From the Howard Hughes Medical Institute and Departments of Physiology and Microbiology, Immunology, and Molecular Genetics, Molecular Biology Institute, UCLA, Los Angeles, California 90095-1662
Received for publication, January 12, 2003, and in revised form, February 13, 2003
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ABSTRACT |
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Insertion and folding of polytopic
membrane proteins is an important unsolved biological problem. To study
this issue, lactose permease, a membrane transport protein from
Escherichia coli, is transcribed, translated, and inserted
into inside-out membrane vesicles in vitro. The protein is
in a native conformation as judged by sensitivity to protease, binding
of a monoclonal antibody directed against a conformational epitope, and
importantly, by functional assays. By exploiting this system it is
possible to express the N-terminal six helices of the permease
(N6) and probe changes in conformation during insertion
into the membrane. Specifically, when N6 remains attached
to the ribosome it is readily extracted from the membrane with urea,
whereas after release from the ribosome or translation of additional
helices, those polypeptides are not urea extractable. Furthermore, the
accessibility of an engineered Factor Xa site to Xa protease is reduced
significantly when N6 is released from the ribosome or more
helices are translated. Finally, spontaneous disulfide formation
between Cys residues at positions 126 (Helix IV) and 144 (Helix V) is
observed when N6 is released from the ribosome and inserted
into the membrane. Moreover, in contrast to full-length permease,
N6 is degraded by FtsH protease in vivo, and
N6 with a single Cys residue at position 148 does not react
with N-ethylmaleimide. Taken together, the findings
indicate that N6 remains in a hydrophilic environment until
it is released from the ribosome or additional helices are translated
and continues to fold into a quasi-native conformation after insertion
into the bilayer. Furthermore, there is synergism between
N6 and the C-terminal half of permease during assembly, as
opposed to assembly of the two halves as independent domains.
Most inner membrane proteins in Escherichia coli are
targeted to the membrane by the signal recognition particle
(SRP)1 pathway and insert
into the membrane via the Sec machinery (1-4). It has been suggested
that bacterial SRP (Ffh protein and 4.5 S RNA) binds to the hydrophobic
region of nascent membrane proteins, and subsequently, the
ribosome-nascent chain complex and SRP interact with the SRP receptor
(FtsY) at the membrane surface. Recently, it has been proposed that
FtsY is a primary membrane-docking site for the ribosome, and SRP binds
to nascent protein after FtsY-ribosome binding (5). The Sec machinery
is comprised of SecY, SecE, and SecG proteins in the cytoplasmic
membrane (6-9) and contributes to the topology of some membrane
proteins (10). It has been shown (11, 12) that the Sec machinery and
YidC exist as a complex, and several inner membrane proteins interact
with YidC during insertion. Thus, YidC appears to play a key role in
inner membrane protein insertion (12). SecD, SecF, and YajC form
another heterotrimeric complex that binds to the SecYEG complex
(13). In addition, it has been reported (14) that YidC interacts
directly with SecDF rather than SecYEG, which forms an even larger
complex with SecYEG. Furthermore, SecA, a cytosolic protein with ATPase activity, is also important for insertion of inner membrane proteins with large periplasmic loops (15, 16). Although some details regarding
the mechanism of integral membrane protein insertion have been
elucidated, there are still many issues that require resolution,
particularly those related to insertion of polytopic membrane
proteins. Examples include folding of intermediates, interaction
between helices during translation/insertion, the order and timing of
helices exiting from the translocon into the lipid bilayer, and
topological determinants.
In an effort to study some of these issues we have chosen to use the
lactose permease of E. coli (LacY) as a model system. LacY
is a member of the Major Facilitator Superfamily (17) and catalyzes the
coupled stoichiometric translocation of galactosides and H+
(symport) (18). The protein has been solubilized from the membrane and
purified to homogeneity in a completely functional state (19, 20) and
is a 12-helix bundle with the N and C termini on the cytoplasmic face
of the membrane (21-23). In addition, LacY is physiologically (24) and
structurally a monomer in the membrane (25-27). Analysis of an
extensive library of mutants, particularly Cys replacement mutants
(28), in conjunction with a battery of site-directed biochemical and
biophysical techniques has led to the formulation of a
tertiary-structure model (29) as well as a hypothesis for the mechanism
of lactose/H+ symport (30).
Like most inner membrane proteins, LacY is inserted into the membrane
co-translationally (31, 32), and insertion involves SRP and FtsY (33,
34) as well as SecY (35). Phosphatidylethanolamine (PE) is also
important for the late maturation of LacY and required for proper
assembly and function (36). PE can even reverse misfolding of LacY
topology after insertion into membranes devoid of PE (37). However, the
mechanism by which LacY is inserted into the membrane and folds into
its final tertiary conformation is far from clear.
