From the Program of Cellular Biotechnology, Institute
of Biotechnology, University of Helsinki, Viikinkaari 9, 00710 Helsinki, Finland, ¶ Consiglio Nazionale delle Ricerche Institute
of Neuroscience, Cellular and Molecular Pharmacology Section, Via
Vanvitelli 32-20129 Milano, Italy, and the ** Faculty of
Pharmacy, University of Catanzaro "Magna Graecia," 8021 Roccelletta di Borgia (Catanzaro), Italy
Received for publication, October 7, 2002, and in revised form, November 11, 2002
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ABSTRACT |
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C-tail-anchored proteins are defined by an
N-terminal cytosolic domain followed by a transmembrane anchor close to
the C terminus. Their extreme C-terminal polar residues are
translocated across membranes by poorly understood post-translational
mechanism(s). Here we have used the yeast system to study translocation
of the C terminus of a tagged form of mammalian cytochrome
b5, carrying an N-glycosylation
site in its C-terminal domain (b5-Nglyc).
Utilization of this site was adopted as a rigorous criterion for
translocation across the ER membrane of yeast wild-type and mutant
cells. The C terminus of b5-Nglyc was rapidly
glycosylated in mutants where Sec61p was defective and incapable of
translocating carboxypeptidase Y, a well known substrate for
post-translational translocation. Likewise, inactivation of several
other components of the translocon machinery had no effect on
b5-Nglyc translocation. The kinetics of
translocation were faster for b5-Nglyc than for
a signal peptide-containing reporter. Depletion of the cellular ATP
pool to a level that retarded Sec61p-dependent
post-translational translocation still allowed translocation of
b5-Nglyc. Similarly, only low ATP
concentrations (below 1 µM), in addition to cytosolic
protein(s), were required for in vitro translocation of
b5-Nglyc into mammalian microsomes. Thus, translocation of tail-anchored
b5-Nglyc proceeds by a mechanism different from
that of signal peptide-driven post-translational translocation.
C-tail-anchored proteins
(TA1 proteins) constitute a
class of integral membrane proteins that are held in the phospholipid bilayer by a single segment of hydrophobic amino acids close to the C
terminus, the entire functional N-terminal portion facing the cytosol.
A large number of TA proteins have been described in animal,
plant, and fungal cells, where their roles range from regulation of
apoptosis to enzyme catalysis, translocation of newly synthesized
polypeptides across membranes, vesicular traffic (see Refs. 1 and 2 for
reviews), and control of gene expression (3). Consistent with these
different functions, TA proteins are found on a variety of membranes,
such as those of the endoplasmic reticulum (ER) and of the Golgi
complex, the plasma membrane and the mitochondrial outer membrane.
Mitochondrial targeting occurs directly from the cytosol (4), whereas
TA proteins located in membranes of the exo- and endocytic pathways are
first inserted into the ER and then transported to their destinations
by membrane traffic (5-8).
A point that remained controversial for many years was whether TA
proteins are bona fide transmembrane proteins, with the C-terminal residues exposed at the exoplasmic side of the membrane, or
rather have a hairpin configuration with the hydrophobic stretch looping back to the cytosolic face of the bilayer. The difficulty in
distinguishing between these alternatives was because of the small
number of polar residues downstream to the hydrophobic domain, so that
traditional approaches, such as protection from proteolysis or
accessibility to antibodies, did not yield clear results (see Ref. 1).
In the past few years, however, the use of recombinant proteins with
N-glycosylation sites appended at the C terminus has allowed
unequivocal demonstration that TA proteins can acquire transmembrane
topology by translocating their C terminus across the ER membrane (6,
9-11). Using glycosylation as a criterion for translocation, a variety
of different C-terminal sequences, deriving from cytosolic or
exoplasmic portions of different proteins, have been shown to be
translocated, indicating that the translocation process is not very
selective with regard to the residues that are transferred to the ER lumen.
The transmembrane segment of TA proteins emerges from the ribosome only
upon completion of translation; thus, translocation of the C terminus
must be post-translational, as has been directly demonstrated (6, 11).
However, the molecular mechanism underlying this process has not been
clarified so far. Studies addressing this question have employed
in vitro systems in which tight (alkaline pH-resistant)
association of the protein under study to added membranes or
proteoliposomes was followed (6, 12, 13). However, tight binding of TA
proteins to lipid bilayers without translocation of the C terminus can
occur in artificial systems, as has been demonstrated for cytochrome
b5 (14, 15).
The Sec61 translocon has been proposed as the most likely candidate for
translocation of the C terminus of TA proteins across the ER membrane
(16-18). This heterotrimeric complex, conserved in higher eukaryotes
and yeast, is composed of the multispanning protein Sec61p that forms
an aqueous channel and two small proteins (called Sbh1p and Sss1p in
the yeast Saccharomyces cerevisiae and Sec61 A second translocon, previously thought to operate in co-translational
translocation only, is present in the yeast ER membrane. It consists of
the Sec61p homolog Ssh1p, the Shb1 homolog Sbh2p, and Sss1p (27).
Recent evidence indicates that it is also involved in
post-translational translocation as well as retrotranslocation (28).
Thus, this complex could also be involved in TA protein insertion.
