From the School of Biology, Centre for Biomolecular Sciences,
Biomolecular Sciences Building, University of St. Andrews, North Haugh,
St. Andrews KY16 9ST, United Kingdom and the School of
Cell and Molecular Biosciences, The Medical School, University of
Newcastle, Framlington Place,
Newcastle upon Tyne NE2 4HH, United Kingdom
Received for publication, November 15, 2002, and in revised form, January 7, 2003
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ABSTRACT |
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During co-translational protein import into the
endoplasmic reticulum ribosomes are docked onto the translocon. This
prevents inappropriate exposure of nascent chains to the cytosol and,
conversely, cytosolic factors from gaining access to the nascent chain.
We exploited this property of co-translational translocation to examine the mechanism of polypeptide cleavage by the 2A peptide of the foot-and-mouth disease virus. We find that the scission reaction is
unaffected by placing 2A into a co-translationally targeted protein.
Moreover, the portion of the polypeptide C-terminal to the cleavage
site remains in the cytosol unless it contains its own signal sequence.
The pattern of cleavage is consistent with the proposal that the
2A-mediated cleavage reaction occurs within the ribosome itself. In
addition, our data indicate that the ribosome-translocon complex
detects the break in the nascent chain and prevents any downstream
protein lacking a signal sequence from gaining access to the
endoplasmic reticulum.
Positive-strand RNA viruses typically encode polyproteins that are
cleaved by viral or host-encoded proteinases (proteolytic processing)
to produce mature, individual proteins (reviewed in Refs. 1 and 2).
Alternatively proteins may be generated by translational effects such
as ribosomal frameshifting or read-through of "leaky" stop codons.
Such programmed alterations of translation are not virus-specific but
widespread (although rare) mechanisms of gene expression (reviewed in
Refs. 3 and 4). In foot-and-mouth disease virus
(FMDV)1 and some other
picornaviruses the oligopeptide (~20 amino acid) 2A region of the
polyprotein mediates cleavage at its own C terminus to release it from
the 2B region. 2A is also active when placed between reporter proteins
and, therefore, cleavage requires no viral (proteinase) sequences
outside this short peptide (5). Similarly, no host proteinases are
known that cleave the 2A/2B site.
Scission of 2A-containing polyproteins requires the correct protein
rather than mRNA sequence (6). However, synthetic peptides containing 2A and "2A-like" sequences from other viruses do not autoproteolyse (7). Furthermore, on translation in vitro the portion of a polyprotein N-terminal to 2A typically accumulates in
excess over the C-terminal portion. This imbalance is not because of
protein degradation or nonspecific transcription/translation termination (6, 8). Modeling of 2A and 2A-like sequences indicates that
the majority of each peptide can form an amphipathic helix whereas the
amino acids immediately preceding the "cleavage" site (-NPG Although published data support the proposal that 2A acts within the
ribosome no direct evidence has been provided for this. Intraribosomal
cleavage would not require cytosolic factors such as proteinases, and
demonstration of this requires translation of 2A-containing proteins in
a situation where cytosolic factors have no access to the nascent
chain. Such "screening" occurs during co-translational
translocation into the endoplasmic reticulum (ER). Here the nascent
chain is shielded from cytosolic factors by first the ribosome, then
the translocation apparatus, before being partitioned into the ER
lumen. Establishment of co-translational translocation requires the
signal recognition particle (SRP; reviewed in Refs. 9 and 10). SRP
binds cis-acting hydrophobic signal sequences at the N
terminus of nascent ER-targeted proteins as they emerge from the
ribosome and concomitantly slows translation by the ribosome, a
phenomenon termed elongation arrest (11-13). This ensures that
ribosome-nascent chain complexes targeted to the translocon by SRP
arrive with a short length of cytosolically exposed nascent chain. Once
the ribosome-translocon junction is established the ribosomal nascent
chain exit site lies directly over the translocon (14-16). This
junction is sufficiently tight to protect the nascent chain from
externally added proteases (17, 18) and even excludes small ions (19,
20). Thus comparing the translation products of 2A-containing proteins
with and without SRP-dependent signal sequences will reveal
if 2A functions without the influence of cytosolic factors.
The translocon has been proposed to have a signal sequence recognition
function independent from that of targeting factors such as SRP (21,
22). Co-translational translocation of a 2A-containing polypeptide will
result in amino acids C-terminal to 2A being presented to the
translocon immediately after they have emerged from the ribosome.
Therefore if 2A is active in a co-translationally targeted protein we
reasoned that we could use this to ask two interdependent questions.
