1 University of Cambridge, CIMR and Department of Clinical Biochemistry,
Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, UK
2 Department of Animal Biology, University of Illinois at Urbana-Champaign, IL
61801, USA
* Author for correspondence (e-mail: kbr20{at}cam.ac.uk)
Accepted 4 April 2003
![]() |
Summary |
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Key words: Sec61 channel, ER lipid composition, Secretion, Antarctic fish, Antarctic yeast
![]() |
Introduction |
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Protein translocation across biological membranes is dependent on
temperature and membrane lipid composition
(Baker et al., 1988;
Leheny and Theg, 1994
;
Nilsson et al., 2001
). In
mesophilic organisms this process is strongly inhibited by low temperatures,
yet 80% of life on earth exists at temperatures below 4°C, suggesting that
the protein translocation machinery of these organisms has been adapted to
permit function in the cold (Baker et al.,
1988
). Protein translocation channel subunits from psychrophiles
have not been characterized so far. In soluble cold-adapted enzymes, the
observed amino-acid changes in many cases lead to increased flexibility of
hinge regions, which allow the enzymes to undergo conformational changes
necessary for activity at low temperatures
(Gianese et al., 2001
;
Russell, 2000
).
Transmembrane proteins intimately interact with the lipids of the membrane
into which they are integrated, and their function is often dependent on
membrane lipid composition (Fyfe et al.,
2001). Nilsson and coworkers showed recently that increasing the
concentration of cholesterol in dog pancreas microsomes, which putatively
increases membrane rigidity, strongly inhibits translocation of proteins into
the microsomal lumen (Nilsson et al.,
2001
). Membrane lipid composition is known to vary with the body
temperature of organisms (Hazel,
1995
; Logue et al.,
2000
). Membranes from cold-adapted animals usually contain higher
proportions of unsaturated fatty acids, resulting in increased membrane
fluidity to offset the rigidifying effects of the cold
(Hazel, 1995
). Thus membranes
of organisms from different thermal environments may have similar fluidities
when measured at their respective body temperatures, as seen in brain synaptic
vesicles from an Antarctic fish, temperate fishes and mammals
(Logue et al., 2000
).
Cold-adaptation of the ER translocation machinery may therefore also be a
function of the physical structure of the lipid bilayer in which it is
integrated.
In this study, we try to elucidate some of the complex structure-function
relationships of the protein translocation channel in the ER membrane and its
lipid environment by comparing protein translocation efficiencies into the ER,
Sec61 protein primary structures and ER lipid compositions of select organisms
from extremely cold habitats and their mesophilic counterparts. Specifically,
we examined post-translational protein translocation into the ER of Antarctic
and mesophilic yeasts over a range of temperatures to establish that
psychrophilic yeasts are indeed more efficient at translocation in the cold.
Although several factors required for protein translocation across the ER
membrane may be cold sensitive, we focused our subsequent analysis on the
pore-forming component of the protein translocation channel, Sec61p or
Sec61, because channel opening is essential for protein entry into the
secretory pathway and has recently been shown to involve a conformational
change in the channel (Beckmann et al.,
2001
). We obtained and sequenced SEC61 cDNAs from
Antarctic yeast in order to identify amino-acid changes potentially
responsible for improved protein translocation channel function at low
temperature. In order to better distinguish between changes due to
phylogenetic divergence and those due to cold adaptation, we further sequenced
SEC61 cDNAs from Antarctic and Arctic fishes. Fish have substantially
longer generation times than yeast and are therefore expected to have a lower
degree of sequence divergence from mesophilic SEC61, and thus fewer
changes that are not related to function. To examine Sec61 channel function in
vertebrates, we post-translationally translocated a truncated, ribosome-bound,
secretory protein into the ER of an Antarctic fish, a temperate fish, and a
mammal over a temperature range. In addition, we assessed the potential
influence of lipid bilayer properties on protein translocation across the ER
membrane by comparative analyses of lipid composition and membrane order of
liver microsomes from vertebrates spanning a wide range of body temperatures,
from Antarctic fish to mammals.
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Materials and Methods |
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Preparation of liver microsomes, lipid analysis, and fluidity
measurements
Liver microsomes from Antarctic Dissostichus mawsoni (caught and
held at -1.6°C), trout (acclimated to 10°C), carp (25°C) and rat
were prepared as described previously
(Logue et al., 1998). Membrane
fluidity was determined by measurements of fluorescence anisotropy using
1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe
(Logue et al., 2000
). Total
lipid extraction and fatty acid analysis were performed using established
methods (Logue et al., 1998
).