In previous studies we used a mutant version of cytochrome
b5 to study the trafficking of TA proteins in
mammalian cells (4, 11, 29). This mutant b5,
called b5-Nglyc, is tagged at its C terminus
with a sequence corresponding to the first 19 amino acids of bovine
opsin. The tag provides an N-glycosylation consensus site
that is used efficiently in vivo as well as in a cell-free system. Here we have taken advantage of S. cerevisiae
mutants to study in vivo whether the Sec61 translocon
complex is involved in translocation of the C terminus of TA proteins,
using glycosylation of b5-Nglyc as a rigorous
criterion for translocation. We find that efficient translocation
occurs under conditions in which Sec61p, Sec62p, Sec63p, or BiP/Kar2p
as well as other proteins functioning in ER translocation, Sbh1p/2p and
Lhs1p, are non-functional for signal peptide-driven translocation. In
addition, translocation of b5-Nglyc has low
energy requirements both in vivo and in a mammalian
cell-free system. These results indicate that post-translational translocation of the C-terminal tail of TA proteins occurs by a
mechanism distinct from signal peptide-driven translocation, most
likely without the participation of the Sec61 complex.
Construction of Recombinant Plasmids and of Yeast
Strains--
The opsin-tagged cytochrome b5
cDNA containing a consensus sequence for N-glycosylation
(b5-Nglyc; Ref. 11) in pGEM4 was amplified by
PCR with primers conferring EcoR1 and XbaI sites at its 5'- and 3'-extremities, respectively. These two sites were used
to place the cDNA between the SUC2 promoter and the
ADH1 terminator in the Escherichia coli-yeast
shuttle vector pFL26, resulting in plasmid pKTH5013, or in the high
copy number episomal vector pYES2 (Invitrogen), resulting in plasmid pKTH5014.
The yeast strains used in this study are listed in Table I. Strains
sec61-3 (H1399), sec63-1 (H1404),
kar2-159 (H1424), lhs1 (H1425),
sec62-101 (H1474), sec63-201 (H1475),
sbh1 (H1520), and sbh1sbh2 (H1521) were created
by integrating the b5-Nglyc gene from plasmid
pKTH5013 into the LEU2 locus of the parental strains H257
(CSY42, Ref. 30), H259 (RSY153, Ref. 31), H22 (MS174, Ref. 21), H823
(see below), H681 and H682 (DNY70 and DNY66, respectively, Ref. 32),
H1503 and H1504 (H1047 and H1239, respectively, Ref. 33). Strains
SEC61-His6 (H1415), sec61-41 (H1417), and sec18-1 (H1641) were created by transformation of the
parental strains H1016 (RSY1293, Ref. 34), H1018 (RSY1295, Ref. 34), and H996 with plasmid pKTH5014. Strain H996 was constructed by integrating plasmid pKTH4660 containing the
HSP150
Yeast cells were grown overnight in synthetic complete medium
containing 2% glucose at 24 °C in shaker flasks until early logarithmic phase. Expression of b5-Nglyc was
induced by shifting the cells to medium containing 2% raffinose or
0.1% glucose. Both conditions resulted in a similar up-regulation of
b5-Nglyc synthesis.
Western Blot Analysis and Endoglycosidase H (Endo H)
Assay--
Cells were mechanically lysed with glass beads in NET
buffer (0.05 M Tris-HCl, pH 8.0, 0.4 M NaCl, 5 mM EDTA, 1% Triton-X-100, 100 µg/ml aprotinin),
containing 2% SDS and 2 mM phenylmethylsulfonyl fluoride
(Sigma). For EndoH treatment, the lysis was performed in non-denaturing
buffer (80 mM CH3COONa, pH 5.2, 2%
Triton-X-100, 0.1 M
Samples, treated or not with Endo H, were analyzed by SDS-PAGE (12%
gels) followed by blotting onto Hybond-CTM extra (Amersham
Biosciences) as previously described (38). b5-Nglyc was detected by ECL (Amersham
Biosciences) using an anti-opsin monoclonal antibody (mAb) (Dr. Paul
Hargrave, University of Florida, Gainesville, FL; Ref. 39) and
anti-mouse horseradish peroxidase-conjugated antibodies (Santa-Cruz
Biotech), diluted 1:2000 and 1:10 000, respectively.
Metabolic Labeling and Immunoprecipitation--
Metabolic
labeling of yeast cells in synthetic complete medium lacking methionine
and cysteine with 20 µCi of [35S]methionine/cysteine/ml
(Amersham Biosciences), cell lysis, and immunoprecipitations were
performed as previously described (38). In pulse-chase experiments,
cycloheximide (CHX) was added in the chase medium at 30 µg/ml. Where
indicated, tunicamycin (TM) from Sigma at 10 µg/ml was added 15 min
prior to labeling and included during the pulse-chase incubation.
b5-Nglyc, carboxypeptidase Y (CPY), or
Hsp150 In Vitro Translation/Translocation of
b5-Nglyc, and ATP Depletion--
In vitro
transcription of b5-Nglyc cDNA from the SP6
promoter, translation of the transcript in the reticulocyte lysate
system (Promega), post-translational incubation of undiluted
translation mix with dog pancreas microsomes (DPMs), and SDS-PAGE
autoradiography analysis of the products were carried out as previously
described (11). To test the effect of lysate proteins on
post-translational glycosylation, translation mixtures were diluted
either in buffer or in lysate as indicated in Fig. 7. The final
concentrations of ions, dithiothreitol, nucleotides (ATP and GTP from
Sigma), phosphocreatine, and creatine kinase (Sigma) in the samples
diluted in buffer were kept at the levels of the initial translation
mix as specified by Promega. The diluted samples were then supplemented with equal amounts of DPMs.
