Opinion
TRENDS in Biochemical Sciences Vol.27 No.5 May 2002
Membrane proteins:
shaping up
Chen-Ni Chin, Gunnar von Heijne and
Jan-Willem L. de Gier
Over recent years, much progress has been made in the identification and
characterization of factors involved in the biosynthesis of integral membrane
proteins of the helix-bundle type. In addition, our knowledge of membrane protein
structure and the forces stabilizing helix–helix interactions in a lipid environment
is expanding rapidly. However, it is still not clear how a membrane protein folds
into its final form in vivo, nor what constraints there are on the folded structure
that results from the mechanistic details of translocon-mediated assembly rather
than simply from the thermodynamics of protein–lipid interactions.
Gunnar von Heijne
Jan-Willem L. de Gier*
Dept Biochemistry and
Biophysics, Stockholm
University, SE-106 91
Stockholm, Sweden.
*e-mail: degier@
dbb.su.se
Chen-Ni Chin
Dept Molecular
Biophysics and
Biochemistry, Yale
University, Bass 429,
266 Whitney Avenue,
New Haven, CT 06520,
USA.
It is now beyond doubt that membrane proteins
require the assistance of a translocation/insertion
machinery for their assembly [1,2], although there
might be some exceptions [3]. This realization has
mainly resulted from studies using the eukaryotic
endoplasmic reticulum (ER) membrane and the
bacterial plasma membrane as model systems. At the
same time, recent structural and biophysical studies
have revealed a wealth of information about the
principal architectures of membrane proteins and
some aspects of the physical chemistry involved in
lipid–protein interactions and helix–helix packing in a
membrane environment [4–8]. However, it is still
unclear how membrane proteins fold in vivo and to
what extent biophysical studies of peptide binding and
helix–helix interactions in detergent or artificial lipid
bilayers reflect what happens during insertion into the
ER or the bacterial plasma membrane (Fig. 1).
Our objective here is to focus attention on the poorly
explored area bridging the gap between the initial
biosynthetic steps and the final structure of membrane
proteins of the helix-bundle class. Thus, we do not
address the folding of β-barrel membrane proteins,
which only occur in the outer membranes of Gramnegative bacteria, mitochondria and chloroplasts. We
first briefly review the basic translocation machineries
in the ER and the bacterial plasma membrane. We also
discuss recent studies addressing the physical
chemistry of protein folding in a membrane
environment. In the final section, we try to bring these
two areas together and discuss possible models for the
steps that connect the initial membrane insertion and
the final structure of a membrane protein.
Translocon
Membrane proteins of the helix-bundle class are
targeted to the ER membrane in eukaryotic cells and to
the plasma membrane in bacterial cells by similar
pathways [9]. Briefly, the signal recognition particle
http://tibs.trends.com
231
(SRP) binds to the N-terminal signal sequence (or first
transmembrane segment) of a membrane protein when
it becomes exposed outside the ribosome. Upon making
contact with the SRP receptor, the SRP dissociates from
the nascent chain, which then enters the translocon – a
membrane-embedded molecular machine that
mediates both the translocation of unfolded globular
proteins across the membrane and the integration of
membrane proteins into the membrane [10]. During
the ensuing translocation process, the ribosome
remains attached to the translocon, allowing a coupled
translation–translocation reaction [11–13].
The translocons in the eukaryotic ER membrane
and the bacterial plasma membrane have similar
compositions. The heterotrimeric core of the eukaryotic
translocon, the Sec61p complex, consists of the integral
membrane proteins Sec61α, Sec61β and Sec61γ. The
TRAM protein is also part of the translocon and is
important for the translocation of some, but not all,
membrane proteins [10]. Electron microscopy studies
suggest that three to four Sec61 heterotrimers form the
functional translocation channel, whereas recent
cryoelectron microscopy suggests that three Sec61
heterotrimers form the translocation channel [13,14].