Remarkably, co-expression of LacY in two contiguous, non-overlapping
fragments with a covalent discontinuity in either cytoplasmic or
periplasmic loops leads to complementation resulting in resistance of
the fragments to proteolysis and functional LacY (38-41). Furthermore, a salt bridge between Asp-237 (Helix VII) and Lys-358 (Helix XI) plays
an important role in membrane insertion (42, 43), indicating that Helix
VII must interact with Helix XI before insertion of LacY into the
bilayer. In addition, the relatively long middle cytoplasmic loop is
important for functional expression, possibly acting as a time delay to
allow the N-terminal six helices (N6) to clear the
translocon before the last six helices are inserted (44). To analyze
these phenomena more completely, as well as the insertion mechanism of
LacY, an in vitro transcription/translation/insertion system
is described in which LacY appears to be inserted in a native,
functional conformation. In this study, we focus on insertion of the
N-terminal half of LacY (N6).
Materials--
[35S]Met,
[125I]protein A, and
N-[14C]ethylmaleimide (NEM) were obtained from
Amersham Biosciences. Factor Xa protease was obtained from New England
Biolabs (Beverly, MA). Site-directed rabbit polyclonal antiserum
against a dodecapeptide corresponding to the C terminus of LacY (45)
was prepared by Berkeley Antibody Co. (Richmond, CA). Monoclonal
antibody (mAb) 4B11 was prepared as described (46). Avidin-conjugated
horseradish peroxidase was purchased from Pierce.
6'-(N-dansyl)-1-thio- Strains and Plasmids--
E. coli T184
[lacI+O+Z Preparation of Inside-out (ISO) and Right-side-out (RSO)
Vesicles--
ISO membrane vesicles were prepared as described (56)
with minor modifications (57). ISO membrane vesicles were obtained from
the 40% sucrose (w/w) layer after centrifugation through a stepwise
sucrose gradient (50, 45, 40, 35, and 30%; w/w) in 50 mM
potassium phosphate (KPi; pH 7.5), 1 mM EDTA.
ISO vesicles were frozen in liquid N2 in 50 mM
KPi (pH 7.5), 10% glycerol, 8.5% sucrose and stored at
In Vitro
Transcription/Translation/Insertion--
In
vitro synthesis of LacY was carried out in the presence or absence
of ISO membrane vesicles (0.2 mg/ml total protein) at 30 °C using
the S-30 cell extract from the EcoProT7 system from Novagen
(Madison, WI) as described by the manufacturer with the minor
modifications described here. The cell extract was centrifuged briefly,
and the supernatant was used after the addition of 0.2-0.5 units/µl
of RNase inhibitor (Promega, Madison, WI).
Transcription/translation/insertion reactions were initiated by the
addition of DNA template. Incubation times and total volume of reaction
mixtures were varied as specified. For antibody binding assays, ligand
protection against NEM labeling, and lactose/Dns6-Gal
counter-flow, 0.2 mM unlabeled Met was used instead of
[35S]Met. After incubation, the mixture was collected
from the 50% sucrose cushion by centrifugation at 350,000 × g for 10 min (when the sample was less than 100 µl) or
220,000 × g for 40 min. The ISO vesicle-enriched
fraction was aspirated from the surface of the 50% sucrose cushion,
diluted 3-4 times with 50 mM KPi (pH 7.5), and
harvested by centrifugation at 350,000 × g for 10 min. Samples were solubilized in 1%
dodecyl- Factor Xa Protease Digestion--
ISO vesicles with specified
in vitro synthesized LacY were collected from the surface of
the 50% sucrose cushion, diluted, and pelleted by ultracentrifugation
as described above. The pellet was resuspended in 20 mM
Tris-HCl (pH 7.5), 100 mM NaCl, 2.0 mM CaCl2, harvested by centrifugation, and resuspended in the
same buffer without or with 1% DDM as indicated. Samples were
subjected to the Factor Xa protease digestion as described (53).
4B11 Binding to Membranes--
Aliquots of the given membrane
vesicle preparations (0.1 mg of membrane protein) suspended in 100 mM KPi (pH 7.5), 10 mM
MgSO4, 10% bovine serum albumin, 1.0 M sucrose
(Buffer A) were mixed with 10 µl of mAb 4B11 ascites fluid (61) and
incubated at room temperature for 30 min. The vesicles were harvested
by centrifugation, washed once in the Buffer A without bovine serum
albumin, and resuspended in the Buffer A. [125I]ProteinA
was added, and the samples were incubated for 30 min at room
temperature. After centrifugation, the vesicles were washed in Buffer A
without bovine serum albumin and resuspended in 1% Triton X-100. Bound
radioactivity was measured in a liquid scintillation counter.
