(Received for publication, November 30, 1995)
From the
The C-terminal globular head of the lollipop-shaped 1
protein of reovirus is responsible for interaction with the host cell
receptor. Like the N-terminal fibrous tail, it has its own
trimerization domain. Whereas N-terminal trimerization (formation of a
triple
-helical coiled coil) occurs at the level of polysomes
(i.e. cotranslationally) and is ATP-independent, C-terminal
trimerization is a posttranslational event that requires ATP.
Coprecipitation experiments using anti-Hsp70 antibodies and truncated
1 proteins synthesized in vitro revealed that only
regions downstream of the N-terminal
-helical coiled coil were
associated with Hsp70. Hsp70 was also found to be associated with
nascent
1 chains on polysomes as well as with immature
postribosomal
1 trimers (hydra-like intermediates with assembled
N termini and unassembled C termini). These latter structures were true
intermediates in the
1 biogenetic pathway since they could be
chased into mature
1 trimers with the release of Hsp70. Thus,
unlike N-terminal trimerization, C-terminal trimerization is Hsp70- and
ATP-dependent. The involvement of two mechanistically distinct
oligomerization events for the same molecule, one cotranslational and
one posttranslational, may represent a common approach to the
generation of oligomeric proteins in the cytosol.
There is now increasing evidence that the folding and assembly of proteins in vivo are mediated by other proteins known as molecular chaperones(1, 2, 3, 4) . In eukaryotes, some of these proteins were initially identified as stress or heat-shock proteins since their expression in cells is inducible by a variety of cellular stresses including heat(5, 6) . It is now known that they are also constitutively expressed and play essential roles in promoting the correct folding and assembly of newly synthesized proteins in the cytosol (7, 8, 9) as well as in the transport of proteins across membranes of mitochondria(10) , chloroplasts(11) , and endoplasmic reticulum(12, 13) . A number of chaperone families have been described, including Hsp90 (HtpG in prokaryotes)(14, 15) , Hsp70 (DnaK in prokaryotes)(4, 16) , Hsp60 (GroEL in prokaryotes)(17, 18, 19) , Hsp40 (DnaJ in prokaryotes) (9, 10, 11, 12, 13, 14, 15, 16) , and Hsp10 (GroES in prokaryotes)(17, 18, 19, 20) , of which members of the Hsp70 and Hsp60 families are the best studied. Hsp70 proteins function as monomers or dimers, whereas Hsp60 proteins adopt a characteristic double toroid structure, with each toroid possessing seven identical subunits. These two chaperones are believed to function as a relay team in the processing of nascent or unfolded polypeptides, which involves a series of ATP-dependent binding and release events, with the eventual generation of the correctly folded and assembled products(16) . Recent evidence suggests that Hsp70 preferentially binds to short extended peptides possessing alternating hydrophobic residues(21) , a finding consistent with the view that chaperones interact with exposed hydrophobic regions of unfolded proteins that are otherwise buried in native proteins. The relative lack of more stringent recognition requirements apparently contributes to the promiscuous nature of substrate binding seen with all classes of chaperones.
Considerable information on chaperone function has been obtained from in vitro refolding experiments using purified components. However, the extent to which observations from these studies can be extrapolated to reflect the actual sequence of events that direct nascent proteins to their native conformations is unclear. Experiments involving the direct analysis of nascent chains are therefore necessary in order to reveal the actual mechanisms of chaperone function. In this regard, Beckmann et al.(7) were the first to demonstrate that nascent polypeptides in the mammalian cytosol associate with Hsp70 and postulated that this may represent a requirement of subsequent folding and/or assembly of polypeptides. More recently, Frydman et al.(9) used in vitro translated firefly luciferase to demonstrate the highly organized chaperone machinery involved in the successful folding of this protein.
The in vitro translation system has also been used extensively in our
laboratory to reveal the mechanisms of folding and oligomerization of
the reovirus cell attachment protein 1, a trimeric protein
located at the 12 vertices of the icosahedral virion (22, 23, 24, 25, 26) . The
1 trimer is highly asymmetric, with an N-terminal fibrous tail
that is anchored to the virion and a C-terminal globular head that
interacts with the cell
receptor(27, 28, 29, 30, 31, 32, 33) .
These two structurally distinct domains are separated by a short
protease-sensitive region(32, 34) . Evidence from in vitro translation studies has revealed that these two
domains are generated by independent trimerization events(26) ,
with N-terminal trimerization preceding C-terminal trimerization. The
core of the N-terminal trimerization domain is the N-terminal one-third
of the protein, which is highly
-helical and contains an extended
heptad repeat of hydrophobic residues(35, 36) ,
endowing this region with the intrinsic propensity to form a triple
coiled coil. During
1 biogenesis, N-terminal assembly takes place
cotranslationally (i.e. on the polysome), involving
neighboring nascent chains on those ribosomes that have traversed past
the midpoint of the S1 mRNA encoding
1. (
)Interestingly, this process is intrinsically
ATP-independent, suggesting the lack of chaperone involvement.
