after the membrane protein leaves the translocon.
Permeability control during membrane
protein integration
Each membrane protein has one or more polypeptide stretches that end up in the cytosol. This complicates the maintenance of the permeability barrier because large cytoplasmic
domains cannot be accommodated within the ribosome. The
ribosome would therefore not be able to constantly maintain the tight ribosome–translocon junction because at some
point a cytoplasmic domain must be released into the cytosol
instead of being transported to the luminal side of the
membrane.
Liao et al. [17] demonstrated using fluorescent nascent
chains and collisional quenching that the permeability barrier
is maintained during co-translational integration of a signalcleaved single-spanning membrane protein by a complex
molecular choreography that involves a co-ordinated and
highly regulated sequence of interactions between the ribosome, the translocon and BiP. These interactions ensure that
the pore is always closed at one end or the other, and sometimes at both ends simultaneously (Figure 3). The cytoplasmic
end of the pore is sealed by the ribosome when the nascent
chain is directed into the lumen [17], while the luminal
end is sealed, either directly or indirectly, by BiP when the
cytoplasmic domain of the membrane protein is moving into
the cytosol [18]. In this way, a nascent polypeptide can be
threaded into the ER membrane without compromising its
permeability barrier.
One startling feature of this process is that the pore is closed
by the action of BiP long before the TM (transmembrane) segment in the nascent chain reaches the translocon (Figure 3B).
Photoreactive probes in the TM sequence reacted covalently
only with ribosomal proteins, not with translocon proteins,
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when the pore was first sealed on the luminal side of the
membrane [17]. Thus the ribosome, not the translocon, first
recognizes the nascent chain as a membrane protein and initiates the conversion of the operational mode of the translocon from translocation to integration. The unexpectedly
early timing of this conversion presumably results from the
need to orchestrate the correct distribution of polypeptide to
one side of the ER membrane or the other before the TM
sequence reaches the translocon.
How does the ribosome identify the nascent chain as a
membrane protein? Since a TM sequence is the diagnostic
structural feature of every membrane protein, the ribosome
is presumably able to recognize the presence of a non-polar
TM sequence in the nascent chain. Liao et al. proposed that
the ribosome is able to detect a hydrophobic TM α-helix
inside the nascent chain tunnel of the ribosome [17]. This
Protein Synthesis and Quality Control at the Endoplasmic Reticulum
speculation has recently been examined experimentally using
a new experimental approach, and the results support such a
mechanism (C.A. Woolhead, P.J. McCormick and A.E.
Johnson, unpublished work).
Unresolved issues
Much still needs to be done to understand how cells are able to
maintain the integrity of a membrane while proteins are being
transported through or into it. For example, do the ribosome
and translocon alternate closing off the translocon pore
as a multi-spanning membrane protein is co-translationally
integrated into the bilayer? If so, what triggers the system to
switch from sealing one end to the other, and when does that
occur? Do TM sequences that orient in opposite directions
in the membrane move through the ribosome and/or enter
the translocon via different pathways? How does BiP effect
pore closure? These are only a few of the important structural
and mechanistic questions that remain to be elucidated. Yet
progress is being made, as evidenced by discoveries made
about the nature of BiP involvement in pore closure using
BiP mutants and other approaches (N.N. Alder, Y. Shen, J.L.
Brodsky, L.M. Hendershot and A.E. Johnson, unpublished
work).
At present, nothing is known about how the permeability
barrier of the ER membrane is maintained when misfolded
or unassembled proteins are retro-translocated from the ER
lumen into the cytoplasm for degradation by the proteasome
[19]. Similarly, no experiments have addressed membrane
permeability during post-translational protein translocation
at ER membranes in yeast, during protein translocation and
integration at the mitochondrial inner membrane, or during
protein export through the bacterial membrane. Yet such
experiments are within reach, as evidenced by the successful
import of fluorescently labelled proteins into mitochondria
(H.M. Cargill and A.E. Johnson, unpublished work) and
the successful transport of fluorescently labelled proteins
into bacterial inverted membrane vesicles (A.L. Karamyshev
and A.E. Johnson, unpublished work). It will be both fun
and intellectually challenging to discover how cells are able
to carry out the very complex processing of proteins at
membranes without compromising membrane integrity. The
relevant molecular mechanisms must be coupled and very
sophisticated, since the need to maintain the membrane’s
permeability barrier is superimposed on the already difficult
mechanical manoeuvring of the substrate polypeptides during
translocation and especially integration.
Our protein trafficking research is supported by NIH grants R01 GM
26494 and R01 GM 64580 and by the Robert A. Welch Foundation.
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Received 9 June 2003
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