[14C]NEM Labeling of Single Cys-148 LacY
Synthesized and Inserted in Vitro--
Reactivity of Cys-148 with
[14C]NEM was determined in the absence and presence of
substrate (62). Single Cys-148 LacY with a His6 tag at the
C terminus was synthesized in vitro at 30 °C for 1 h
as described. ISO vesicles (0.1 mg of total protein in 50 µl) were
incubated in the absence or presence of 100 mM
Lactose/Dns6-Gal
Counter-flow--
Lactose/Dns6-Gal counter-flow assays
were carried out as described (63). ISO vesicles with in
vitro synthesized LacY were isolated from a 50% sucrose cushion
and pre-equilibrated with 20 mM lactose at 4 °C
overnight (~50 mg/ml membrane protein). Pre-equilibrated vesicles
(~0.1 mg of membrane vesicles) were diluted into a cuvette containing
200 µl of 50 mM KPi (pH 6.6), 6 µM Dns6-Gal. Fluorescence was recorded an
SLM-Aminco 8100 spectrofluorimeter (excitation, 340 nm; emission, 500 nm).
In Vitro Transcription/Translation/Insertion
of LacY--
LacY was synthesized in vitro directly from a
plasmid by using an E. coli S30 extract and
[35S]Met in the presence or absence of ISO membrane
vesicles. To distinguish between membrane-associated proteins and
aggregated LacY, ISO vesicles were fractionated by centrifugation on a
50% sucrose cushion and subjected to SDS/PAGE. The major
35S-labeled product exhibits an molecular mass of about 33 kDa, which is typical of LacY (i.e. the molecular mass of
LacY is ~45 kDa, but it regularly exhibits an molecular mass of ~33
kDa on SDS/PAGE) and is detected in the membrane fraction exclusively when ISO membrane vesicles are present during transcription/translation (Fig. 1A). The band does not
appear in the absence of plasmid lacY template DNA or the
S30 extract alone (data not shown).
Integral membrane proteins are not extracted by 5 M urea or
at alkaline pH, and accordingly, LacY synthesized in vitro
in the presence of ISO vesicles fractionates with the membranes even after treatment with urea or alkali (Fig. 1, B and
C). Thus, LacY synthesized in vitro appears to
integrate into the membrane. Furthermore, when in vitro
transcription/translation is carried out in the presence of liposomes
or RSO vesicles rather than ISO vesicles, little or no
membrane-associated LacY is observed (Fig. 1D). The findings
confirm previous observations (64) showing that LacY insertion not only
requires other membrane components (e.g. the SecYEG complex)
but that insertion must occur vectorially from the cytoplasmic surface
of the membrane.
To examine the topology of LacY inserted in vitro, the
accessibility of engineered Factor Xa protease sites in a cytoplasmic or periplasmic loop to Factor Xa protease was tested. LacY with a
cytoplasmic Factor Xa site at the N terminus of a biotin acceptor domain (BAD) (51) in loop VI/VII was transcribed/translated in
vitro in the presence of ISO vesicles and digested with Xa protease in the absence or presence of DDM (Fig.
2A). Clearly, in the absence
of DDM, the band corresponding to full-length permease is completely
digested, and a band corresponding to N6 as well as a band
corresponding to C6 plus the BAD is observed. In contrast, ISO vesicles with in vitro synthesized LacY containing three
tandem Factor Xa protease sites in periplasmic loop VII/VIII require solubilization with DDM to achieve complete digestion (Fig.
2A). The data indicate that LacY transcribed, translated,
and inserted into ISO vesicles in vitro retains the topology
observed in the native membrane (i.e. cytoplasmic loops are
exposed to the external surface of ISO vesicles and vice versa for
periplasmic loops).
Monoclonal antibody 4B11 binds to a discontinuous epitope on the
cytoplasmic surface of the membrane that contains determinants from
both loops VIII/IX and X/XI (61). Binding of 4B11 to LacY synthesized
and inserted into ISO vesicles in vitro is essentially identical quantitatively to LacY synthesized in vivo (Fig.
2B), suggesting that the in vitro product likely
has native tertiary structure (36). This contention is supported by
experiments shown in Fig. 3A
in which lactose/Dns6-Gal counter-flow (47, 63) was tested
in ISO vesicles containing LacY synthesized and inserted in
vitro. As shown, when ISO vesicles with LacY are equilibrated with
lactose and rapidly diluted into buffer containing
Dns6-Gal, a transient increase in fluorescence is observed
at 500 nm. However, if saturating concentrations of lactose are added to the external medium, no change in fluorescence is observed (47).