First, where does cleavage take place? Second, can the translocon
discern the presence or absence of a signal sequence on a nascent chain
presented to it directly by the ribosome and in vivo? If
cleavage does not take place inside the ribosome the portion of the
protein downstream of 2A will arrive in the lumen of the ER. However,
if the nascent chain cleaves within the ribosome a gap will occur in
the polypeptide. The translocon may then "detect" this
discontinuity in the nascent chain as it does the normal termination of
translation, closing, and excluding the downstream protein from the ER.
In contrast addition of a signal sequence to the N terminus of protein
downstream of the 2A site and expression in vivo should
result in reopening of the translocon and translocation of this protein
into the ER.
We chose the yeast Saccharomyces cerevisiae for these
studies. We demonstrate that the FMDV 2A sequence is functional in
yeast, and that targeting through the SRP-dependent
co-translational translocation pathway does not impair the cleavage
reaction. In addition the released downstream product remains in the
cytosol unless it contains its own signal sequence. Thus nascent chain cleavage takes place before the C-terminal portion of the protein initiates translocation and therefore within the ribosome. These data
provide significant support for the proposal that 2A modifies the
activity of the ribosome to promote scission of the nascent chain (6,
7) and are consistent with the notion that the translocon itself
examines nascent chains for the presence of signal sequences.
Strains, Constructs, and General Methods--
Yeast strains were
JDY6 (MATa/
Mouse monoclonal anti-GFP were from Roche Molecular Biochemicals, sheep
polyclonal anti-2A antibodies were raised against a peptide
corresponding to the FMDV 2A sequence (QLLNFDLLKLAGDVESNPG). Anti-CPY,
Kar2p, and Pho8p antibodies were as previously described (25).
In Vitro Transcription-Translation--
Coupled
transcription/translation reactions were performed as per the
manufacturer's instructions (Promega). Briefly, rabbit reticulocyte
lysates (10 µl) were mixed with [35S]methionine (10 µCi; Amersham Biosciences) and 0.1 µg of unrestricted plasmid DNA
and incubated at 30 °C for 90 min.
Microscopy--
For visualization of GFP cultures were
concentrated to 5-10 A600/ml, mixed 1:1 with
molten 0.8% (w/v) low melting point agarose, and spotted onto slides.
Vacuolar membranes were visualized with FM4-64 (Molecular Probes Inc.)
(28). Cells were prepared for immunofluorescence (29) using anti-GFP
and anti-Kar2p antibodies at 1:50 and 1:10,000 dilution, respectively.
Alexa 488 and 594 dye-coupled fluorescent secondary antibodies
(Molecular Probes Inc.) were used at 1:200. Live and fixed cells were
viewed and images were captured using a Zeiss Axiovert 200 microscope
equipped with Plan-Apochromat ×100 1.4NA DIC objective, Zeiss Axiocam
monochrome camera, and Zeiss Axiovision software using Zeiss filter
sets 10 (GFP and Alexa 488) and 31 (FM4-64 and Alexa 594). Phase images were collected in the 4,6-diamidino-2-phenylindole channel.
Cell Labeling and Fractionation--
Cell labeling with
[35S]LPromix (Amersham Biosciences),
preparation of non-native extracts and immunoprecipitation were as
described (25). Quantification was carried out using a Fuji BAS1500
PhosphorImager and TINA software (Raytest). To allow comparison between
cleaved and uncleaved species, values obtained were divided by the
number of labeled amino acids in each. Native extracts were prepared by
breaking cells using zirconium beads (Bio-Spec Products) in a ribolyzer
(Hybaid) with two pulses on setting 5.5 for 20 s with 100 A600 units of cells in 1 ml of 20 mM
HEPES·KOH, pH 7.4, 1 mM EDTA, 0.8 M sorbitol.
The lysate was spun at 500 × g for 5 min to remove
nonbroken cells and at 100,000 × g for 1 h in a Beckman SW50.1 rotor to produce the cytosol fraction (supernatant). The
pellet of the second spin was resuspended in 1 ml of 20 mM HEPES·KOH, pH 7.4, 1 mM EDTA, 150 mM KOAc,
2.1 M sucrose, overlaid with 1 ml of the same buffer at 1.9 M sucrose and 3 ml without sucrose and spun for 5 h at
190,000 × g in a Beckman SW50.1 rotor yielding a
single interface/membrane band. Cytosol and membrane fractions were
adjusted to 15% (w/v) trichloroacetic acid, proteins were recovered by
centrifugation in a microcentrifuge and washed in acetone before
electrophoresis on SDS-PAGE gels.