Microsomes for translocation experiments: dog pancreas microsomes were a gift
from Stephen High (University of Manchester). Carp microsomes were prepared
from livers of animals acclimated to 17°C.
Yeast strains and microsomes
The Saccharomyces cerevisiae wild-type strain used in this study
was RSY255 (Stirling et al.,
1992). Antarctic yeasts Cryptococcus adeliensis
(CBS8351), C. antarcticus (CBS7687), and mesophilic C.
laurentii (CBS942) were obtained from the CBS culture collection,
Utrecht, The Netherlands (Garancis et al.,
1970
; Scorzetti et al.,
2000
; Vishniac and Kurtzman,
1992
). Microsomes were prepared from cells grown to
OD600 of 3.0-5.0 in YPD at 15°C (Antarctic species) or 24°C
(C. laurentii, S. cerevisiae); under these conditions, the generation
times were 10 hours for C. antarcticus and C. adeliensis, 2
hours for C. laurentii and 1.7 hours for S. cerevisiae.
Cells were lysed by liquid nitrogen lysis, and microsomes isolated by
differential centrifugation as described elsewhere
(Lyman and Schekman,
1995
).
Electron microscopy
C. antarcticus, C. adeliensis grown at 15°C and C.
laurentii grown at 24°C to OD600 of 4.0 in YPD were fixed
in 0.1 M cacodylate and processed for standard electron microscopy.
Translocation assays
Into yeast microsomes: in vitro translated, radiolabelled mutant
alpha-factor precursor (pgp
f; 4 µl=106 cpm per 20
µl reaction) was translocated into yeast microsomes in the presence of ATP
and an ATP-regenerating system at the indicated temperatures for 15 minutes.
Microsomal protein in the reactions (2-5 µg) was limiting for translocation
under these conditions. Reactions were terminated by addition of an equal
volume 20% trichloro-acetic acid, protein samples analyzed by SDS-PAGE on 18%
acrylamide/4 M urea gels, and individual bands were quantified with a
phosphorimager (Packard). Translocation was defined as a percentage of
signal-cleaved alpha-factor precursor. Protease-protection of the signal
cleaved form was at least 90%.
Into vertebrate microsomes: plasmid pSPB4 was linearized within the
preprolactin coding sequence using PvuII and a truncated mRNA
encoding the N-terminal 86 amino acids of preprolactin transcribed using SP6
polymerase (Siegel and Walter,
1988). PPL86 was synthesized in reticulocyte lysate (Promega) at
25°C for 15 minutes in the presence of [35S]-methionine.
Translations were transferred to ice and the translation terminated by adding
cycloheximide to a final concentration of 250 µM. Limiting amounts of
membranes (2 µg protein) from D. mawsoni, C. carpio or C.
familiaris were added to 15 µl in vitro translations, and reactions
were incubated for 10 minutes at the indicated temperatures (targeting). Then
puromycin was added to 1 mM final concentration, and incubation continued for
10 minutes at the indicated temperatures (translocation). Membranes were
sedimented (4 minutes, 4°C, 16,000 g), taken up in SDS
sample buffer and radiolabelled proteins resolved by SDS PAGE and analyzed
using a phosphorimager (Perkin Elmer, Cambridge, UK). Translocation was
assessed by quantifying signal-cleaved PL56. For protease protection,
sedimented membranes were resuspended in 0.1 mg/ml Proteinase K and incubated
on ice for 20 minutes. The signal-cleaved form was, on average, 85%
protease-protected in these experiments with no significant differences
between species. The signal-sequence-containing PPL86 associated with the
membranes was >80% protease sensitive.
Quantitative immunoblotting
Equal amounts of D. mawsoni liver or dog pancreas microsomes were
resolved on 12.5% SDS gels, transferred to nitrocellulose and Sec61
detected using anti-dog Sec61
antibodies (S. High) at 1:1000 dilution,
followed by [125I]-Protein A (Amersham) and quantitation using a
phosphorimager.
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Results |
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Fig. 1 shows protein
translocation into microsomes from S. cerevisiae and C.
adeliensis grown at 24°C and 15°C, respectively. The upper panel
shows a phosphorimager scan of an SDS-gel of cytosolic precursor
(pgp
f) and translocated, signal-cleaved
gp
f; the
lower panel shows the percentage of tranlocation at different temperatures
normalized to 20°C (Fig.