Furthermore, single Cys-148 LacY synthesized and inserted in
vitro exhibits significant ligand protection against alkylation by
NEM (Fig. 3B). Although background labeling is relatively
high, NEM labeling is inhibited significantly and reproducibly by
Insertion of N6--
The N-terminal half of LacY
contains six transmembrane helices and a long cytoplasmic C terminus
corresponding to most of the middle cytoplasmic loop (38, 40).
N6 is not stably expressed in vivo unless
C6 is expressed simultaneously. However when N6 containing a stop codon is synthesized in vitro in the
presence of ISO vesicles, the polypeptide is not extracted by urea
(Fig. 4, lanes 1 and
2). Surprisingly, N6 truncated at position 226 and lacking a stop codon so that ribosomes remain attached is partially
extracted from the membrane by urea (lane 3), and urea extraction is increased in the presence of 150 mM KCl (data
not shown). The data suggest that the translation intermediate,
N6 with an attached ribosome, is located in a relatively
hydrophilic environment. Interestingly, the quantity of urea-soluble
product is decreased by the addition of puromycin, which causes release of the polypeptide from the ribosome before urea extraction (lane 4). LacY truncated at position 312 and lacking a stop codon which contains 9 transmembrane helices followed by a bound ribosome is no
longer susceptible to urea extraction even before treatment with
puromycin (lanes 5 and 6). Thus, ribosome binding
to LacY per se does not appear to affect susceptibility to
urea extraction. The data are consistent with the interpretation that
the N-terminal half of LacY does not fully insert into the lipid
bilayer before ribosome release or subsequent synthesis and insertion
of two of the C-terminal six helices.
Ribosome Attachment and Folding of N6--
Either
full-length LacY or N6 with two tandem Factor Xa protease
sites in cytoplasmic loop IV/V synthesized and inserted in vitro are partially proteolyzed after 2 h of digestion at
4 °C in the presence of DDM (Fig. 5,
compare lanes 1 and 2 with lanes 6 and
7, respectively). In contrast, N6 truncated at
position 207 or 226 and lacking a stop codon so that ribosomes remain
attached is digested completely by Xa protease (compare lanes
3 and 4 with 8 and 9,
respectively). However, LacY truncated at position 312 with attached
ribosomes is resistant to digestion (compare lanes 5 and
10). Although it is anticipated that the tandem Factor Xa protease sites in loop IV/V of LacY synthesized and inserted in vitro should be digested in ISO vesicles by Xa protease, little or
no proteolysis is observed when the construct is synthesized in
vivo in ISO vesicles, and DDM solubilization is
required.2 Possibly, loop
IV/V is occluded, which accounts for this behavior. In any case, in the
presence of detergent at room temperature, the constructs synthesized
in vitro are completely proteolyzed (see lanes 4 and 5 in Fig. 6). The results
suggest that N6 attached to the ribosome does not fold
sufficiently well to protect the Factor Xa sites in loop IV/V from
proteolysis. Although not shown, the addition of puromycin to
N6 with attached ribosomes increases sensitivity to
proteolysis by Xa protease. Thus, insertion of N6 into the
bilayer leads to more efficient folding of this polypeptide fragment.
Glu-126 (Helix IV) and Arg-144 (Helix V) are in close proximity and
charged-paired (65-67), and two Cys residues at these positions spontaneously form a disulfide bond (53). N6 containing
mutant E126C/R144C and two tandem Factor Xa protease sites in loop IV/V with or without a stop codon (i.e. without or with attached
ribosomes) were synthesized in vitro and examined for
disulfide bond formation by studying proteolysis by Xa protease (53)
(Fig. 6). With full-length LacY or N6, significant
cross-linking is observed, as evidenced by the presence of bands
corresponding to the intact polypeptides after Xa protease digestion
(lanes 1 and 2). In contrast, N6
truncated at position 226 without a stop codon exhibits no band
corresponding to the intact polypeptide (lane 3). Moreover,
in the presence of reducing agents, dithiothreitol and mercaptoethanol,
no bands corresponding to the intact proteins are observed (lanes
4-6). Even in the presence of iodine, N6 with
attached ribosomes does not exhibit cross-linking (data not shown).
Thus, insertion of the polypeptides into the lipid bilayer is necessary
to achieve sufficiently close proximity between positions 126 and 144 for disulfide formation to occur.