The FMDV 2A Sequence Functions during Co-translational
Translocation--
Two targeting pathways to the ER operate
side-by-side in yeast, the SRP-dependent co-translational
route and an SRP-independent post-translational route (25, 30). The
hydrophobicity of the signal sequence determines which route is used
and changing the signal sequence of a protein can target it into a
different pathway. To examine whether 2A functions in yeast, and
further during co-translational translocation, we tested a series of
constructs (Fig. 1A) each encoding the same core artificial polyprotein consisting pro-
In vitro translation reactions using rabbit reticulocyte
lysate confirmed that 2A was active in the context of the new
artificial polyproteins (Fig. 1B). Similar to GUS-2A-GFP
(lane 1) most of the translation products were of sizes
corresponding to cleavage products
(ss
Next we examined the proteins produced from the constructs in
vivo in yeast. As the N-terminal portions of
DN
To confirm that 2A is functional when shielded from the cytosol it was
necessary to demonstrate that DN
To verify that polyproteins were targeted by the expected pathways,
DN Protein Sequences following 2A Are Excluded from the ER
Lumen--
As discussed above if a nascent 2A-containing polypeptide
"cleaves" within the ribosome an expectation might be that protein C-terminal to the cleavage site remains in the cytosol. We therefore examined the localization of the GFP portion of
DN
A caveat to the above experiment is that a proportion of the
DN
Taken together the results of these experiments confirm that cytosolic
fluorescence in cells expressing DN 2A Is Active in a Pho8-2A-GFP Fusion--
We sought to extend
examination of co-translationally targeted 2A-containing proteins.
Although SRP-mediated elongation arrest slows translation and thus
reduces the amount of each nascent chain exposed to the cytosol,
translation and translocation are not immediately coupled for all
individual polypeptides targeted by SRP in vivo. This
stochastic aspect to the coupling of targeting and translocation was
revealed in experiments in which ubiquitin was inserted into
proteins targeted to the ER by SRP, the "ubiquitin translocation
assay" (13, 37). These were translocated into the ER intact if
ubiquitin was >30 amino acids from the signal sequence. However, if
ubiquitin was closer than this, a proportion of the proteins were
proteolytically processed by ubiquitin-dependent proteases
C-terminal to ubiquitin, indicating cytosolic exposure of the
ubiquitin. The signal sequence to 2A distance in
DN
We therefore fused 2A-GFP and 2A*-GFP (in these cases
lacking a C-terminal HDEL motif) to the 566-amino acid vacuolar type II
membrane protein Pho8 (Fig.
5A). Examination of cells
expressing Pho8-2A-GFP revealed, as with DN A Signal Sequence after 2A Is Recognized Allowing Translocation of
the Released C-terminal Protein--
A prediction of the "gating"
model for translocon function is that if a substrate is presented to it
directly by the ribosome it should recognize signal sequences as well
as prevent nonsignal sequence containing proteins from accessing the
ER-lumen. We modified two of our initial substrates
(DN The 2A sequence of FMDV directs production of separate proteins
from one polyprotein open reading frame in a variety of higher eukaryotic and plant systems. As such it has great potential as a
biotechnological tool, reducing the need for multiple vectors (e.g. Refs. 39-42). Previously the activity of FMDV 2A had
not been examined in yeast. We found that it is active in
vivo in yeast (Fig. 2). Thus, whatever features of higher
eukaryotic cells allow 2A to promote scission of the polypeptide chain
at the 2A C terminus are conserved to this "simpler" organism. This
opens up the possibility of examining, in a genetically tractable
system, how 2A functions and what trans-acting factors are
required for or influence the cleavage reaction. A similar
approach has been successfully applied to analysis of ribosomal
frameshifting (43), indicating that such examination of alterations to
normal ribosome function in yeast is feasible.
The proportion of 2A-containing proteins that were cleaved in yeast was
similar to that seen previously in in vitro translation reactions, and the 2A-dependent reaction was unaffected by
sequestering the nascent chain from cytosolic factors. As discussed
above although SRP-dependent targeting functions similarly
in yeast to higher eukaryotes (13, 44), translation and translocation
are not efficiently coupled at short nascent chain lengths (37). Both SRP-dependent substrates used in our analysis are of
sufficient length to expect tight coupling between targeting and
translocation for the great majority of individual protein chains. Thus
if cytosolic factors were required for 2A-dependent
cleavage, a significant reduction of the proportion of these proteins
that were cleaved would have been expected. Instead the efficiency of
cleavage was maintained when the protein containing it was
co-translationally translocated and we conclude that extraribosomal
cytosolic factors such as proteinases are not necessary for the 2A reaction.
Determination of the fate of the separated C-terminal (GFP) portions of
2A-containing polyproteins yielded information both on (i) where
cleavage took place and (ii) the gating function of the translocon.