The core of the prokaryotic Sec translocase consists
of SecY and SecE [15–17], which are homologous to
Sec61α and Sec61γ, respectively [1]. Four SecYE
heterodimers oligomerize to form the functional
translocation channel. It has been suggested that
oligomerization of the SecYE heterodimers can be
induced by SecA, a peripheral subunit of the Sec
translocase that drives polypeptide chains into and
through the SecYE protein translocation channel [18].
In vitro studies of membrane protein folding
Turning from the early to the late stages of the
membrane-protein folding process, it is generally
recognized that overall hydrophobicity is the main
driving force for the integration of α-helical
transmembrane segments into the lipid bilayer [19].
According to the two-stage model, the folding pathway of
helical membrane proteins can be conceptually divided
into two kinetically distinct stages [20]: (1) individually
stable, transmembrane helices are formed in the
hydrophobic region of the lipid bilayer; (2) these helices
associate with each other to form a specific tertiary
structure. Possible driving forces for helix–helix
association in the lipid bilayer are van der Waals
interactions (i.e. close packing) and interhelical polar
interactions including hydrogen bond and electrostatic
interactions [4]. Folding of extra-membranous
polypeptide segments and the binding of cofactors can
also help to bring transmembrane helices together.
Studies using the single-transmembrane-span
protein glycophorin A (GpA) as a model system have
provided a first glimpse of the physical chemistry of
helix–helix interactions. GpA forms stable homodimers
in the detergent SDS that can be detected by gel
electrophoresis. Using this simple detection method in
combination with saturation mutagenesis, the
0968-0004/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0968-0004(02)02082-0
232
Opinion
TRENDS in Biochemical Sciences Vol.27 No.5 May 2002
Assembly and folding in vivo
(a)
(b)
?
Ti BS
Fig. 1. From nascent
chain to folded protein.
Membrane proteins (blue)
are synthesized on
ribosomes (yellow) that
are attached to the
translocon (green). The
steps between the initial
docking of the ribosome
to the translocon (a) and
the final folding of the
membrane protein (b) are
largely unknown.
dimerization motif LIxxGVxxGVxxT was identified in
the GpA transmembrane helix [21–24]. The NMR
structure of the GpA dimer further revealed a righthanded helix–helix crossing, and showed that the Gly
residues at the interface allow close van der Waals
contacts between the two helices [25]. In the available
three-dimensional structures of membrane proteins,
Gly often occurs at the helix–helix crossing points,
where it provides a good van der Waals packing surface
[26], and GpA-type dimerization motifs are statistically
over-represented in transmembrane sequences [27].
A careful examination of the GpA dimer interface in
combination with a survey of known protein structures
suggests that weak Cα–H --- O hydrogen bonds are
responsible for the specificity of the GxxxG motif [28].
Helix–helix association in detergent micelles and
lipid bilayers has also been shown to be promoted by
hydrogen bonding between the sidechains of Asn,
Asp, Gln and Glu residues [5,6]. The free energy
of association mediated by a pair of these polar
residues is comparable to that of the entire GpA
dimerization motif [6].
Concerning the role of extramembranous
polypeptide segments in the folding process, in vitro
studies of the folding of bacteriorhodopsin have
uncovered kinetic effects of sequence changes in the
loops between the transmembrane helices on both the
rate-limiting apoprotein-folding step and on the
covalent attachment of the retinal [29], although it is
not known how general such effects are and to what
extent they influence folding in vivo.
Similar to soluble proteins, cofactor binding is
sometimes needed to reach the fully folded state. The
binding of retinal to bacteriorhodopsin [30], and of
chlorophylls to the light-harvesting complex II [31],
are perhaps the best examples of this for membrane
proteins. Recent data also suggest that lipids can
have a more specific role in membrane protein folding
than merely providing a solvent. Thus,
phosphatidylethanolamine has been shown to assist
the folding of the Escherichia coli inner membrane
protein lactose permease by interacting with
non-native folding intermediates [32].
http://tibs.trends.com
The basic physical chemistry of membrane protein
folding is thus beginning to be understood, but it is
still unclear whether the simple two-stage model is
directly applicable to the complex process of
membrane protein assembly in vivo or whether the
translocon itself significantly affects membrane
insertion, helix–helix interactions and the overall
folding reaction. Two extreme views are: (1) that
transmembrane helices leave the translocon one by
one, driven simply by the thermodynamics of lipid
partitioning, and thus that the two-stage model
applies as is; and (2) that helices initially pack
together inside the translocation channel and move
out en bloc, in which case the so-far-uncharacterized
internal environment in the translocation channel
might exert a decisive influence on the final structure.