Although the C terminus possesses its own independent trimerization domain, its assembly into a globular head requires the prior trimerization of the N terminus(26) , which presumably serves to bring the three C termini into close proximity to each other for interaction. Also, unlike N-terminal trimerization, which can tolerate relatively large alterations(26, 38) , C-terminal trimerization is under stringent control, which requires that the C-terminal halves of all three subunits be intact(26, 32, 39) . The global nature of this trimerization event is compatible with a posttranslational assembly mechanism, as opposed to a cotranslational one as seen with N-terminal trimerization. A pertinent question would be whether the two trimerization processes also differ in terms of ATP and chaperone involvement.
In the present study, we demonstrate that trimerization
of the C-terminal globular head indeed occurs posttranslationally, and
is an ATP-dependent process. In addition, we find that nascent 1
chains and immature, but not mature,
1 trimers are transiently
associated with Hsp70 and that this association is strictly limited to
regions downstream of the N-terminal
-helical coiled coil. We
conclude that distinct Hsp70/ATP-dependent and -independent
oligomerization and folding domains can coexist within the same
protein.
To follow the fate of the proteins synthesized, reaction mixtures were subjected to ultracentrifugation at 96,000 rpm for 1.5 h at 4 °C (TLA 100.1 rotor, Beckman TL-100 tabletop ultracentrifuge) to pellet ribosomes. The supernatants that had no translation activity were then incubated at 37 °C for various durations indicated and then analyzed by SDS-PAGE.
Figure 1:
Posttranslational C-terminal
trimerization of protein 1. Full-length reovirus S1 transcripts
were translated in rabbit reticulocyte lysate in the presence of
[
S]methionine for 10 min (pulse) and then
subjected to ultracentrifugation at 4 °C to pellet the polysomes.
The supernatants, which contained no translation activity, were then
incubated at 37 °C for various durations (chase). Aliquots from
both the pulse and chase samples were incubated in SDS-containing
protein sample buffer at either 4 °C (at this temperature both N-
and C-terminal trimers were stable), or 37 °C (at this temperature
the N-terminal trimer, but not the C-terminal trimer, was stable).
SDS-PAGE was carried out at 4 °C.
Figure 2:
Effect of ATP deprivation on C-terminal
trimerization of protein 1. Full-length S1 transcripts were
translated for 10 min and subjected to ultracentrifugation to pellet
the polysomes. The supernatant was then incubated (chased) at 37 °C
for various durations indicated either in the presence (+) or
absence (-) of apyrase (40 units/ml). Chase samples were
incubated in protein sample buffer at 4 °C for 30 min prior to
SDS-PAGE.
Figure 3:
Association of Hsp70 with polysome-bound
1 chains. Full-length S1 transcripts were translated in vitro for 10 min, and the reaction mixture was subjected to
centrifugation through a 10-45% sucrose gradient as described
under ``Materials and Methods.'' Peak polysomal fractions
were pooled (lane 1), concentrated, and immunoprecipitated
with the Hsp70-specific serum (lane 2) or with the preimmune
serum (lane 3). All samples were boiled prior to
SDS-PAGE.
Since the hydra-like folding intermediate represents the
precursor to mature 1, and since C-terminal trimerization is
ATP-dependent, it was of interest to determine whether this hydra-like
folding intermediate might also be associated with Hsp70. We therefore
translated
1 for 20 min, a time at which both the hydra-like
folding intermediate and the mature form of
1 were present in the
translation reaction, and examined their association with Hsp70 by
coimmunoprecipitation with the Hsp70-specific serum. Immunoprecipitates
were subjected to SDS-PAGE under nondissociating conditions in order to
resolve the various forms of
1 (Fig. 4). The results show
that in addition to monomeric
1, the hydra-like folding
intermediate was also associated with Hsp70. Importantly, the mature
and functional form of
1 was not found to be in association with
Hsp70. These results suggest that Hsp70 may be playing a dynamic role
in
1 trimerization.
Figure 4:
Association of Hsp70 with the 1
hydra-like intermediate. Apyrase was added to a 20-min
1
translation reaction, which was then diluted and microcentrifuged. The
supernatant (lane 1) was immunoprecipitated with the
Hsp70-specific serum (lane 2) or with the preimmune serum (lane 3). Immunoprecipitates were incubated in protein sample
buffer at 4 °C prior to SDS-PAGE (also carried out at 4 °C) to
allow for the detection of both the hydra-like intermediate and the
mature trimer.
Figure 5:
Chase of Hsp70-associated 1 to
mature
1. A, full-length S1 transcripts were translated
in the presence of [
S]methionine for 7 min and
subsequently chased with excess (16 mM) unlabeled methionine.