ATP was depleted from lysates either with apyrase or with a
hexokinase/glucose trap. For apyrase treatment, 7 µl of lysate containing in vitro-translated
b5-Nglyc were incubated for 10 min at 30 ° C
with 2.5 µl of a solution containing 3.75 units of apyrase (grade VI;
Sigma) before addition of 0.5 µl of DPM suspension (Promega).
Controls received 2.5 µl of water instead of the apyrase solution.
For depletion with hexokinase (Sigma) and glucose, 4.5-µl aliquots of
lysate containing in vitro-translated b5-Nglyc were supplemented with 0.6 µl of a
solution containing 0.25 M glucose, 0.3 mg/ml CHX, and
varying concentrations of hexokinase (0, 4.2, 21, or 210 µg/ml).
After incubation for 10 min at 30 °C, the samples received 0.6 µl
of DPMs and were further incubated for 1 h at 30 °C. To test
the effect of the hexokinase/glucose trap on translation, 4.5-µl
aliquots of lysate lacking the b5-Nglyc transcript were treated as above with glucose and varying
concentrations of hexokinase (but without CHX), then supplemented with
the synthetic transcript and incubated for 1 h at 30 °C. To
determine the ATP concentration, 1-µl aliquots of
hexokinase/glucose-treated lysates were diluted in ice-cold water and
assayed by the luciferin-luciferase procedure. 20 µl of diluted
lysate samples were mixed with 100 µl of a 20 mg/ml solution of
combined luciferase-luciferin from Sigma. Light emission was recorded
after 20 s with the use of a Lumat LB 9501 luminometer (Berthold
Detection Systems, Pforzheim, Germany). ATP concentrations were
calculated by comparison with a standard curve.
Morphological Methods--
Indirect immunofluorescent staining
of yeast cells was carried out as before (40), using anti-opsin mAbs
(1:100) and/or porin antiserum (1:100), a kind gift from Dr. Harald
Pichler (University of Graz, Austria), and fluorescein-conjugated
anti-mouse (1:100) and/or rhodamine-conjugated anti-rabbit (1:100)
antisera (Santa Cruz Biotechnology). Transmission electron microscopy
was performed as previously described (41).
ER Targeting and N-Glycosylation of b5-Nglyc--
A
cDNA coding for a mammalian cytochrome b5
variant with an opsin tag and a consensus site for
N-glycosylation close to the C terminus (designated
b5-Nglyc; see Ref. 11 and Fig.
1A) was placed under the
glucose-regulated SUC2 promoter and integrated into the
genome of a yeast strain with normal Sec61p function (H1415; see Table
I for yeast strains) and of the
temperature-sensitive mutant sec61-3 (H1399). In the latter
cells, the channel protein Sec61p of the Sec61 translocon is defective
at 37 °C. After induction of expression of
b5-Nglyc by incubation of the cells in
glucose-deficient, raffinose-containing medium for an hour at the
permissive temperature (24 °C), the cells were lysed and subjected
to SDS-PAGE followed by Western blotting and detection of the
recombinant protein with anti-opsin mAb. Parallel samples were digested
with Endo H to remove N-glycans prior to gel
electrophoresis. In the normal (Fig. 1B, lane 1),
as well as in the mutant (lane 3) cells, ~25 and ~20-kDa
proteins were detected in the absence of Endo H treatment, whereas
after digestion only the ~20-kDa protein was detected (lanes
2 and 4). The parental strains lacking the recombinant gene revealed no signal (not shown). Thus, the C-terminal domain of
most of the b5-Nglyc molecules had been
translocated into the ER lumen and glycosylated.
ER localization was confirmed by indirect immunofluorescent staining.
In cells with normal Sec61p (H1415), induced to express b5-Nglyc, staining for the recombinant protein
(Fig. 2, a and d)
was visible in the perinuclear and peripheral regions, which correspond
to the perinuclear and cortical ER, respectively (42). No staining was
present in vacuoles, revealed by differential interference contrast
optics (panel b). Co-staining of the sample of
panel d with anti-porin antibodies resulted in a pattern
typical for mitochondria (panel c), clearly different from
that obtained with the anti-opsin mAbs, confirming that most of the
b5-Nglyc was specifically targeted to the ER as
occurs in mammalian cells (4, 11).
The yeast ER has been shown to proliferate up to 20 pairs of stacked
membranes encircling the nucleus (so-called karmellae) upon
overexpression for 24 h of rat cytochrome
b5 (43). Therefore, we checked by electron
microscopy whether the ER of our b5-Nglyc expressing cells was normal or underwent a similar
proliferation. Comparison of Fig.
3, A and B shows
that after 2 h of induction, within the time window of our
biochemical experiments (panel B), the cortical and
perinuclear components of the ER appeared indistinguishable from
those of uninduced cells (panel A). After 6 h of
induction, some proliferation of the ER appeared to have started
(Fig. 3C), whereas after 22 h membrane stacks similar
to those described by Vergères et al. (43) were
clearly visible (Fig. 3D).