Kinetics could also be important. Thus, even if a
particular segment of a nascent polypeptide chain
thermodynamically prefers a transmembrane location
to a water-exposed location, it could be pushed through
the translocon too rapidly for proper equilibration and
end up not spanning the membrane. Conversely, a
non-hydrophobic segment that would not insert across
the membrane on its own could be forced into such a
disposition by rapidly integrating flanking
transmembrane helices with strong orientational
preferences. Because reorientation of already inserted
transmembrane helices and translocation of polar
flanking regions should, in most cases, be extremely
slow, one can imagine that kinetically trapped states
would be common among membrane proteins. In fact,
stably integrated membrane proteins have already
been engineered to prevent hydrophobic segments that
normally span the membrane from doing so [33,34],
and to force polar segments that would normally
prefer an aqueous environment to insert as
transmembrane segments [35].
So, what is currently known about the Sec61p and
SecYE translocation channels and the lateral
movement of transmembrane segments from the
channel into the lipid bilayer? A recent cryoelectronmicroscopy-based 15 Å reconstruction of a nascentchain–ribosome–Sec61p complex suggests that the
channel is narrow and might only accommodate a
single transmembrane segment at a time [13].
However, this particular structure represents a
snapshot of the translocon in action. Other studies
indicate that the size of the translocation channel is
flexible and might reach a diameter of ~40–60 Å, and
that the shape and composition of the translocon can
differ depending on the tasks it has to perform
[10,14,36]. One study, in which urea–salt extraction was
used to assay membrane integration, even suggests
that the translocation channel can accommodate a fully
synthesized, multiple-spanning membrane protein
before its expulsion into the lipid bilayer [37].
Whatever the exact nature of the translocation
channel, it is nevertheless clear that, during the
translocation of a membrane protein, hydrophobic
Opinion
Acknowledgements
Our work was supported
by grants from the
European Molecular
Biology Organization, the
Swedish Foundation for
Strategic Research and
the Swedish Research
Council to J.W.d.G., and
from the Swedish Cancer
Foundation and the
Swedish Research
Council to G.v.H.
TRENDS in Biochemical Sciences Vol.27 No.5 May 2002
transmembrane segments get trapped in the
translocon and, at some stage, move laterally into the
lipid bilayer. Glycosylation sites have been engineered
to measure the number of residues in a nascent chain
that are needed to span the distance between the
ribosomal P-site and the lumenal oligosaccharide
transferase enzyme; studies of these suggest that
segments that will eventually form transmembrane
helices can already adopt a compact, possibly α-helical,
conformation in the ribosome-translocon channel, well
before they become exposed to lipids [38,39]. For an
engineered, single-spanning model membrane protein,
it has further been shown by cross-linking that the
transmembrane region, before it enters the lipid
bilayer, contacts first Sec61α and then TRAM [10]. It
was therefore suggested that TRAM recognizes and
helps to transfer transmembrane segments from the
core translocon into the lipid bilayer [10].
More recently, the nature of the transmembrane
segment of another engineered single-spanning
membrane protein (a derivative of the E. coli inner
membrane protein leader peptidase) was shown to have
a profound influence on its interactions with membrane
components. A strongly hydrophobic transmembrane
segment became sequestered within a lipid
environment almost as soon as it entered the translocon,
whereas a mutated, slightly less hydrophobic
transmembrane segment could still be cross-linked to
TRAM and Sec61α under the same conditions. Based on
these results, it was proposed that transmembrane
segments equilibrate between the interior of the
translocon and the surrounding lipid environment
depending on their hydrophobicity. The authors further
suggested that TRAM plays a crucial role in the
retention of mildly hydrophobic transmembrane
segments in the interior of the translocon [40].