N6 Is a Substrate for Membrane Protease
FtsH--
N6 is stable when co-expressed with
C6; however, N6 is unstable and degraded when
expressed in the absence of C6. FtsH, a membrane-bound
metalloprotease with ATPase activity, is responsible for degradation of
certain integral membrane proteins (68). Therefore, expression of
N6 in an FtsH temperature-sensitive mutant strain was
examined (Fig. 7A) because the
mutant lacks FtsH protease activity at high temperature (69). When
expressed in wild type cells (FtsH+), N6 is not
observed (lane 1). However, when co-expressed with C6, bands corresponding to N6 and
C6 are observed (lane 2). Interestingly, when
N6 is expressed in the FtsH mutant at the non-permissive temperature, a clear band corresponding to N6 is observed
(lane 3). At the non-permissive temperature, co-expression
of N6 with C6 in the FtsH mutant also leads to
a significant increase in the amount of each polypeptide observed.
Clearly, N6 is a substrate for FtsH and requires
C6 for stable expression in vivo.
By expressing either full-length single Cys-148 LacY or single Cys-148
N6 in the FtsH mutant, the reactivity of Cys-148 with NEM
can be compared. Although the full-length and N6 constructs are expressed at about the same levels as judged by avidin blots (Fig.
7B, bottom), it is apparent that although Cys-148
labels readily with NEM as a function of time in full-length LacY, the sulfhydryl group is completely unreactive in single Cys-148
N6 (Fig. 7B, top). These data
indicate that N6 synthesized and inserted in
vitro does not have the same tertiary structure as it does in
native, full-length LacY.
Although previous studies (32, 64) indicate that LacY transcribed
and translated in vitro can be inserted into ISO membrane vesicles with the correct topology (36), questions remain as to whether
or not the inserted protein is in a native conformation. Because LacY,
like many other membrane proteins, is resistant to traditional means of
structural analysis, alternative approaches have been developed to
study topology and discern the overall three-dimensional fold (see
Refs. 29 and 30). The studies presented here, which utilize some of
these approaches, provide convincing evidence that LacY synthesized and
inserted into the membrane in vitro is in a native
conformation. (a) By using engineered Factor Xa protease
sites in cytoplasmic or periplasmic loops, the topology of the
polypeptide with respect to the membrane appears to be correct.
(b) A mAb that binds to a discontinuous epitope comprised of
residues in cytoplasmic loops VIII/IX and X/XI (61) binds to LacY
synthesized and inserted in vitro as well as it binds when
LacY is synthesized and inserted in vivo. (c) Cys
residues at positions 126 and 144 in the in vitro system
undergo spontaneous cross-linking as observed in vivo (53).
(d) LacY synthesized and inserted in vitro
exhibits lactose/Dns6-Gal counter-flow and significant
ligand protection against alkylation of Cys-148. Therefore,
transcription, translation, and insertion of LacY into ISO vesicles
in vitro represents a system in which the mechanism of
insertion of a polytopic membrane protein and its folding into a
tertiary conformation can be studied reliably.
Despite conjecture regarding co-translational insertion of
polytopic membrane proteins, it is unclear how many
transmembrane helices are accommodated by the translocon before
insertion into the bilayer. Furthermore, little information is
available regarding whether folding into a tertiary conformation begins
in the translocon or only after migration into the bilayer with the
assistance of chaperones such as PE (36, 37, 70). The observation that the first six helices of LacY are extracted with urea when the ribosome
remains attached, whereas the first eight helices are not, suggests
that the translocon can accommodate at least six transmembrane helices,
a conclusion similar to that drawn from findings obtained with
P-glycoprotein (71). Similarly, an in vitro study of the
polytopic membrane protein MtlA suggests more than one pair of
transmembrane domains are assembled before the exit from translocon
(72). On the other hand, the N6 portion of LacY may not be
fully accommodated by the translocon, and part of N6 may be
in contact with lipid, which would explain why the polypeptide is not
completely extracted with urea (Fig. 3).
Photo cross-linking studies on certain membrane proteins indicate that
the transmembrane domains may indeed interact with lipid during
insertion (72, 73). With LacY, the first or second transmembrane helix
might interact with phospholipid, whereas helices III to VI are still
in the translocon. However, if this is the case, it is unlikely that
the first two helices migrate very far from the translocon. Indeed, it
has been suggested that in some endoplasmic reticulum membrane
proteins, a transmembrane helix can re-enter the translocon after its
initial release into the lipid phase if it has affinity for other
helices still within the translocon (74). Taken as a whole, the data
suggest that the N6 half of LacY must remain near the
translocon after exit into the bilayer as the C6 half is
translated. Once the C6 half exits the translocon, folding
into a final tertiary structure likely occurs within the bilayer,
resulting in a protease-resistant, functional molecule.