Even when polyproteins were co-translationally translocated into the
ER, GFP remained cytosolic if it lacked a signal sequence. This
exclusion was, as far as we could determine from fractionation
experiments, quantitative and our view of how this occurs is shown in
Fig. 6. For GFP to be excluded from the ER the cleavage reaction must have taken place before the N terminus of
GFP was translocated and therefore our data demonstrate that the
proposal that the break in the nascent chain is generated within the
ribosome is correct (6, 7). We suggest that the N terminus of GFP is
treated as a new translocation substrate and is "surveyed" by the
translocon for the presence of a signal sequence. Without a signal
sequence the translocon remains closed and the ribosome-translocon
complex dissociates. When a signal sequence is added to the N terminus
of GFP (substrates DN
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
P-)
form a tight turn (7). A co-translational model for the cleavage
reaction has been proposed (6, 7) in which the conformation of 2A
places strain on the peptidyltransferase center of the ribosome,
re-positioning the peptidyl(2A)-tRNA ester linkage. This steric effect
prohibits nucleophilic attack by the incoming (prolyl)-tRNA amide
nitrogen that normally creates the new peptide bond. Instead, the
N-terminal product is released from the ribosome by hydrolysis of the
peptidyl(2A)-tRNA ester bond. A proportion of ribosomes then cease
translation, while the remainder continue, effectively "initiated"
by the prolyl-tRNA, to produce the downstream product as a discrete
("cleaved") entity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, trp1-
99,
his3-
200, ura3-
99,
leu2-
1, ade2-101,
ciro), JDY13 (trp1, his3,
ura3, ade2, lys2, sec65-1,
MAT
), JDY365 (trp1-
99,
his3-
200, ura3-
99,
leu2-
1, ade2-101,
sec63-201-HIS3, ciro) both (13) and
JDY37 (trp1-1, his3-11, -15,
ura3-1, leu2-3, -112,
ade2-1, can1-100,
pep4
::TRP1). Yeast transformations
were performed by the lithium acetate method (23) and growth media and
temperatures were as indicated. Plasmids were constructed as follows.
Sequences encoding pp
F, ss
F, or DN
F were
amplified from pDJ100 (24) or pJD75 (25) and cloned as
BamHI-XbaI fragments, along with an
XbaI-ApaI fragment encoding the 19-amino acid
2A/2B FMDV sequence from plasmid pMR90 (5) and an
ApaI-NsiI GFP(S65T) fragment with the ER
retention motif HDEL appended to its C terminus into pGEM11zf+
(Promega). This yielded plasmids encoding pp
F-2A-GFP, DN
F-2A-GFP, and ss
F-2A-GFP.
DN
F-2A-Kar2-GFP and ss
F-2A-Kar2-GFP were
constructed in the same way but using a PCR product that contained the
Kar2 signal sequence in addition to GFP. Digestion of these plasmids
with ApaI and ClaI removed the Kar2 signal
sequence allowing it to be replaced with the carboxypeptidase Y
(CPY) signal sequence. To generate noncleavable fusions 2A was replaced
with 2A* incorporating a change that alters the essential proline at position 17 of 2A to alanine (26). Fragments consisting of the whole of
each fusion were transferred to pMW20 (27) for expression in yeast.
PHO8-containing constructs were assembled directly into pMW20 from PHO8 lacking its stop codon amplified from
genomic DNA as an EcoRI-XbaI fragment, 2A or 2A*
and EGFP (Clontech) amplified as an
ApaI-SacI fragment.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
factor-2A-GFP appended by the ER retention motif HDEL. Three variants had the Dap2p signal sequence (DN
F-2A-GFP), the native
-factor signal sequence (pp
F-2A-GFP), or no signal sequence
(ss
F-2A-GFP). DN
F is a well characterized
SRP-dependent translocation substrate (25), whereas pp
F
is translocated post-translationally relying on cytosolic chaperones
for this, indicating cytosolic exposure (31-33). Without a signal
sequence ss
F remains in the cytosol. A requirement for cytosolic
factors in 2A-dependent cleavage would be revealed by
cleavage of pp
F-2A-GFP and ss
F-2A-GFP but not DN
F-2A-GFP. If cytosolic factors are not required then
all constructs would be cleaved. Controls were (i) the same three
polyproteins with a nonfunctional 2A variant (26) termed 2A* hereafter,
(ii) GFP alone preceded by the signal sequence from the ER lumenal hsp70 orthologue Kar2p (recognized by both ER targeting pathways) and
appended by HDEL (Kar2-GFP), and (iii) the previously characterized GUS-2A-GFP fusion (6).
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Fig. 1.
2A fusion constructs. A,
constructs are drawn N to C terminus, left to right, with the
N-terminal portions of the fusions in pale gray and the
C-terminal GFP black. Signal sequences are in dark
gray, active 2A is a clear box, whereas the inactive
mutant (2A*) is indicated by a cross. For details of how
constructs were assembled, see "Experimental Procedures."