In E. coli, transmembrane segments likewise first
get trapped in the Sec translocon and then move from
the translocation channel into the lipid bilayer [41].
Cross-linking studies using the single-spanning
protein FtsQ and the multiple-spanning protein MtlA
have provided important clues to the lipid
partitioning of transmembrane segments [42–44].
The earliest interactions of the transmembrane
segment of FtsQ with the membrane involve both
SecY and lipids. When the nascent chain gets longer,
the transmembrane segment of FtsQ can be crosslinked to the plasma membrane protein YidC and to
lipids, whereas its hydrophilic N-terminal tail is still
close to SecA and SecY. This suggests that YidC is
close to the Sec translocase during membrane protein
insertion and facilitates the lipid partitioning of
transmembrane segments [42,43]. Indeed, YidC can
associate with the Sec translocase, and depletion of
YidC hampers the assembly of membrane proteins
[45,46]. Similar cross-linking studies with MtlA have
shown that YidC can simultaneously accommodate
more than one transmembrane segment at the
protein–lipid interface [44], suggesting that helix
bundles can form before their full release into the
http://tibs.trends.com
233
lipid bilayer. Although YidC and TRAM show no
obvious sequence similarity, the resemblance in their
interactions with nascent membrane proteins
suggests a shared, yet poorly characterized, function
in facilitating the transit of transmembrane segments
from the translocon into the lipid bilayer [47].
Finally, it has been proposed that pairs of closely
spaced helices (‘helical hairpins’) constitute an
important ‘insertion unit’that is handled as a single
entity by the translocon [48,49]. If this is correct,
helix–helix interactions are likely to become established
before the helical hairpin leaves the translocon. The
accumulated knowledge about the sequence
determinants for helical-hairpin formation is broadly
consistent with the constraints on protein structure
imposed by a non-polar environment [50], providing
another strong argument for the existence of a nonpolar and possibly lipid-rich environment in or very
near the protein-conducting channel in the translocon.
It is not entirely clear whether the threshold
hydrophobicity for the membrane incorporation of
transmembrane helices observed in vivo is the same as
for a purely thermodynamic partitioning of peptides
into a lipid bilayer, although recent calculations suggest
that they are not very different [51].
Conclusion
The studies discussed above suggest that the onehelix-at-a-time insertion model applies to very
hydrophobic transmembrane segments and singlespanning membrane proteins, whereas more-polar
helices remain inside the translocon, possibly waiting
either for chain termination or for the appearance of
suitable partner helices with which to associate. The
time is thus ripe for studies that aim to define the
kinetics of helix integration, and the immediate
environment along the lateral ‘escape route’ from the
translocation channel that is available for
transmembrane segments of different hydrophobicity
and located at different separations from
neighbouring, potentially interacting helices.
Perhaps the most interesting result of such studies
will be a better understanding of the degree to which
the translocon itself and the kinetics of the process
influence membrane protein structure, over and
beyond the constraints imposed by the lipid bilayer.
Although they are not immediately apparent in the
known membrane protein structures, we
nevertheless contend that such influences exist. An
early example is the recent observation that the
sequence requirements for the formation of a helical
hairpin with a lumenally oriented connecting loop are
clearly different from those promoting the formation
of a hairpin with a cytoplasmically oriented
connecting loop [52]. There is no obvious asymmetry
in the properties of the ER lipid bilayer that could
explain this difference and hence there is good reason
to believe that it is dictated by details of the
translocon-mediated assembly process rather than by
the thermodynamics of protein–lipid interactions.
234
Opinion
TRENDS in Biochemical Sciences Vol.27 No.5 May 2002
The cellular processes that ensure proper
delivery, membrane insertion and folding of
integral membrane proteins seem, in many
respects, to lead to structures that are at the global
free-energy minimum and yet, when studied closely,
also seem to leave room for kinetically trapped
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