1). Translocation into S. cerevisiae microsomes is most
efficient at 20°C; translocation at 10°C is approximately 1.8-fold
lower, and translocation at 0°C is 3.1-fold lower than at 10°C,
suggesting that the activity of the Sec61 channel in S. cerevisiae is
optimal at its growth temperature in the wild. There is no further increase in
translocation into S. cerevisiae ER at higher temperatures (data not
shown) (Baker et al., 1988
).
The relative protein import into C. laurentii microsomes was similar
to import into S. cerevisiae microsomes at the same temperatures
(data not shown). In contrast, protein translocation into microsomes derived
from the Antarctic Cryptococcus species, C. antarcticus and C.
adeliensis, was approximately equal at 20°C and 10°C, and
decreased by only a factor of 2 between 10°C and 0°C
(Fig. 1; C.
antarcticus data not shown). Microsomes prepared from Antarctic yeast
grown at 6°C showed no further improvement of translocation at low
temperature. The absolute amount of translocation per µg of microsomal
protein varied between microsome preparations, but the ratios of translocation
at different temperatures remained identical.
|
Electron micrographs were taken, and a representative micrograph for each Cryptococcus species is shown in Fig. 2. Compared with its congeners, C. antarcticus has unusually large mitochondria, which can occupy a substantial fraction of the cell volume (Fig. 2, top). By contrast, the mesophilic C. laurentii and the psychrotolerant C. adeliensis display no substantial morphological differences (Fig. 2). On the basis of qualitative assessment of images taken from large numbers of cells at low magnification, all three species contain comparable amounts of ER cisternae. Taken together our results indicate that Antarctic yeast are more efficient at protein translocation across the ER membrane at low temperature than their mesophilic counterparts.
|
Primary structure of Sec61p in Antarctic yeast
Given the relatively high translocation efficiency at low temperature
through the Sec61 channel into the ER of Antarctic yeast, we asked whether
Sec61p had acquired amino-acid changes that may be adaptive to function in the
cold. We obtained partial sequences of SEC61 from C.
antarcticus and C. adeliensis and compared them with the
SEC61 sequence from C. laurentii. Overall, the
SEC61 sequence was relatively divergent between mesophilic and
Antarctic Cryptococcus species (84% amino acid identity over the 187 amino
acid region sequenced); differences appear to have accumulated in the loop
between transmembrane domains 7 and 8 (76% identity over 58 amino acids), but
it is not immediately obvious which changes are of functional
significance.
Comparison of SEC61 genes from mesophilic and cold-adapted
vertebrates
To identify amino-acid changes in Sec61p that are potentially adaptive to
function in the cold, we analyzed SEC61 cDNAs from cold-adapted organisms with
substantially longer generation times than yeast. SEC61 cDNAs from
liver of four southern/Antarctic cold-water fish species (Notothenia
angustata, Pagothenia borchgrevinki, Harpagifer antarcticus, Dissostichus
mawsoni) and three northern/Arctic ones (Hemitripterus americanus,
Gadus ogac, Boreogadus saida) were obtained and sequenced, and translated
sequences aligned with SEC61 sequences from the temperate water
fishes Fugu rubripes, Oncorhynchus mykiss (rainbow trout) and
Danio rerio (zebra fish) and a mammal (Mus musculus, mouse)
(Fig. 3). The Sec61
protein sequence and length across these highly divergent vertebrate taxa are
remarkably conserved (Fig. 3).
Compared to the mouse sequence we observed a number of fish-specific
amino-acid changes in Sec61
: V14A, I15V and K29R occur in the
cytoplasmic N-terminus of Sec61
. S128A and T152I are located in the
center of transmembrane domains 3 and 4, respectively. M207T is
membrane-proximal in the cytoplasmic loop between transmembrane domains 4 and
5. The I249V exchange is in the center of transmembrane domain 6, and A310T,
L316F, S319N, E356D and V363I occur in the ER-lumenal loop between
transmembrane domains 7 and 8. The last two changes, A430G, and L434M, are
located towards the lumenal end of transmembrane domain 9. Only a small number
of amino-acid changes seem to be specific for cold-water fishes
(Fig. 3). These are located at
the ER lumenal end of transmembrane domain 8 (V363A/G/S) and in the loop
region between transmembrane domain 7 and 8 (T327A, S328T and/or S329T, G339A,
Y344F or L345F). Although the majority of these changes are conservative
substitutions, the cluster of changes occurring in the loop region between
transmembrane domains 7 and 8 of Sec61
may be functionally significant.
