Intracellular Retention of Procollagen within the Endoplasmic
Reticulum Is Mediated by Prolyl 4-Hydroxylase*
Adrian R.
Walmsley
,
Margaret R.
Batten,
Usha
Lad, and
Neil J.
Bulleid§
From the School of Biological Sciences, 2.205 Stopford Building,
University of Manchester, Manchester M13 9PT, United Kingdom
 |
ABSTRACT |
The correct folding and assembly of proteins
within the endoplasmic reticulum (ER) are prerequisites for subsequent
transport from this organelle to the Golgi apparatus. The mechanisms
underlying the ability of the cell to recognize and retain unassembled
or malfolded proteins generally require binding to molecular chaperones within the ER. One classic example of this process occurs during the
biosynthesis of procollagen. Here partially folded intermediates are
retained and prevented from secretion, leading to a build up of
unfolded chains within the cell. The accumulation of these partially
folded intermediates occurs during vitamin C deficiency due to
incomplete proline hydroxylation, as vitamin C is an essential co-factor of the enzyme prolyl 4-hydroxylase. In this report we show
that this retention is tightly regulated with little or no secretion
occurring under conditions preventing proline hydroxylation. We studied
the molecular mechanism underlying retention by determining which
proteins associate with partially folded procollagen intermediates within the ER. By using a combination of cross-linking and sucrose gradient analysis, we show that the major protein binding to
procollagen during its biosynthesis is prolyl 4-hydroxylase, and no
binding to other ER resident proteins including Hsp47 was detected.
This binding is regulated by the folding status rather than the extent of hydroxylation of the chains demonstrating that this enzyme can
recognize and retain unfolded procollagen chains and can release these
chains for further transport once they have folded correctly.
 |
INTRODUCTION |
The biosynthesis of multi-subunit proteins entering the secretory
pathway is regulated at the endoplasmic reticulum
(ER)1 where the individual
subunits are synthesized and their assembly is coordinated. This
regulation ensures that unassembled subunits are prevented from being
transported out of the ER and are either degraded or maintained in an
assembly-competent state by interacting with ER resident proteins (1).
The mechanism underlying this "quality control" appears to involve
the binding of unassembled subunits to a variety of ER proteins until
assembly occurs. The assembled complex is then released and can be
transported from the ER. Such a mechanism has been likened to affinity
chromatography, with the "matrix" being the resident proteins in
the ER and the selective interactions occurring via oligosaccharide
side chains (2), hydrophobic regions in the protein (3), or free thiol residues (4).
To investigate further the generality of these mechanisms of retention,
we have studied the folding, assembly, and secretion of procollagen.
The folding and assembly of procollagen occurs in a vectoral manner;
trimerization of individual polypeptides called the pro-
-chains
occurs via type-specific association of their C-propeptides (5, 6).
This association facilitates the formation of a stable nucleus of
triple helix at the C-terminal end of the triple helical domain (7)
that is subsequently propagated to the N terminus in a zipper-like
fashion (8). The prerequisite for the formation of a triple helix that
is stable at physiological temperatures is the 4-hydroxylation of a
certain proportion of proline residues within the triple helical domain
of the pro-
-chains (9), a modification catalyzed by the ER resident
enzyme prolyl 4-hydroxylase (P4H) (10).
Evidence that the procollagens of fibril-forming collagens are subject
to a retention mechanism has been provided by studies on type I
procollagen synthesized by chick fibroblasts (11, 12). It was
demonstrated that the rate of secretion of the protein is significantly
depressed when the formation of the triple helix is prevented either by
the incorporation of helix-destabilizing proline analogues into the
pro-
-chains or by the inhibition of hydroxylation of the
pro-
-chains. Furthermore, point mutations in the genes for the
pro-
-chains of type I procollagen which cause the human disease
osteogenesis imperfecta have been shown to disrupt the folding of the
triple helix and result in the retention and subsequent degradation of
the mutant protein within the cell (13, 14).