GUS, -glucuronidase; pp
F,
prepro-
-factor; DN, the dipeptidyl aminopeptidase
B N-terminal cytosolic tail and signal anchor region;
ss
F, prepro-
-factor lacking its signal
sequence; Kar2, the signal sequence of Kar2p. B,
activity of 2A constructs in coupled in vitro transcription
and translation reactions. Reactions were carried out in rabbit
reticulocyte extract using plasmids encoding the fusions shown and the
proteins labeled with [35S]methionine. Reaction products
were analyzed by SDS-PAGE and fluorography. Full-length proteins
(uncleaved) and cleavage products are indicated.
F-/pp
F-/DN
F-2A and GFP; lanes 2,
4, and 6) indicating that 2A was active.
Immunoprecipitation with anti-GFP and anti-2A antibodies confirmed the
identity of the proteins (data not shown). As expected, equivalent
reactions in which 2A*-containing polyproteins were synthesized
(lanes 3, 5, and 7) yielded
predominantly full-length fusion proteins and minor, lower molecular
weight products likely from internal initiation of translation.
F-2A-GFP and pp
F-2A-GFP encode secreted
-factor
peptides we did not expect them to be stable. We therefore
pulse-labeled cells expressing the various proteins with
[35S]methionine/cysteine and isolated 2A- and
GFP-containing species from cell lysates by immunoprecipitation (Fig.
2A). N- and C-terminal portions of ss
F-2A-GFP, pp
F-2A-GFP, and
DN
F-2A-GFP (lanes 3, 5, and
7) were immunoprecipitated from the lysates along with some
uncleaved full-length proteins. Thus the FMDV 2A sequence is functional
in yeast cells. Pulse-chase analysis (e.g. Fig. 2B) revealed that the proportion of full-length protein to
cleavage product remained constant during the chase period, the amount of both reducing similarly over time, presumably because of turnover. Therefore, similar to the situation in other systems (5)
2A-dependent cleavage is closely coupled to protein
synthesis, taking place rapidly and only during the labeling period. On
the assumption that 2A antibodies immunoprecipitated full-length and
cleavage products with equal efficiency, we determined the approximate percentage of each protein that was cleaved. Averages of two
independent experiments gave 88% for DN
F-2A-GFP, 82%
for pp
F-2A-GFP, and 76% for ss
F-2A-GFP, similar to the
cleavage efficiency determined previously using in vitro
transcription/translation for a fusion protein containing this 2A
sequence (26).
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Fig. 2.
2A is active in yeast. A,
wild type yeast cells carrying plasmids encoding the fusions indicated
were grown overnight at 30 °C in media containing raffinose as the
sole carbon source. Expression of the fusion proteins was induced by
addition of galactose to 2% (w/v). After 5 h cells were labeled
with [35S]methionine/cysteine, extracts were made and
immunoprecipitations performed with anti-GFP (upper panel)
or anti-2A (lower panel) antibodies on either untreated
extracts (lanes 1-8) or following treatment with
endoglycosidase F (lanes 9-14). The immunoprecipitated
proteins were analyzed by SDS-PAGE and fluorography, full-length
(uncleaved) and cleavage products are indicated with dots to
the left of the lanes. Note that the glycosylated products
in lanes 5-8 run at the same size as the anti-2A Ig heavy
chain and thus are not as clearly resolved as other species.
B, pulse-chase analysis. Cells expressing
DN F-2A*-GFP (lane 1) or
DN
F-2A-GFP (lanes 2-5) were labeled as in
A, and then excess cold methionine and cysteine were added.
Samples were removed at this time (the zero time point), and
subsequently at the times indicated and immunoprecipitations were
carried out as in A with anti-GFP antibodies. C,
pathway specificity of constructs. Cells as indicated expressing either
DN
F-2A*-GFP or pp
F-2A*-GFP were treated as in
A except that sec65-1 cells were incubated at
23 °C and then at 37 °C for 30 min prior to labeling. Antibodies
used in each immunoprecipitation were anti-GFP (upper two
panels), anti-Pho8p, or anti-CPY as indicated and relevant
portions of each autoradiogram are shown.
F-2A-GFP was indeed targeted to the ER, and through the expected SRP-dependent
co-translational targeting pathway. As pp
F-derived sequences in the
polyproteins contain sites for N-linked glycosylation the
proteins will be glycosylated if they enter the ER lumen. Treatment of
cell lysates with endoglycosidase F prior to immunoprecipitation
increased the mobility of both full-length DN
F-2A-GFP
and pp
F-2A-GFP fusion proteins and the N-terminal
DN
F-2A and pp
F-2A fragments derived from these
fusions in SDS-PAGE. This confirmed that they had been glycosylated and
hence translocated into the ER (Fig. 2A, compare lanes
5-8 with 11-14). In contrast the mobility of the
ss
F-containing proteins were not affected by endoglycosidase
treatment (compare lanes 3 and 4 with
9 and 10) indicating that, as expected, these remained in the cytosol. As GFP is not modified on entering the ER the
fate of the GFP portions of the fusion proteins could not be assessed
by this method.