The differential sensitivity of engineered Factor Xa protease sites in
cytoplasmic loop IV/V to cleavage in N6 with attached ribosomes versus fully translated N6 suggests
that the conformation of the two polypeptides differs. The observation
that fully translated N6 is not extracted with urea whereas
a significant amount of the translocation intermediate is urea-soluble
(Fig. 4) implies that a folding event occurs after N6 exits
the translocon. Cross-linking data with Cys residues at positions 126 (Helix IV) and 144 (Helix V) also support the interpretation that
N6 folds after insertion into the bilayer (Fig. 5).
However, it seems unlikely that N6 synthesized and inserted
in vitro has the same tertiary structure as it does in
native, full-length LacY since N6 is clearly sensitive to
proteolysis by FtsH protease in the absence of C6 but
stabilized in its presence (Fig. 7A). Furthermore,
N6 with a single Cys residue at position 148 does not react
with NEM (Fig. 7B).
Unlike the two-dimensional projection map of OxlT (75), which indicates
that each helix in the two halves of the protein occupy
symmetry-related positions, neither the Na+/H+
antiporter NhaA (76, 77), the Na+/sugar symporter MelB
(78), nor LacY (29) exhibit such symmetry. Rather, in these transport
proteins, helices from the N- and C-terminal halves of the polypeptides
interdigitate. Thus, it is not surprising that N6 in LacY
probably does not insert into the bilayer in a native conformation,
although some of the structural features of the native, full-length
protein are observed. This being the case, it seems clear that folding
of LacY into a native, functional conformation must occur in the
bilayer after both halves of the protein have exited the translocon, a
conclusion consistent with the finding that PE acts as a molecular
chaperone in the folding of LacY into its final native conformation
(36, 37).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside
(Dns6-Gal) was synthesized as described (47). All other
materials were obtained from commercial sources.
Y
(A)
rpsL, met
,
thr
, recA, hsdM,
hsdR/F',
lacIqO+ZD118(Y+A+)]
(48) was used for the preparation of membrane vesicles. E. coli AR796 [MC4100 zhd::Tn10
zgj-3198::Tn10kan] (49) or E. coli AR797 [AR796 ftsH1(Ts)] (49) were used as
indicated for in vivo expression of the N6 and
C-terminal half LacY (C6). The lacY cassette gene in plasmid pT7-5 was used for in vitro synthesis of
wild type LacY. Plasmids encoding LacY with three tandem Factor Xa protease sites in loop VII/VIII (50), LacY with a Factor Xa protease
site in loop VI/VII (51, 52), and LacY with two tandem Factor Xa
protease sites in loop IV/V (53) were constructed as described.
Plasmids pN6 (54) and pC6 (55) were used for in vivo expression. Plasmid encoding N6 with two
tandem Factor Xa protease sites between Helix IV and V was constructed
in the same manner as pN6 (54).
80 °C. RSO membrane vesicles were prepared by osmotic lysis as
described (58, 59), suspended in 100 mM KPi (pH
7.5), 10 mM MgSO4, frozen in liquid
N2, and stored at
80 °C.
-D-maltopyranoside (DDM) and then subjected to
SDS/12% PAGE in the absence of reducing reagent. The gel was dried,
exposed to a PhosphorImager screen, and visualized by a Molecular
Dynamics Storm 860 PhosphorImager. Where indicated liposomes (0.2 mg/ml
total lipid) or RSO membrane vesicles (0.2 mg/ml total protein) were
used instead of ISO vesicles. Liposomes were prepared from total
E. coli lipid as reported (60). DNA templates were used at
40-80 µg/ml. For synthesis of truncated LacY, PCR products were used
as DNA template. The sense primer for truncated LacY was
5'-GCGAGGCCCAGCTGGCTTATCG. Construction of genes encoding mutants
truncated at position 207, 226, or 312 utilized the same sense primer,
and the antisense primer was 5'-TGCCGAATGGCAGGCACCTACCGC, 5'-GTGACAAAAACCACAGTTTTG, or 5'-CGCTGAGGTGGCGAACGATG, respectively.
-D-galactopyranosyl-1-thio-
-D-galactopyranoside as indicated for 5 min at room temperature. Labeling was initiated by
adding 12 µl of [14C]NEM (40 mCi/mmol) at a final
concentration of 0.5 mM, and the vesicles were incubated
for 30 min at room temperature. Reactions were quenched by addition of
10 mM dithiothreitol. The vesicles were solubilized with
2% DDM, and the samples were mixed with Talon resin
(Clontech; Palo Alto, CA) equilibrated with 50 mM KPi (pH 7.5), 0.2% DDM, 10 mM
imidazole. After a 20-min incubation at room temperature, the resin was
washed twice with 1 ml of equilibration buffer. Samples were quantified
by the scintillation spectrometry.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
In vitro transcription,
translation and insertion of LacY. A, LacY was
synthesized in vitro in the absence or presence of ISO
membrane vesicles (10 µg of protein). 35S-Labeled
translation products from a 1-h incubation were carefully placed on a
50% sucrose cushion and centrifuged. The pellet and inner membrane
fraction were analyzed. B, reaction components were
fractionated as described. The inner membrane fraction was incubated
with 5 M urea for 20 min on ice in 50 mM
KPi (pH 7.5), and the membranes were harvested by
centrifugation. The supernatant fraction from the urea wash
(sup) was precipitated with 10% trichloroacetic acid.