The functional importance of this region has been demonstrated by the fact
that S. cerevisiae mutants carrying mutations in this loop of Sec61p
are defective in protein translocation across the ER membrane
(Stirling et al., 1992
;
Zhou and Schekman, 1999
). Thus
the cluster of subtle amino-acid changes in this ER-lumenal loop of cold-water
fish Sec61
may represent adaptive changes that improve protein
translocation through the Sec61 channel in the cold.
|
Post-translational protein translocation into the ER of Antarctic and
temperate vertebrates at low temperature
To test the temperature dependence of protein translocation into the ER of
cold-water fish, we prepared microsomes from the liver of the Antarctic
teleost D. mawsoni (habitat temperature -1.8°C) and the liver of
the temperate carp C. carpio (acclimation temperature 17°C), and
compared post-translational translocation of a ribosome-bound truncated
radiolabelled secretory protein consisting of the N-terminal 86 amino acids of
preprolactin (PPL86) into the fish microsomes and into dog pancreas microsomes
over a temperature range. As shown in Fig.
4A, protein translocation into dog pancreas microsomes was optimal
at 25°C and did not increase significantly at higher temperature as shown
previously [light grey bars, not shown and
(Nicchitta and Blobel, 1989)].
Secretory protein import into dog prancreas microsomes at 0°C was minimal
(Fig. 4A). In contrast, protein
import into D. mawsoni microsomes was efficient at 0°C, and
approximately equal at all temperatures tested (black bars,
Fig. 4A). Protein import into
carp microsomes was more efficient than into dog pancreas microsomes at
0°C, but in contrast to the D. mawsoni ER, protein import into
the carp ER had an optimum in the range between 10-20°C
(Fig. 4A; data not shown).
Translocated, signal-cleaved PL56 sedimenting with the membranes was, on
average, 85% protease-protected in these experiments, confirming translocation
into the ER lumen. In contrast, membrane-associated signal-sequence containing
PPL86 was over 80% protease sensitive, suggesting association with the
cytoplasmic face of the microsomes. The remaining 15-20% constitute either
substrate that was translocated, but not signal-cleaved, or incomplete
dissociation of PPL86 from ribosomes. There were, however, no differences in
the amounts of protease-resistant, membrane-associated PPL86 between
species.
|
To investigate whether increased protein import at low temperature into
D. mawsoni microsomes at low temperature was related to the number of
protein translocation channels, we analyzed the amount of Sec61 in
D. mawsoni and dog microsomes by quantitative immunoblotting. We
found that our membrane preparations contained comparable amounts of
Sec61
(Fig. 4B). The
comparatively high protein import efficiency of D. mawsoni membranes
at low temperature must therefore be due to improved channel function.
Lipid composition of ER membranes in the Antarctic teleost
Dissostichus mawsoni
The identical length and similar amino-acid composition of the
transmembrane domains of Sec61 from all vertebrates examined
(Fig. 3, boxed) suggests that
the thickness of the ER membranes of cold-water fishes is similar to those of
mesophilic organisms. We therefore compared the lipid compositions of liver
microsomes from Antarctic D. mawsoni, temperate-water fishes trout
and carp and a mammal, the rat. Total lipid fatty acid compositions of liver
microsomes from these vertebrate species are shown in
Table 1. As generally found in
such comparative studies, the cold-adapted or cold-acclimated species had a
higher proportion of unsaturated fatty acids in their membranes than temperate
or warm-bodied species (Fig.
5A). Microsomes from the Antarctic fish in particular contained an
increased percentage of monounsaturated fatty acids (MUFA), compared to trout,
carp and rat with increasingly higher body temperatures
(Table 1).
|
|
Despite the substantial differences in membrane fatty acid composition, the membrane order of liver microsomes derived from these vertebrates measured by DPH anisotropy at a given temperature (21°C) were near identical for D. mawsoni, trout, carp and rat (Fig. 5B). Our results indicate that no adaptation has taken place to increase the fluidity of ER membranes from species with low habitat temperatures.