Translocation of the C Terminus of de Novo Synthesized
b5-Nglyc across the ER Membrane of Translocation-deficient
Mutants--
Next we studied whether ER translocation of newly
synthesized b5-Nglyc was blocked when the Sec61
channel was inactivated for signal peptide-driven translocation at
37 °C in the sec61-3 mutant (H1399). Sec61-3
cells were preincubated for 30 min in 2% raffinose to induce
b5-Nglyc expression, either at the restrictive or at the permissive temperature, then labeled with
[35S]met/cys for 5 min, and chased for 20 min. At both
temperatures the major immunoprecipitated form of
b5-Nglyc corresponded to the ~25-kDa
glycosylated form (Fig. 4A,
lanes 1 and 3). When the incubation at the
restrictive temperature was performed in the presence of TM to inhibit
N-glycosylation (Fig. 4A, lane 2),
only the ~20-kDa polypeptide could be immunoprecipitated, confirming that the band at 25 kDa corresponds to the N-glycosylated
form of b5-Nglyc. When this experiment was
repeated in a sec61-41 mutant (strain H1417, Table I),
which is cold-sensitive for translocation (34), similar results,
showing efficient glycosylation of b5-Nglyc, were obtained both at 17 and 24 °C (data not shown). When
b5-Nglyc was expressed in a sbh1
strain (H1520) lacking the Sec61 translocon component Sbh1p, it again
was glycosylated, as well as in a sbh1 sbh2 double deletant
that also lacked Sbh2p, a component of the alternate Ssh1 translocon
complex (data not shown).
The Sec63 complex (Sec62p, Sec63p, Sec71p plus Sec72p) and the lumenal
chaperones Kar2p/BiP and Lhs1p are required for signal peptide-driven
post-translational ER translocation (19, 25, 44, 45). Therefore, we
integrated our b5-Nglyc cDNA under the
SUC2 promoter into the genome of yeast strains harboring
temperature-sensitive mutations in the SEC63, SEC62,
or KAR2 genes and of a strain lacking the LHS1
gene (37). A pulse-chase experiment carried out with a
temperature-sensitive sec63-1 mutant showed that
b5-Nglyc was again translocated equally
efficiently at the restrictive and permissive temperatures (Fig.
4A, lanes 4-6). Similar results were obtained
for the temperature-sensitive kar2-159 (H1424) and lhs1 (H1425) mutants (data not shown).
Inactivation of the Sec61 channel is lethal, whereas inactivation of
proteins required for signal peptide-driven post-translational translocation only, such as Sec63p or Sec62p, can be tolerated (32).
The results on the sec63-1 mutant of Fig. 4A were
confirmed by using constitutive sec63-201 (H1475) and
sec62-101 (H1474) mutants. Again, the mutations did not
prevent translocation of the C terminus of
b5-Nglyc (Fig. 4B, lanes
1-4).
Comparison of Post-translational Translocation of
b5-Nglyc and Carboxypeptidase Y (CPY)--
To study
whether the observed glycosylation of b5-Nglyc
in the translocation-deficient mutants could be explained by leakage of
the mutants, we compared the fate of b5-Nglyc to
that of an internal control, the vacuolar protease CPY, a protein that
is post-translationally translocated in a
Sec61/62/63-dependent manner (32) As demonstrated before
(46) and shown here for reference, in normal cells CPY is rapidly
translocated into the ER, appearing after a 5 min pulse of radioactive
amino acids mainly as a primary N-glycosylated 67-kDa form
with a minute portion present as the cytosolic, unglycosylated, 59-kDa
polypeptide (Fig. 5A,
lane 7). Thereafter, the glycosylated form is transported
via the Golgi, where the glycan extension causes the apparent molecular
mass to increase to 69 kDa (lane 8), to the vacuole
where removal of the pro-peptide results in the mature 61-kDa form
(lane 9).
In the constitutive sec62-101 and sec63-201
mutants, it has previously been demonstrated that CPY accumulates
in the cytosolic form (32). Here, we have compared the fates of
b5-Nglyc and CPY in the conditional
translocation-defective mutants, sec61-3 and
sec63-1. After preincubation and pulse-labeling for 5 min at the restrictive temperature, one half of each lysate was exposed to
anti-opsin mAbs to immunoprecipitate b5-Nglyc
(Fig. 5A), while CPY was immunoprecipitated from the other
half (Fig. 5B). In sec63-1 (lane 2)
and sec61-3 (lane 3) cells, which were induced
for b5-Nglyc expression, CPY occurred mostly as
the untranslocated 59-kDa form, as in a sec63-1 strain
lacking the recombinant gene (lane 1). Similar results were
obtained for these same strains under conditions where
b5-Nglyc expression was not induced (lanes
4-6). In contrast, b5-Nglyc was
glycosylated in sec63-1 (Fig. 5B, lane
2) and sec61-3 (lane 3) cells. No signal
was obtained in the sec63-1 strain lacking the recombinant
gene (lane 1) or in any of the three strains when induction
in low glucose was omitted (lanes 4-6). We conclude that
under conditions in which pre-pro-CPY remained in the cytosol because
of a non-functional translocon channel or Sec63p, the C terminus of
b5-Nglyc was rapidly translocated into the
ER.
Kinetics of Translocation of b5-Nglyc and
HSP
Translocation of Hsp150 ATP and Cytosolic Protein Requirement for b5-Nglyc
Translocation into Mammalian Microsomes--
We further investigated
the energy requirements for post-translational translocation of
b5-Nglyc in the mammalian reticulocyte lysate
cell-free system supplemented with dog pancreas microsomes (DPMs). As
shown in Fig. 7A, lane
1, when in vitro-translated b5-Nglyc was incubated with DPMs about half of
the radioactively labeled protein was glycosylated, as indicated by the
presence of the ~25-kDa band, in agreement with our previous results
(4, 11). However, if the sample was exposed to apyrase to hydrolyze ATP
before incubation with membranes, post-translational glycosylation did
not occur (Fig. 7A, lane 2). To more precisely
define the ATP requirement, we used the hexokinase/glucose trap as an
alternative procedure for energy depletion. Samples were preincubated
for 10 min with 30 mM glucose and varying concentrations of
hexokinase to obtain different degrees of ATP depletion. ATP levels
obtained after the preincubation were monitored directly with the
luciferin-luciferase assay and are reported below the lanes of Fig.