F-2A*-GFP and pp
F-2A*-GFP were expressed in wild
type yeast and strains defective in either co-translational
(sec65-1) (34, 35) or post-translational
(sec63-201) (36) translocation. Fig. 2C shows the
results of immunoprecipitations with anti-GFP (top 2 panels)
and control (anti-CPY and Pho8p) antibodies from extracts of these
cells pulse-labeled with [35S]methionine/cysteine. As
expected sec65-1 cells revealed a defect in the
glycosylation and thus translocation of the co-translationally translocated Pho8p, but not the post-translational substrate CPY (compare lane 2 with the wild type in lane 1). A
significant proportion of DN
F-2A*-GFP immunoisolated
from the sec65-1 cell extract was also untranslocated,
whereas all pp
F-2A*-GFP was translocated. The results obtained with
sec63-201 cells were opposite to those obtained with
sec65-1 cells, these revealing defects in translocation of
CPY and pp
F-2A*-GFP but not Pho8p or DN
F-2A*-GFP
(lane 3). Thus the pathway specificity of the pp
F and
Dap2p signal sequences were maintained. We conclude that
DN
F-2A-GFP is co-translationally translocated and,
therefore, that cytosolic factors are not necessary for
2A-dependent cleavage.
F-2A-GFP by fluorescence microscopy. As the GFP was
appended by the ER retention motif HDEL, we expected an ER localization
pattern (Fig. 3D, Kar2-GFP) if
the released GFP was translocated, and a cytosolic signal if the
protein was not (Fig. 3C, ss
F-2A-GFP). Cells
expressing DN
F-2A-GFP revealed cytosolic fluorescence
(Fig. 3A), as did the post-translationally targeted
pp
F-2A-GFP (Fig. 3B), the major observable exclusion
being from the vacuole in both cases. Thus even though translation and
translocation are coupled for DN
F-2A-GFP, the released
GFP did not gain access to the ER. This indicates both intraribosomal
cleavage of the 2A containing protein and recognition of the gap in the
nascent chain by the translocon.
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Fig. 3.
GFP cleaved from
DN F-2A-GFP is cytosolic.
Phase images, and the fluorescence signals of GFP and vacuolar
membranes stained with FM4-64 were captured and processed from live
cells as described ("Experimental Procedures"). Cells were grown
and protein expression was induced as described in the legend to Fig. 2
before mounting on slides. Cells expressed DN
F-2A-GFP
(A), pp
F-2A-GFP (B), ss
F-2A-GFP
(C), or Kar2-GFP (D).
F-2A-GFP polyprotein does not cleave and some intact
(uncleaved) fusion protein remains in cells. Intact, translocated
DN
F-2A-GFP fusion protein should reside in the ER
because of the ER retention (HDEL) motif at its C terminus. Thus we
expected some ER fluorescence in cells expressing
DN
F-2A-GFP regardless of the localization of the
released GFP. As we did not see this (Fig. 3A), this raised the possibility that the signal from the uncleaved
DN
F-2A-GFP in the ER was masked by the strong
cytoplasmic GFP signal. If this were the case then some released GFP
could also have been translocated and its ER fluorescence again hidden.
We therefore carried out two further experiments. First we examined
cells expressing the noncleaving pDN
F-2A*-GFP and
pp
F-2A*-GFP fusions. Unexpectedly no GFP signal was detected,
despite robust expression of these proteins (data not shown). However,
immunofluorescence using antibodies against GFP revealed that the
translocated DN
F-2A*-GFP and p
F-2A*-GFP proteins were
localized to the ER as expected (Fig. 4,
A and B). This explained the lack of ER signal
from intact DN
F-2A-GFP protein in Fig. 3A.
Second, we separated extracts of cells expressing the various fusions
into cytosolic and membrane fractions. Fig. 4C shows that
GFP released from DN
F-2A-GFP and pp
F-2A-GFP was cytosolic, whereas intact DN
F-2A*-GFP was exclusively in
the membrane fraction, consistent with its ER localization by
immunofluorescence (Fig. 4A).
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Fig. 4.