ppt, pellet. C, the inner membrane fraction from
the 50% sucrose cushion was subjected to alkali extraction at pH 11.3 or treatment with 5 M urea. All samples were precipitated
with 10% trichloroacetic acid. D, in vitro
translation was carried out with liposomes, ISO membrane vesicles, or
RSO membrane vesicles as indicated, and the vesicles were isolated by
centrifugation on a 50% sucrose cushion and washed with 5 M urea.
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Fig. 2.
Confirmation of LacY synthesized and inserted
in vitro. A, Factor Xa protease
cleavage of 35S-labeled mutants with engineered Xa sites.
The membrane topology of in vitro synthesized and inserted
LacY was studied by introducing Factor Xa protease sites into
cytoplasmic loop VI/VII or periplasmic loop VII/VIII. These mutants
were transcribed and translated for 1 h in vitro. After
the isolation of ISO membrane vesicles from the reaction mixture,
Factor Xa protease was added to the membrane suspension in the presence
or absence of DDM. Proteolysis was carried out for 12 h at
4 °C, and the samples were subjected to SDS/PAGE. Arrows
indicate the position of intact LacY (with the BAD in the first
four lanes and without in the second four lanes).
B, mAb 4B11 binding. mAb 4B11 binds to a cytoplasmic epitope
in LacY comprised of residues from loops VIII/IX and X/XI. ISO vesicles
containing LacY synthesized and inserted in vivo were
prepared as described (61) and purified as described under
"Experimental Procedures." In vitro synthesis of LacY
was carried out for 2 h at 30 °C in the presence of ISO
membrane vesicles and purified as described. Both ISO vesicles
preparations were then assayed for 4B11 binding as described under
"Experimental Procedures." -LacY, ISO vesicles from
E. coli T184 cells containing no LacY; in vivo
LacY, ISO vesicles from E. coli T184 expressing
LacY; in vitro LacY 1 and 2, LacY with
2 Xa protease sites in cytoplasmic loop IV/V or single Cys-148 LacY,
respectively.
-D-galactopyranosyl-1-thio-
-D-galactopyranoside. The data as a whole support the contention that LacY synthesized and
inserted in vitro is in a native, functional
conformation.
View larger version (19K):
[in a new window]
Fig. 3.
Function of LacY synthesized and inserted
in vitro. A,
lactose/Dns6-Gal counter-flow. Experiments were carried out
as described under "Experimental Procedures." Fluorescence at 500 nm was recorded (excitation, 340 nm) as described. Reactions were
initiated by diluting preloaded ISO vesicles with LacY synthesized and
inserted in vitro into a cuvette with or without 20 mM lactose. B, NEM labeling of single Cys-148
LacY synthesized and inserted in vitro in the absence or
presence of
-D-galactopyranosyl-1-thio-
-D-galactopyranoside
(TDG). Experiments were carried out as described under
"Experimental Procedures."
View larger version (62K):
[in a new window]
Fig. 4.
Urea extraction of in vitro
synthesized and inserted [35S]N6
without or with attached ribosomes. In vitro synthesis
and insertion was carried out for 30 min at 30 °C. As indicated, 1.5 mM puromycin was added to the reaction mixtures, and
incubation was continued for an additional 10 min at 30 °C. Samples
that were not incubated with puromycin were placed on ice. Vesicles
were treated with 5 M urea for 20 min on ice and
centrifuged. The supernatants (sup) were aspirated carefully
and precipitated with 10% trichloroacetic acid. Both the pellet
(ppt) and supernatant fractions were solubilized in 1% DDM
and subjected to SDS/PAGE and phosphorimaging. The pellet samples were
exposed overnight, and the supernatant samples were exposed for 2 days.
The number 226 or 312 refers to the last amino
acid in the truncated LacY.
View larger version (80K):
[in a new window]
Fig. 5.