7B. As shown in panel B, lanes 1-4,
if hexokinase/glucose treatment was carried out before
translation, production of b5-Nglyc was already
reduced with the lowest concentration of hexokinase (0.4 µg/ml) and
was completely abolished at higher concentrations. In contrast, when the energy-depleting treatment was carried out after translation but
before addition of DPMs, glycosylation occurred at all concentrations of hexokinase (Fig. 7B, lanes 5-8). Significant
glycosylation, albeit with somewhat reduced efficiency, also occurred
with the highest amount of enzyme (25 µg/ml, lane 8),
resulting in an ATP concentration as low as 0.2 µM at the
start of the post-translational incubation with DPMs. This ATP
concentration was totally non-permissive for protein synthesis (Fig.
7B, lanes 1-4) and was ~4 orders of magnitude lower than that of intracellular ATP under physiological conditions.
It has been reported that binding of another TA protein, VAMP-1B, to
mitochondrial outer membranes can occur without ATP if cytosolic
chaperones are absent (48). We tested a possible role of chaperones in
our system by comparing the glycosylation of b5-Nglyc obtained in an undiluted translated
sample (Fig. 7C, lane 1) with that obtained after
dilution of the sample in lysate (Fig. 7C, lanes
2 and 3) or in buffer (lanes 4 and
5). The concentrations of ions, nucleotides, and components
of the energy-regenerating system (creatine phosphate and creatine
kinase) were held constant, and all samples received the same amount of
DPMs. As shown in Fig. 7C, both with a 1:5 and a 1:10
dilution of the lysate in buffer (lanes 4 and 5),
glycosylation of b5-Nglyc was severely impaired,
indicating that its delivery to ER membranes in a
translocation-competent form requires at least one cytosolic protein.
In recent years it has become clear that TA proteins can
translocate their C-terminal residues across the ER membrane. However, the mechanism of membrane penetration has remained unsolved. The transmembrane domains (TMD) of TA proteins resemble classic
signal-anchor sequences of type II monotopic proteins, with the
difference that they emerge from the ribosome only upon termination of
translation and thus must interact with the target membrane
post-translationally. Because the Sec61 translocon can operate in both
co- and post-translational modes (19), it seemed plausible that it
could be involved in translocation of TA protein tails as well, as
proposed by Tyedmers et al. (16) and Wattenberg and Isenmann
(17). This hypothesis was supported by the observations that (i) TA
proteins localized to compartments of the secretory pathway must first
insert into the ER (5-8)and (ii) glycosylation sites at the C terminus
of TA proteins are good substrates for oligosaccharyltransferase (6,
9-11), an enzyme complex that is closely associated with the
translocon (49). The finding that another translocon-associated enzyme,
signal peptidase, can cleave a modified TA protein further strengthened
this idea (18). The translocon hypothesis was, however, not supported
by data obtained from in vitro binding assays
that showed that carbonate-resistant binding of synaptobrevin to
mammalian or yeast microsomes can occur in the absence of Sec61p function (6, 13). In these in vitro experiments, however, no
evidence for translocation of the C terminus of synaptobrevin was presented.
In the present study we have employed a rigorous assay for
translocation, based on utilization of an N-glycosylation
consensus sequence engineered to the C terminus of mammalian cytochrome b5 (b5-Nglyc; see scheme
in Fig. 1). We have taken advantage of the yeast system to assess in
ultrastructurally normal living cells a possible role of the Sec61
translocation machinery in the translocation of the C terminus of
b5-Nglyc. The large number of mutant yeast
strains utilized here included conditional and constitutive mutants or
knock-out strains defective in the translocon protein Sec61p
(sec61-3 and sec61-41) or accessory proteins
Sbh1p (sbh1) and Sec62p (sec62-101), in Sbh2p
(sbh2, a component of an alternate yeast translocon), in the
ER lumenal chaperones Bip/Kar2p (kar2-159) and Lhs1p
(lhs1), and in the co-chaperone Sec63p (sec63-1 and sec63-202). None of these mutations or deletions had
detectable effects on the translocation of the tail of
b5-Nglyc, which was rapidly glycosylated in all
the strains at permissive and restrictive temperatures, under
conditions in which translocation of CPY was abolished. CPY harbors a
signal peptide driving post-translational translocation (32) and was
used as an internal control to monitor eventual leakage of the mutants.
The most likely interpretation of these results is that translocation
of TA protein tails occurs by a mechanism not involving the Sec61 (or
the alternate Ssh1p) translocon machinery. However, another possibility
is that the tails are inserted into the ER membrane via the Sec61 pore
but using a novel function of Sec61p that is not compromised by the
sec61-3 and sec61-41 mutations. Indeed, Sec61
appears to be a multifunctional protein. So far, three activities have
been assigned to it: the two types of signal peptide-dependent ER entry and dislocation by which
short-lived or misfolded proteins are translocated from the ER lumen
back to the cytosol for ubiquitinylation and proteasomal degradation (20, 50). The proteins to be dislocated differ markedly from those
entering the ER lumen because they have lost the signal peptide and
undergone co- and post-translational modifications and folding.
Accordingly, distinct TMDs of Sec61p appear to operate in ER entry and
dislocation. Deletion analysis showed that TMD2 has specific functions
in post-translational translocation, whereas TMD3 is needed for
efficient dislocation (51). It is interesting to note that the
sec61-41 mutation (V134I), which did not affect ER
insertion of b5-Nglyc, resides in TMD3. It
blocks entry at low temperature and dislocation at all temperatures
(34). Thus, should the Sec61 complex have a role in TA protein
translocation, it must be independent from the known entry and
dislocation functions.