A and B, localization
of the noncleaved polyproteins DN F-2A*-GFP and
pp
F-2A*-GFP to the ER. Wild type cells transformed with plasmids
encoding either of the noncleaved proteins DN
F-2A*-GFP
(A) or pp
F-2A*-GFP (B) were grown and protein
expression was induced as described in the legend to Fig. 2. Cells were
then fixed and proteins were localized by indirect immunofluorescence
using either anti-GFP or anti-Kar2p antibodies as described
("Experimental Procedures"). In the merged image GFP is shown in
green, Kar2p in red. C and
D, fractionation of cells extracts. Cells expressing the
indicated proteins were fractionated as described ("Experimental
Procedures") and probed with antibodies against GFP, Sec61p, or
phosphoglycerate kinase (PGK). Bound antibodies were
revealed by enhanced chemiluminescence (Amersham Biosciences). Relevant
portions of exposed films are shown: full-length
DN
F-2A*-GFP and GFP released from fusions containing
active 2A.
F-2A-GFP is an accurate reflection of the subcellular localization of GFP released by
cleavage of this protein. The translocon therefore "perceives" the
gap in the nascent chain and closes, effectively and quantitatively excluding the signal sequence-deficient GFP from the ER.
F-2A-GFP is significantly greater than that required
to prevent a ubiquitin-containing protein from being processed (210 amino acids compared with ~100 amino acids for a ubiquitin-containing
protein). We therefore believed that translation and translocation of
DN
F-2A-GFP would be coupled when the 2A peptide was
synthesized. However, we considered it important to demonstrate that an
SRP-dependent protein with a significantly greater distance
between the signal sequence and 2A (and hence a statistically greater
chance of tight coupling between translation and translocation for all
nascent chains) would still cleave efficiently.
F-2A-GFP, a
strong cytoplasmic fluorescence (Fig. 5B). In contrast,
Pho8-2A*-GFP was localized to the vacuolar lumen (Fig. 5C).
Pho8p is a vacuolar membrane protein, but its maturation involves
removal of a C-terminal propeptide (38). Thus GFP fluorescence within
the vacuolar lumen was not surprising for Pho8-2A*-GFP. To confirm that
Pho8-2A*-GFP reached the vacuole intact we expressed it in a processing
protease-deficient (pep4) strain. In this case the GFP
signal was largely coincident with the vacuolar membrane (Fig.
5D). Pulse labeling and imunoprecipitation with anti-GFP and
anti-2A antibodies (Fig. 5E) confirmed that Pho8-2A-GFP
cleaved efficiently (96% in the experiment shown) whereas, as
expected, Pho8-2A*-GFP did not. Thus positioning 2A in the context of a
co-translationally translocated protein of much greater length than
DN
F-2A-GFP had no effect on the cleavage reaction and
these data confirm the findings with the shorter protein and our
conclusion that 2A-dependent cleavage is
intraribosomal.
View larger version (44K):
[in a new window]
Fig. 5.
2A is active in Pho8-2A-GFP.
A, constructs drawn as in Fig. 1A.
B-D, localization of GFP. Wild type cells transformed with
plasmids encoding Pho8-2A-GFP (B) or Pho8-2A*-GFP
(C) and pep4 cells transformed with Pho8-2A*-GFP
(D) were grown, protein expression was induced as in Fig. 2
and images captured from them as in Fig. 3 are shown. GFP,
vacuolar membranes stained with FM4-64, merged images and phase images
are shown for each. In the merged images GFP is green and
FM4-64 is red. E, Pho8-2A-GFP is cleaved. Cells
from the same cultures as in B and C were labeled
with [35S]methionine/cysteine, extracts made from them
and proteins immunoprecipitated as in Fig. 2 using the antibodies are
shown. Positions of the intact fusion proteins and cleaved portions are
indicated.
F-2A-GFP and ss
F-2A-GFP) to contain either the
Kar2 or CPY signal sequence after the 2A site. Expression of these
proteins in yeast resulted, in all cases, in released GFP being
translocated and thus associated with the membrane rather than
cytosolic fraction of cell extracts (Fig. 4D). With the
exception of a proline at their N terminus, Kar2-GFP and CPY-GPF
released from these proteins are structurally normal translocation
substrates. As the N-terminal portions of ss
F-2A-Kar2-GFP and
ss
F-2A-CPY-GFP are not targeted to the ER the ribosomes synthesizing them would be expected to be free in the cytosol. Thus for
the C-terminal fragments released from these polyproteins we concluded
that the proline at their N terminus had no effect on either targeting
to or translocation into the ER. For the equivalent fragments released
from DN
F-2A-Kar2-GFP or DN
F-2A-CPY-GFP we could not distinguish whether the translocon had opened directly on
presentation of Kar2-GFP/CPY-GFP by the ribosome or whether the
ribosome had dissociated and then been re-targeted. However, as
discussed below, we consider it likely that these proteins may be
recognized directly by the translocon without the need for
re-targeting.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
F-2A-Kar2-GFP and
DN
F-2A-CPY-GFP) the translocon recognizes this and
reopens. In addition to indicating that cytosolic proteases are not
responsible for the cleavage reaction the cytoplasmic location of GFP
released from ER-targeted proteins refutes other models of 2A activity such as one 2A sequence acting in trans on another nascent
2A-containing protein to cleave it, or 2A requiring release from the
ribosome and folding before it becomes active in cis. Each
of these possibilities would result in cleavage within the ER lumen,
and thus the GFP portion of the polyproteins would have been found in
the ER.