Our data suggest that the protein translocation channel in the ER of Antarctic fish has to assemble and function in membranes that have an increased unsaturated fatty acid content, but are substantially more rigid than mammalian ERs at the body temperature of the fish. We conclude that membrane fluidity is not limiting for protein translocation through the Sec61 channel in the cold.
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Discussion |
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We found an increase in unsaturated fatty acids in the ER membrane of
Antarctic fish, as expected, but surprisingly no changes in fluidity compared
to microsomes from fishes with more temperate habitats or a warm-blooded
mammal (Fig. 5B). This finding
is in sharp contrast to the large membrane fluidity differences in brain
synaptic vesicles from the same four species measured with the same technique
(Logue et al., 2000).
Antarctic fish brain membranes are more fluid at any given temperature than
rat brain membranes; as a result fluidity of fish brain membranes at
-1.8°C (body temperature of the fish) is comparable to that of rat brain
membranes at 37°C (body temperature of the rat), a phenomenon known as
homeoviscous adaptation (Logue et al.,
2000
). Not all membranes of organisms with low habitat
temperatures show homeoviscous adaptation
(Cossins et al., 1978
); thus
the lack of it in D. mawsoni liver microsomes, although unexpected,
is not unique. A reasonable inference from our data would be that increased
membrane rigidity at low temperature is not a primary obstacle to Sec61
channel assembly and function. Our observation is in good agreement with that
of Leheny and Theg, who found that protein import into chloroplasts at low
temperature was not limited by membrane fluidity, but rather by ATP supply
(Leheny and Theg, 1994
). The
identical length and conserved protein sequence of the 10 transmembrane
domains of Sec61
(Fig.
3) suggest that the protein-lipid interactions between
Sec61
and the ER membrane lipids may be similar in cold-adapted fishes
and mammals regardless of the membrane lipid composition. This notion is
supported by recent work from Mitra and colleagues (personal communication),
who have shown that transmembrane proteins affect the structure of their local
environment within the lipid bilayer, which in turn suggests that there is no
optimal match between proteins and lipids in biological membranes, but that
proteins may generate lipid microdomains that support their individual
structure and function.
The lowest habitat temperatures experienced by the fishes whose
SEC61 genes we sequenced (temperate species: D. rerio
20°C; O. mykiss 4°C; New Zealand species: B. variegatus,
N. angustata 4°C; North Atlantic species: H. americanus
1°C; Antarctic species: H. antarticus -1.0°C, D. mawsoni,
P. borchgrevinki, P. devriesii -1.8°C; Arctic species: B. saida,
A. glacialis, G. ogac -1.8°C) are significantly lower than the body
temperature of the mouse (M. musculus 37°C). The fish-specific
amino-acid changes that we observed in Sec61
(Fig. 3,
Fig. 6A) in positions which
except for A310, L316 and S319 are completely conserved in mammals, may
therefore all reflect adaptations to lower temperature, particularly in light
of the fact that protein translocation into the ER of a temperate fish had a
much lower temperature optimum than translocation into dog ER
(Fig. 4A, dark grey bars versus
light grey bars). An alternative explanation is that these changes are not
adaptive but inherited from the common ancestor to these fish taxa. These
fishes are phylogenetically highly divergent and belong to three different
superorders and therefore do not share a recent common ancestor
(Cheng, 1998
). The superorder
Ostariophysi (to which zebrafish belong) and the order Salmoniformes (to which
trout belong) arose both in the early cretaceous (140 million years ago), and
the DNA sequences of Antarctic and Arctic Sec61
are equally (about 85%)
identical to both trout and zebrafish. Fugu rubripes is a tropical
fish that belongs to the same superorder as the Antarctic D. mawsoni
(Acanthopterygii) and evolved after the Antarctic and Arctic species that we
examined. Analysis of the Fugu Sec61
amino-acid sequence revealed that
Fugu does not have the amino-acid substitutions that we identified as
cold-specific at positions 327, 328 and 339, and, like mouse and zebrafish,
contains a large hydrophobic amino acid (F) at position 362
(Fig. 3). Thus the common amino
acid substitutions among the unrelated cold-water fishes
(Fig. 3,
Fig. 6A) are likely to have
occurred independently as an adaptation to the chilling of their respective
habitats over geologic times.
|
The amino-acid changes that we observed specifically in teleosts with low
habitat temperatures are subtle and cluster in the loop between transmembrane
domains 7 and 8 of Sec61 (Fig.