At various times indicated (with the first time point being the time of
addition of unlabeled methionine), chase samples (expression)
were subjected to sequential immunoprecipitation: anti-Hsp70 serum
followed by mAb G5 (a) or mAb G5 followed by anti-Hsp70 serum (b). All the immunoprecipitates were boiled in protein sample
buffer prior to SDS-PAGE. B, full-length S1 transcripts were
translated for 11 min in the presence of
[
S]methionine. The reaction mixture was then
immunoprecipitated with the anti-Hsp70 serum. After repeated washes in
a buffer containing 150 mM NaCl and 25 mM Tris (pH
7.4), the immunoprecipitates were resuspended in fresh rabbit
reticulocyte lysate and incubated at 37 °C. At the times indicated,
aliquots were taken from the suspension and analyzed by SDS-PAGE under
nondenaturing conditions (4 °C
preincubation).
Figure 6:
Association of 1 deletion mutants
with Hsp70. A, full-length (FL) and various
3`-terminally truncated S1 transcripts encoding proteins lacking
30-294 amino acids at the C terminus (d30 to d294) were
translated in vitro for 6 min. Reactions were either analyzed
directly by SDS-PAGE (expression) or immunoprecipitated with
either the anti-Hsp70 serum (i) or the preimmune serum (pi) and then analyzed by SDS-PAGE. All samples were boiled
prior to electrophoresis. Molecular size markers (in kilodaltons) are
indicated to the left. B, S1 deletion mutants
encoding the C-terminal half of
1 (III & IV,
containing amino acids 223-455) or portions thereof (III, containing amino acids 223-364; and IV,
containing amino acids 365-455) were translated in vitro as above and similarly analyzed.
Studies on the structure/function relationships of the
reovirus cell attachment protein 1 have led to a number of
interesting revelations. Earlier sequence analysis suggests that the
N-terminal portion of
1 is an
-helical coiled coil, while
the C-terminal portion exists as a globular head(35) . Such a
prediction was subsequently confirmed by electron microscopic studies
that showed purified
1 as a lollipop-shaped structure with a
fibrous tail topped by a globular
head(27, 28, 30) . Similar structures have
been found to project from the surfaces of virus particles, with the
globular heads being most distal from the virions(28) . That
the globular head and the fibrous tail indeed represent the C- and
N-terminal portions, respectively, of
1 has been confirmed by
biophysical analysis of the two fragments (representing the C- and
N-terminal halves) generated by trypsin digestion of purified
1(23) . It was further shown that the C-terminal portion
of
1 harbors the conformation-dependent receptor binding domain (29, 32, 39, 44) and that the
N-terminal portion possesses intrinsic oligomerization and virion
anchoring
function(24, 27, 31, 33, 37) .
Biochemical and biophysical evidence suggests that intact protein
1, as well as the N- and C-terminal tryptic fragments, are all
trimeric (23) and that trimerization of
1 is accompanied
by extensive conformational changes necessary for its cell attachment
function(25) . Of particular significance was the subsequent
demonstration that the N- and C-terminal halves of
1 each
possesses its own trimerization domain (26) , leading us to
further characterize the two trimerization events in terms of
temporality relative to translation and of ATP/chaperone requirements.
N-terminal trimerization of 1 has recently been found to be a
cotranslational event that is intrinsically ATP-independent, suggesting
the lack of chaperone involvement.
This is not likely the
case for C-terminal trimerization, which appears to be global and thus
necessarily occurs posttranslationally. The global nature of this
process is suggested by the following observations. First, the deletion
of as few as four amino acids from the C terminus totally abrogates the
cell binding function of
1(32) , as does the single
substitution of certain conserved amino acids at the C-terminal half of
1(39) . In both cases, the N-terminal half of the protein
remains intact (trimeric and protease-resistant), whereas the
C-terminal half is grossly misfolded (unassembled and
protease-sensitive). Second,
1 heterotrimers comprised of two
wild-type subunits and a mutant subunit with deletion or substitution
at the C terminus are invariably nonfunctional and manifest C-terminal
misfolding(26) . These observations have led to the prediction
that trimerization of the C terminus can proceed only when the C
termini of all three subunits are intact, and is accordingly a
posttranslational and global event. That this is in fact the case is
clearly demonstrated in the present study by following the fate of
1 intermediates in the postribosomal fractions. C-terminal
trimerization therefore contrasts sharply with N-terminal trimerization
in both temporality and stringency. The involvement of Hsp70 and
possibly other chaperones is another feature that demarcates these two
events.
Hsp70 interacts with nascent 1 chains, but this
interaction is confined to regions downstream of the N-terminal
extended
-helix and occurs with progressively higher affinity
toward the C terminus. The lack of Hsp70 binding sites on the
N-terminal one-third of
1 correlates with the ATP-independent
nature of N-terminal trimerization when this portion of the protein
alone is expressed.