View larger version (15K):
[in a new window]
Fig. 6.
Co-translational translocation and
intraribosomal nascent chain cleavage by 2A. A model is presented
to incorporate the findings in this paper into established models of
translocation. A, as 2A is synthesized by the ribosome the
cleavage reaction separates 2A from the 2B proline residue creating a
gap in the nascent chain (26). B, the first portion of the
cleaved polyprotein passes across the ER membrane and the translocon is
closed, at least partly by the action of Kar2p (oval at
bottom of translocon in B) (53). At this point or
soon after the portion of the cleaved polyprotein C-terminal to the
cleavage site engages with the translocon and is examined for the
presence of a signal sequence. C and D, depending
on whether a signal sequence is present on the downstream protein the
translocon either reopens to allow passage of the downstream protein
into the ER or remains closed, resulting in dissociation of the
ribosome into the cytosol (46).
The model above and in Fig. 6 is consistent with the recent finding
that, upon completion of co-translational translocation in
vitro, ribosomes remain attached to the translocon, competent to
initiate translation of new mRNA species (45, 46). If these ribosomes encode proteins with signal sequences they are
translocated directly into the ER independent of targeting by SRP,
whereas if they do not contain a signal sequence the translating
ribosomes detach and release the protein into the cytosol. This has
been argued to represent a physiologically important mechanism by which the cell ensures correct partitioning of cytosolic and ER-targeted proteins. Assuming that the in vitro experiments (45, 46) reflect in vivo events, then translocation of Kar2-GFP and
CPY-GFP released from DNF-2A-Kar2-GFP and
DN
F-2A-CPY-GFP likely represents direct recognition of
signal sequences in vivo by the translocon. Thus 2A may
provide a means by which we can, for the first time, examine the signal
sequence recognition and gating activities of the translocon in
vivo independent of targeting.
Analysis of 2A and 2A-like sequences has revealed that the FMDV 2A
reaction is far from unique. It is a strategy for creating separate
proteins from one polypeptide chain used by a variety of picornaviruses
and insect viruses, and active 2A-like sequences are also found in
repeated sequences within the genomes of Trypanosoma species
(8, 26, 47). Neither is 2A the only example of a nascent peptide chain
that modifies the activity of the ribosome while within the ribosome
exit tunnel (reviewed in Refs. 48 and 49). Other examples are sequences
within the bacteriophage T4 gene 60 that promote "hopping" of the
ribosome along the mRNA (50), the TnaC leader peptide of the
Escherichia coli tryptophanase operon that, in the presence
of tryptophan, causes ribosomes to stall allowing expression of the
downstream open reading frames (51) and a peptide within SecM that
causes ribosomes to stall unless the protein is engaged with the
protein export machinery. Mutations that suppress the stalling activity
of SecM have been identified in both RNA and protein components of the
ribosome, these mapping to the nascent chain exit tunnel (52). Thus
interactions between the nascent chain and the ribosome can have
significant effects on translation and we expect that our further
analysis may reveal similar interactions important for the activity of the 2A peptide.
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ACKNOWLEDGEMENTS |
---|
We thank members of the Brown laboratory for discussions and suggestions, and Janet Quinn, Simon Whitehall, and Susan Farrington for comments on the manuscript. We are grateful to Davis Ng, Peter Walter, Tom Stevens, Joachim Li, Caroline Shamu, Nils Johnsson, and Reid Gilmore for reagents that were used during this study.
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FOOTNOTES |
---|
* This work was supported by Wellcome Trust Research Career Development and UK Medical Research Council Senior NonClinical Research Fellowships (to J. D. B.) and Wellcome Trust Grant 064814/Z/01/Z (to P. F.).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. Tel.: 44-191-222-7470; Fax: 44-191-222-7424; E-mail: Jeremy.Brown@ncl.ac.uk.
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M211644200
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ABBREVIATIONS |
---|
The abbreviations used are: FMDV, foot-and-mouth disease virus; ER, endoplasmic reticulum; SRP, signal recognition particle; GFP green fluorescent protein, CPY, carboxypeptidase Y.
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