3, Fig. 6A). Note
that trout can live at habitat temperatures similar to those of the New
Zealand notothenioids (lowest winter temperature 4°C), which may explain
why trout Sec61
contains all but one of the amino-acid changes specific
to cold-water fishes in the loop between transmembrane domains 7 and 8
(Fig. 3). These changes are
present in fishes with habitat temperatures both above and below 0°C,
suggesting that there are no specific adaptations in Sec61
for function
at extremely low temperatures (Fig.
3, Fig. 6A). The
majority of these amino-acid positions (S328, G339, Y344, L345, and V363) are
absolutely conserved in Sec61
from warm-blooded vertebrates (mouse,
rat, dog and human; data not shown).
Only two subunits of the Sec61 channel, Sec61 and Sec61
are
essential for protein translocation across the ER membrane
(Finke et al., 1996
;
Kalies et al., 1998
). We found
that Sec61
is completely conserved from the Antarctic fish
Harpagifer antarcticus to mouse at the amino-acid level
(Fig. 6B) and hence cannot
contribute to improved protein translocation at low temperature. Targeting of
nascent chain/ribosome complexes to the ER membrane is mediated by a signal
recognition particle (SRP) and its receptor at the ER membrane
(Johnson and van Waes, 1999
).
When we separated the targeting and translocation reactions in vitro, we found
that targeting was not limiting for translocation into dog or fish microsomes
at low temperature, confirming previous data from Nicchitta and Blobel
(Nicchitta and Blobel, 1989
),
who had addressed this question for translocation into dog pancreas
microsomes. The ER lumenal Hsp70 BiP may contribute to efficient protein
translocation into the ER, although its role in import into mammalian ER is
controversial (Gething, 1999
;
Johnson and van Waes, 1999
).
In S. cerevisiae and in mammalian cells, BiP is inducible by a
variety of stress factors (Gething,
1999
). BiP recognizes hydrophobic patches of folding intermediates
and prevents their aggregation (Rudiger et
al., 1997
). Hydrophobic interactions, however, are relatively weak
at low temperature (Russell,
2000
). Furthermore, liver cells of the Antarctic notothenoid
T. bernacchii do not express stress-inducible Hsp70s
(Hofmann et al., 2000
).
Unfortunately, the available antibodies against mammalian BiP do not
crossreact with the fish protein (K. R. and J.-C. Röper, unpublished). It
is therefore unclear whether Antarctic fishes express BiP at all, but even if
they do, owing to the specificity of BiP for hydrophobic interactions, the
contribution of BiP to protein translocation into the ER and protein folding
at low temperature is likely to be limited. Taken together, these
considerations further support our notion that the amino-acid changes in
Sec61
from cold-water fishes are responsible for the improved protein
translocation into their ER at 0°C
(Fig. 4A). At higher
temperatures, these changes may be detrimental to channel stability, which may
explain why both carp and D. mawsoni membranes translocate less
efficiently than dog membranes at 25°C.
In soluble proteins, increased activity at low temperature often requires
increased flexibility of hinge regions to allow the protein to undergo the
conformational changes necessary for its function
(Gianese et al., 2001;
Russell, 2000
). As a result,
adaptive amino-acid changes are often observed in these hinge regions.
Adaptation to the low temperature of transmembrane channels has not been
studied before. Our observations suggest that, as in soluble proteins, only a
few key positions need to be changed to improve function at low temperature
(Fig. 3 and
Fig. 6A). We know from yeast
genetics that the loop between transmembrane domains 7 and 8 of Sec61
is functionally important; mutations in positions 327 and 341
(Fig. 6A, asterisks) lead to
defects in protein transport through the channel and to cold-sensitive
function of the channel (Stirling et al.,
1992
; Zhou and Schekman,
1999
). Recent structural data suggest that the Sec61 channel
undergoes a conformational change during translocation
(Beckmann et al., 2001
). It is
possible that the amino acids changed in cold-adapted Sec61
denote
hinge regions and that the changes allow the adapted protein to undergo
conformational changes more easily. If so, our data would indicate that the
loop between transmembrane domain 7 and 8 may need to move during protein
translocation into the ER (Fig.
6C).
In summary, our data suggest that membrane fluidity may not be limiting for
protein translocation across the ER membrane at and below 0°C and that a
conformational change involving the loop between transmembrane domains 7 and 8
of Sec61 and key positions throughout the protein may be pivotal for
protein translocation across the ER membrane.
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Acknowledgments |
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