In addition to investigating whether translocon components are required
for TA protein insertion, in the present study we have also analyzed
the energy requirements of the process. On this question, previous
studies with in vitro binding assays have yielded diverging
results. Whereas there is general agreement that synaptobrevin (VAMP)
requires ATP for membrane association (6, 12), other TA proteins seem
to have either lower energy requirements (e.g.
bcl-2 and Nyv1p) or none at all (cytochrome b5) (12, 13). Moreover, in the case of the
mitochondrial outer membrane isoform of synaptobrevin, VAMP-1B, the
need for energy was found to be related to chaperone function. In the
absence of cytosolic proteins, VAMP-1B could bind tightly to added
mitochondria in the absence of ATP (48).
We first examined b5-Nglyc translocation in
yeast cells grown at high density, a condition that results in partial
energy depletion (38, 47), and found that its glycosylation was not affected, whereas translocation of Escherichia coli
To better define the energy requirements for translocation of the tail
of b5-Nglyc, we turned to a mammalian in
vitro translation-translocation system. N-glycosylation
in cell-free systems is based on the utilization of preassembled
oligosaccharyl-dolichol molecules and thus does not in itself require
energy. Therefore, this post-translational modification could be used
to report on translocation of the tail of
b5-Nglyc, also under conditions of energy
depletion. Complete depletion of ATP with apyrase resulted in failure
of translocation of b5-Nglyc into dog pancreas
microsomes. This is in agreement with what has been reported for a
synaptobrevin variant carrying an N-glycosylation consensus
close to the C terminus (6) but contrasts with the widely believed
notion that cytochrome b5 does not require
energy for membrane insertion (12) and illustrates how in
vitro binding data must be interpreted with caution. When ATP
depletion was achieved in a more controlled fashion using a
hexokinase/glucose trap, we observed that translocation of
b5-Nglyc occurred even at extremely low
concentrations of ATP, in agreement with the results we obtained
in vivo in yeast. We also found that dilution of the lysate
severely impaired glycosylation of b5-Nglyc, demonstrating for the first time the involvement of cytosolic proteins
in b5 membrane insertion. Taken together, the
in vitro experiments suggest that cytosolic chaperone(s) may
be needed for delivery of b5 and other TA
proteins to the ER in a translocation-competent form and that the ATP
requirement may be related to chaperone function. However, we cannot
exclude that ATP also has a direct function in translocation.
In conclusion, our results based on in vivo glycosylation of
the C terminus of a cytochrome b5 construct in
yeast mutants, combined with in vitro experiments in the
mammalian system, demonstrate that post-translational translocation of
a TA protein can occur by a mechanism distinct from that of signal
peptide-dependent translocation, characterized by fast
kinetics, low energy requirements, and the participation of at least
one cytosolic protein. The obvious question now is what molecules are
involved in this alternative translocation process, whether only
lipids, protein(s), or both. TA proteins are targeted specifically from
the cytosol to ER or mitochondrial outer membranes, the specificity
depending on residues adjacent to the TMD as well as on features of the
TMD itself (4, 52-54). This specificity argues in favor of proteins
playing a role in the targeting process. Consistently, a proteinaceous
receptor has been implicated in the binding of synaptobrevin and Nyv1p to microsomal membranes (6, 12, 13). However, once a TA protein is
delivered to its target membrane, it is conceivable that its
translocation could occur directly across the lipid bilayer without the
aid of proteins. A combined effort, involving yeast genetics and
biochemistry, will hopefully lead to the elucidation of the molecular
details of this novel translocation process.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and Sec61
in mammals). In conjunction with additional accessory polypeptides, the
Sec61 complex is able to provide all translocation functions of signal
peptide-containing proteins at the ER membrane. It serves as the
channel for both co- and post-translational translocation as well as
for retrotranslocation back to the cytosol of short-lived or malfolded
proteins destined for proteasomal degradation (see Refs. 19 and 20). In
the case of post-translational translocation, more common in yeast than
mammalian cells, the Sec61 channel must interact with Sec62p, Sec63p,
Sec71p, and Sec72p and cooperate with the lumenal Hsp70 chaperone
BiP/Kar2p (21-25). Mammalian orthologues of Sec62p/63p are known (16,
26) and may also be involved in post-translational transport.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-
-lactamase gene (35) into the LEU2
locus of strain H4 (mBy12-6D, Ref. 36). Strain H823 was created by
disrupting the LHS1 gene with a loxP-kanMX-loxP cassette
from strain H486 (37).
-mercaptoethanol, and 2 mM phenylmethylsulfonyl fluoride). The lysates were boiled
for 10 min and treated with endoglycosidase H (Endo H, 20 milli-units) for 4 h at 37 °C.
-
lactamase (38) were immunoprecipitated with anti-opsin
mAbs (1:400), with anti-CPY, or with anti-
-lactamase antisera (both
1:100), respectively. SDS-PAGE of b5-Nglyc and Hsp150
-
-lactamase samples was in 12% gels and of CPY in 8% gels.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (31K):
[in a new window]
Fig. 1.