Additionally, the observation that
stabilization of the trimeric N terminus in the full-length
1
protein is ATP-dependent
can now be explained by the
present finding that sequences immediately downstream of the
-helical coiled coil are recognizable and bound by Hsp70. While
this binding has no effect on the initial cotranslational assembly of
the three N termini, it probably sterically hinders further tightening
of the coiled coil. Upon subsequent ATP-dependent release of Hsp70 from
1 as required for posttranslational C-terminal trimerization, the
three loosely associated strands of the coiled coil are then allowed to
further interact to form the mature fiber. This view therefore portrays
Hsp70 as playing no active role in N-terminal trimerization. Rather, in
carrying out its chaperoning duty of promoting C-terminal folding,
Hsp70 inadvertently delays the N-terminal maturation process.
Since
segments from the C-terminal half, when generated separately, were
found to be independently associated with Hsp70 (Fig. 6), it is
likely that a good portion of the C-terminal half of 1 nascent
chains and intermediate forms is protected by Hsp70. This association
persists as the
1 complex leaves the polysome. It is not known at
present whether other chaperones such as Hsp40 and/or TRiC are involved
prior to this release. However, results from our experiments (not
shown) suggest that the release of Hsp70 from the
1 substrate
requires an additional factor(s) present in the reticulocyte lysate.
Recent evidence from pulse-chase experiments performed in our
laboratory (not shown) indicates that with time, there is a decrease of
Hsp70-
1 association that is concomitant with an increase of
TRiC-
1 association, suggestive of an ordered sequence for the
interaction of these two chaperones with
1. The involvement of
Hsp40 in the
1 folding process is also a likely possibility since
this chaperone has recently been shown to cooperate with Hsp70 and TRiC
in promoting the folding of firefly luciferase in
vitro(9) . Whatever the nature of cochaperones, the almost
exclusive interaction of Hsp70 with the C-terminal half of
1 is
in absolute accord with this second trimerization event being
ATP-dependent. As is the case with monomeric proteins,
1
interaction with these chaperones necessarily involves cycles of
release and rebinding mediated by ATP binding and hydrolysis. Although
speculative, a possible role for cochaperones in
1 trimerization
and folding might be to coordinate an orderly release of Hsp70 from the
1 folding intermediates. In this respect, the present
identification of a transient association between Hsp70 and
1
intermediates represents an important step toward a testable model.
The overall scheme of protein 1 biogenesis is summarized in a
schematic in Fig. 7. Commitment of the
1 N termini to
trimerize occurs at the level of polysomes (depicted as the
cotranslational formation of a triple coiled coil, the most plausible
scenario) and with no ATP or Hsp70 involvement. Binding of Hsp70 (and
possibly other chaperones such as Hsp40 and TRiC) to sequences
downstream of the N-terminal
-helical region prevents tightening
of the coiled coil, and
1 leaves the polysome as an SDS-unstable
trimeric complex. Subsequent ATP-dependent release of the bound
chaperones leads to the second (C-terminal) trimerization event, which
completes the
1 maturation process. Such a strategy of
cotranslational followed by posttranslational oligomerization is highly
efficient since it spares individual subunits the need to search for
their partners in a soluble pool. It would be interesting to see
whether other homooligomeric proteins in the cytosol follow a similar
strategy of biogenesis.
Figure 7:
Model for the biogenesis of the reovirus
1 trimer. Assembly of three
1 nascent chains occurs
cotranslationally at the N terminus. This process does not involve
Hsp70 or ATP and results in the generation of a loose triple coiled
coil after the midpoint of the polysome where Hsp70 begins to interact
with emerging residues, thereby sterically hindering tightening of the
coiled coil. As the triplex moves down the polysome, more Hsp70 becomes
associated with the elongating C termini, preventing their misfolding
and aggregation. The immature trimer leaves the polysome as a complex
comprised of three
1 subunits, Hsp70, and possibly other
chaperones and is SDS-sensitive (
1 migrating as a monomer in
SDS-PAGE even under ``nondissociating'' conditions).
Subsequent ATP-dependent release of Hsp70 presumably provides the
opportunity for the loose coiled coil to quickly snap together
(tightening the coiled coil) while the remaining portions of the three
C termini continue to interact with Hsp70 and other chaperones (TRiC
and Hsp40?). This structure migrates as a retarded trimer (hydra-like
intermediate) in SDS-PAGE under nondissociating conditions. Further
ATP-dependent release and rebinding of Hsp70 and other chaperones leads
to global trimerization and folding of the C terminus, generating
mature
1 with the characteristic lollipop-shaped structure that
migrates as an unretarded trimer in SDS-PAGE under nondissociating
conditions.