Effect of attached ribosomes on conformation
of N6. In vitro synthesized and inserted
full-length LacY, N6, or N6 without a stop
codon at position 207, 226, or 312, all with 2 tandem Xa protease sites
in cytoplasmic loop IV/V, were treated with Factor Xa protease in the
presence of DDM. In vitro synthesis and insertion was
carried out at 30 °C for 1 h. Membranes with
35S-labeled LacY mutants were then purified by
centrifugation as described under "Experimental Procedures."
Vesicles were solubilized in 1% DDM, and Factor Xa protease was added
for 2 h at 4 °C. Arrows indicate the molecular mass
full-length LacY and each truncation mutant. The numbers
denote the position of the last amino acid in the mutants.
View larger version (100K):
[in a new window]
Fig. 6.
Spontaneous cross-linking between Cys
residues at positions 126 and 144 in vitro.
Full-length E126C/R144C LacY, N6, or N6 without
a stop codon, all with Cys residues at positions 126 and 144 containing
2 tandem Xa protease sites in cytoplasmic loop IV/V, were synthesized
and inserted in vitro. Membrane vesicles (10 µg) were
purified on a sucrose cushion, washed by 20 mM Tris-HCl (pH
7.5), and resuspended in 50 µl of 20 mM Tris-HCl (pH
7.5). After incubation for 30 min at room temperature, NEM was added to
a final concentration of 20 mM. Membranes were harvest by
centrifugation and resuspended in 50 µl of 20 mM Tris-HCl
(pH 7.5), 100 mM NaCl, 2.0 mM
CaCl2, 1.0% DDM. The samples were incubated overnight at
room temperature after the addition of 1 µg of Factor Xa protease to
each sample. An aliquot containing 5 µg of membrane protein was
incubated with 10 mM dithiothreitol (DTT) before
SDS/PAGE. The arrow denotes the molecular mass of the
cross-linked product of full-length LacY (lane 1). The
arrowhead denotes the molecular mass of C8
(lanes 1 and 4). The asterisk denotes
the molecular mass of the cross-linked product of N6
(lane 2). ME, mercaptoethanol.
View larger version (34K):
[in a new window]
Fig. 7.
Effect of FtsH protease activity on
expression of N6 or N6/C6 and
reactivity of Cys-148 with NEM in N6. A,
N6 was expressed alone in cells that are wild type for FtsH
protease (E. coli AR796 (ftsH+)) or a
temperature mutant (E. coli AR797 (ftsH1ts)).
Cells were grown at 30 °C in Luria-Bertani broth containing
appropriate antibiotics to an A600 of 0.6 and induced with 0.5 mM
isopropyl-1-thio- -D-galactopyranoside. After 1 h,
the culture was transferred to 42 °C and incubated for 2 h, and
the cells were harvested by centrifugation. The cells were lysed by
sonification, and the membrane fraction was isolated by centrifugation
and subject to SDS/PAGE. Protein bands were identified by Western
blotting; N6 was detected by avidin horseradish peroxidase,
and C6 was detected with anti-C-terminal antibody.
B, full-length single Cys-148 LacY with a BAD in the middle
cytoplasmic loop or N6 single Cys-148 with a BAD at the C
terminus were expressed in E. coli AR797
(ftsH1ts). Top, RSO membrane vesicles were
isolated as described under "Experimental Procedures," incubated
with [14C]NEM for the given times, purified by avidin
affinity chromatography, and subjected to SDS/PAGE and phosphorimaging
exactly as described (see 79). Bottom, Western blots with
avidin horseradish peroxidase of the same samples as those used for NEM
labeling. The asterisk indicates nonspecific bands.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to Eitan Bibi, William Dowhan, and Mikhail Bogdanov for editorial suggestions, K. Nishiyama for advice regarding the in vitro system, and T. Ogura for providing strains AR796 and -797.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant DK51131:08 (to H. R. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: HHMI/UCLA, 5-748
Macdonald Research Laboratories, Box 951662, Los Angeles, CA 90095-1662. Tel.: 310-206-5053; Fax: 310-206-8623; E-mail:
RonaldK@HHMI.UCLA.edu.
Published, JBC Papers in Press, February 16, 2003, DOI 10.1074/jbc.M300332200
2 C. D. Wolin and H. R. Kaback, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
SRP, signal
recognition particle;
N6, N-terminal six helices of lactose
permease;
C6, C-terminal six helices of lactose permease;
LacY, lactose permease;
PE, phosphatidylethanolamine;
NEM, N-ethylmaleimide;
mAb, monoclonal antibody;
Dns6-Gal, 6'-(N-dansyl)-1-thio--D-galactopyranoside;
ISO, inside out;
RSO, right-side out;
KPi, potassium
phosphate;
DDM, dodecyl-
-D-maltopyranoside;
BAD, biotin
acceptor domain.
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