Expression and glycosylation of
b5-Nglyc in yeast. A,
schematic representation of b5-Nglyc. The
cytosolic heme-binding domain (white box), the transmembrane
domain (TMD, gray box), and the lumenal C-terminal regions
of wild type b5 (a) and
b5-Nglyc (b) are shown. The tag
corresponding to the first 19 amino acids of bovine opsin is
underlined and the N-glycosylation site is
indicated by a star. The numbers indicate the
position of the amino acid residues. B, Western blot
analysis of strains transformed with b5-Nglyc
cDNA. Control cells (H1415) and a sec61-3 mutant
(H1399) were incubated for 1 h at 24 °C in 2% raffinose to
induce expression of b5-Nglyc and lysed. One
half of the samples was subjected directly to SDS-PAGE (lanes
1 and 3) and the other after Endo H digestion
(lanes 2 and 4). Western blotting was performed
with anti-opsin mAbs. 25- and 20-kDa forms of
b5-Nglyc are indicated on the
right.
Yeast strains constructed for this study
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Fig. 2.
Immunofluorescent staining of
b5-Nglyc-expressing cells. Control
cells (H1415) were induced in 2% raffinose for 3 h at 24 °C
and stained with anti-opsin mAbs (a) to visualize the
recombinant protein or doubly immunostained with anti-opsin mAbs and
antiporin antibodies to compare the distribution of
b5-Nglyc with that of mitochondria (c
and d). Panels c and d show the same
field of cells viewed with the rhodamine filter for the anti-porin
antibodies and under the fluorescein isothiocyanate filter for
the anti-opsin antibodies, respectively. Arrows indicate the
cortical and perinuclear ER (a and d) and
mitochondria (c). In panel b, the differential
interference contrast image of the cells of panel a is
shown.
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[in a new window]
Fig. 3.
Ultrastructure of the ER in cells induced or
not induced to express b5-Nglyc.
Yeast cells (H1415) were fixed and processed for transmission electron
microscopy before induction (A) or after 2 (B), 6 (C), or 22 h (D) of induction of
b5-Nglyc expression with 2% raffinose at
30 °C. Panel D shows two different images illustrating
formation of karmellae (arrowheads). ER,
endoplasmic reticulum. N, nucleus. V, vacuole.
Bar, 0.5 m.
View larger version (69K):
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Fig. 4.
ER translocation of the C terminus of
b5-Nglyc in translocation mutants.
A, the temperature-sensitive mutants sec61-3
(H1399) and sec63-1 (H1404) were preincubated for 30 min in
2% raffinose, labeled with [35S]methionine/cysteine for
5 min, and chased in the presence of CHX for 20 min. Preincubation,
pulse, and chase were carried out at the indicated temperatures.
B, the constitutive mutants sec62-101 (H1474)
and sec63-201 (H1475) were treated similarly as above,
except that the temperature was 30 °C. In both panels, tunicamycin
(TM) was added 15 min before labeling, as indicated. Cells
were lysed and immunoprecipitated with anti-opsin mAbs prior to
SDS-PAGE analysis. The ~25- and ~20-kDa forms of
b5-Nglyc are indicated.
View larger version (46K):
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Fig. 5.
Inhibition of translocation of CPY in yeast
mutants that allow translocation of
b5-Nglyc. Sec63-1 (H1404)
and sec61-3 (H1399) strains containing the
b5-Nglyc gene and a Sec63-1 (H257)
strain not transformed with b5-Nglyc were
preincubated for 15 min in 0.1% glucose (lanes 1-3 of both
panels) or 2% glucose (lanes 4-9 of panel A and
4-6 of panel B), then labeled with
[35S]met/cys for 5 min at 37 °C. Lanes
1-6: samples were divided in two and immunoprecipitated with
anti-CPY antiserum (panel A) or anti-opsin mAbs (panel
B). Lanes 7-9 of panel A: after
pulse-labeling, samples were chased for the indicated times and then
immunoprecipitated with anti-CPY antiserum. The cytosolic (59 kDa), ER
(67 kDa), Golgi (69 kDa), and vacuolar (61 kDa) forms of CPY
(A) and the 25-and 20-kDa forms of
b5-Nglyc are indicated (B).
-
-Lactamase--
We have previously shown that the
-lactamase portion of a fusion protein, Hsp150
-
-lactamase,
folds in the cytosol to a native-like conformation and is then
translocated into the ER. Penetration across the Sec61 translocon
requires unfolding, and this apparently takes time because the
translocation rate is slow, occurring with a half time of about 5 min
(38, 47) Because the catalytic N-terminal portions of TA
proteins also fold in the cytosol, we compared the translocation
kinetics of Hsp150
-
-lactamase and
b5-Nglyc. Both proteins were expressed in a
sec18-1 mutant where translocated proteins are prevented
from escaping the pre-Golgi compartment at 37 °C, allowing facile
comparison of the cytosolic and ER forms. After induction of
b5-Nglyc expression at the restrictive temperature and pulse-labeling with [35S]met/cys,
immunoprecipitation of half of the cell lysates with
-lactamase
antiserum showed that most of the Hsp150
-
-lactamase was in its
non-glycosylated cytosolic form of 66 kDa and some in the primary
glycosylated ER form of 110 kDa (Fig.
6A, lane 1). After
a 10-min chase, most of the protein was in the ER form (lane
2). In contrast, and in agreement with the results of Fig. 5,
immunoprecipitation of the other half of the lysates with anti-opsin mAb showed that almost all of the b5-Nglyc was
already in the ER form at the end of the pulse (Fig. 6A,
lanes 3 and 4). The rapid rate of glycosylation
of b5-Nglyc in comparison to
Hsp150
-
-lactamase suggests that its translocation is not limited
by a folding/unfolding process.
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Fig. 6.
Translocation kinetics of
b5-Nglyc in yeast cells grown to different
densities. The sec18-1 mutant containing both the
Hsp150 -
-lactamase and the b5-Nglyc genes
(H1641) was preincubated at 37 °C in 2% raffinose for 20 min,
35S-labeled for 5 min, and chased for 10 min at normal cell
density (5 × 107 cells/ml (A) or high cell
density (5 × 108 cells/ml (B). The cell
lysates were divided in two for immunoprecipitation with
-lactamase
antiserum (
-bla) or anti-opsin mAb (
-opsin)
as indicated. In panel B, lanes 1-2, twice as
much radioactive label was used as in the other experiments. The
cytosolic and ER forms of b5-Nglyc (20 and
25 kDa, respectively) and Hsp150
-
-lactamase (66 and 110 kDa,
respectively) are indicated.
-
-lactamase is slowed under high cell
density conditions, apparently because of glucose limitation that
results in reduction of the ATP pool (38). To compare the energy
requirements of b5-Nglyc and
Hsp150
-
-lactamase, the above pulse-chase experiment was
repeated in high cell density conditions. After the pulse, only the
cytosolic form of Hsp150
-
-lactamase could be detected (Fig.
6B, lane 1), and after a 10-min chase most of the
protein still was in the cytosolic form (lane 2). In
contrast, growth under high cell density conditions had no effect on the glycosylation of b5-Nglyc
(lanes 3 and 4). Thus, the TA protein had lower
energy requirements for translocation than signal peptide-containing
Hsp150
-
-lactamase.
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Fig. 7.
Effect of ATP and cytosolic protein depletion
on post-translational glycosylation of
b5-Nglyc in vitro.
A, depletion with apyrase. A synthetic mRNA coding for
b5-Nglyc was in vitro-translated in
the rabbit reticulocyte lysate system in the presence of
[35S]methionine. Aliquots of the lysate were then
incubated in the presence or absence of apyrase as indicated (see under
"Materials and Methods") before addition of DPMs and analysis by
SDS-PAGE autoradiography. B, lanes 1-4, effect
of glucose/hexokinase treatment on translation. Aliquots of
reticulocyte lysate were pretreated with a mixture of glucose (final
concentration 30 mM) and increasing concentrations of
hexokinase (0, 0.5, 2.5, and 25 µg/ml in lanes 1-4,
respectively) before incubation with synthetic
b5-Nglyc mRNA and analysis by SDS-PAGE
autoradiography. Lanes 5-9, effect of glucose/hexokinase
treatment on glycosylation. Aliquots of rabbit reticulocyte lysate
containing in vitro-translated
b5-Nglyc were incubated for 10 min at 30 °C
with glucose as above, hexokinase (0, 0.5, 2.4, and 24, and 0 µg/ml
in lanes 5-9, respectively) and CHX (final concentration 30 µg/ml) and then incubated with (lanes 5-8) or without
(lane 9) DPMs for 1 h at 30 °C before analysis by
SDS-PAGE autoradiography. ATP was assayed in the samples immediately
after the glucose/hexokinase treatment. The values obtained are given
below the lanes. See under "Materials and Methods" for
experimental details. C, effect of lysate dilution on
glycosylation of b5-Nglyc. 4.5 µl of an
undiluted translation mix (lane 1) or of a 1:5 (lanes
2 and 4) or 1:10 dilution (lanes 3 and
5) of the mix in lysate (lanes 2 and
3) or buffer (lanes 4 and 5) were
supplemented with 0.5 µl of DPMs and incubated further for 90 min at
30 °C. In all panels, the ~20- and ~25-kDa unglycosylated and
glycosylated polypeptides are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-lactamase, expressed in S. cerevisiae as a fusion
protein with a post-translationally operating signal peptide, was
drastically slowed. We have previously demonstrated that the
-lactamase fusion protein folds to a native-like protease-resistant
and catalytically active conformation in the cytosol, is then unfolded,
and is thereafter translocated (38, 47). Its slow and energy-sensitive
rate of translocation may be because of the time required for the
folding/unfolding process. The catalytic domain of
b5 also folds in the cytosol, but the rapid rate
of glycosylation observed here suggests that the conformation of the
N-terminal domain does not influence the translocation of the
C-terminal region.
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ACKNOWLEDGEMENTS |
---|
In addition to the colleagues who kindly donated antibodies and yeast strains, we thank Aldo Ceriotti for helpful suggestions and Netta Fatal and Eija Paunola for constructing strains H823 and H996.
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FOOTNOTES |
---|
* This work was supported in part by Associazione Italiana Ricerca sul Cancro (AIRC), Telethon (Grant E734), and the Ministero per la Istruzione, Università e Ricerca (M.I.U.R.- COFIN 2001).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.
§ Supported by the University of Helsinki and the Academy of Finland (Grant 53607).
Present address: Consiglio Nazionale delle Ricerche Istituto
di Biologia e Biotecnologia Agraria, Via Bassini 15-20133 Milano, Italy.
To whom correspondence should be addressed. Tel.:
39-02-50316971; Fax: 39-02-7490574; E-mail: nica@csfic.mi.cnr.it.
§§ A Biocentrum Helsinki fellow.
Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M210253200
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ABBREVIATIONS |
---|
The abbreviations used are: TA, tail-anchored; CHX, cycloheximide; CPY, carboxypeptidase Y; DPMs, dog pancreas microsomes; Endo H, endoglycosidase H; ER, endoplasmic reticulum; mAb, monoclonal antibody; TM, tunicamycin; TMD, transmembrane domain.
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REFERENCES |
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