A number of ER resident proteins have been proposed to mediate the
retention of non-native procollagen within the cell, one of these being
Hsp47, a collagen-binding heat-shock protein (15). Hsp47 has been shown
to bind to a wide range of collagens and procollagens in
vitro (16, 17). It also associates with type I procollagen
in cellulo (18, 19), leading to the suggestion that Hsp47 is a chaperone of procollagen biosynthesis. In addition BiP
and PDI have been individually shown to associate with certain mutant
forms of type I procollagen that were retained within the fibroblasts
of patients with osteogenesis imperfecta (20, 21). Furthermore, it has
been proposed that P4H is a candidate for the ER resident protein
involved in mediating procollagen retention. This was based on the fact
that the binding affinity of this enzyme for procollagen substrates
in vitro was in compliance with the selectivity of the
retention mechanism for procollagens in cellulo (22). More
recently, PDI has been shown to interact specifically with individual
C-propeptide chains prior to trimerization (23) and with assembled type
X collagen (24).
To address the question of which ER resident proteins are involved in
the retention of procollagen, we have prepared stable cell lines
expressing a type III procollagen mini-gene containing either the
authentic C-propeptide or a construct where the C-propeptide has been
replaced with a transmembrane domain. Both chains have been shown
previously to fold and assemble correctly when expressed in the
presence of semi-permeabilized cells (7, 25). These cell lines were
used to investigate the regulation of procollagen secretion and to
identify proteins interacting with partially folded procollagen chains.
The results clearly demonstrate a novel role for a post-translational
modifying enzyme in the regulation of secretion of its substrate, and
the results show that this regulation is not influenced directly by
enzymatic activity.
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MATERIALS AND METHODS |
HT1080 cells (ATCC CCL121) were obtained from the American Type
Culture Collection (Rockville, MD). A mouse monoclonal antibody to
Hsp47 (SPA-470) and a rat monoclonal antibody to GRP94 (SPA-850) were
obtained from StressGen (York, UK). A goat polyclonal antibody to BiP
(N-20) was purchased from Santa Cruz Biotechnology (Mile Elm, Calne,
UK). A rabbit polyclonal antibody to calreticulin antibody was
purchased from Cambridge Biosciences (Cambridge, UK). A rabbit
polyclonal antibody to type III procollagen was prepared as described
previously (25). Rabbit polyclonal antibodies to PDI, ERp57, and to
calnexin were prepared as described previously (26-28). A mouse
monoclonal antibody specific for the
-subunit of prolyl
4-hydroxylase was a gift from Professor Kari Kivirikko (University of
Oulu, Finland), and a mouse anti-Myc monoclonal antibody (9E10) was
obtained from Calbiochem (Nottingham, UK)
Plasmid Construction--
The plasmid p
1(III)
1 containing
the sequence encoding the prepro-
1(III)
1 chain of type III
1
procollagen had been constructed previously (29). The
SpeI/ClaI fragment excised from p
III
1 containing the prepro-III
1 sequence was cloned into the
XhoI/AccI endonuclease-treated pCIneo
vector to create the construct pCIneoIII
1. The plasmid
HA-trans was constructed previously (7). A Myc-tagged version of
p
1(III)
1 was prepared by introducing the Myc tag between the end
of the signal sequence and the beginning of the coding region using
standard polymerase chain reaction techniques as described previously
(30). A 300-base pair XhoI/KpnI fragment was
excised from HA-trans and subcloned into
XhoI/KpnI cut p
1(III)
1-Myc to generate
HA-trans-Myc. The entire coding region was excised with NotI
and subcloned into pIRES1hyg (CLONTECH,
Basingstoke, Hampshire, UK) to generate pIREShyg-HA-trans-Myc.
Cell Culture--
All cell lines were maintained in Dulbecco's
modified Eagle's medium with 110 µg ml
1 pyruvate
supplemented with 0.3 mg ml
1 glutamine and 10% (v/v)
fetal calf serum (complete medium).
Construction of Clonal Cell Lines--
HT1080 cells at 50-80%
confluence were suspended by treatment with trypsin and pelleted by
centrifugation. Harvested cells were washed twice in phosphate-buffered
saline (PBS) and resuspended in complete medium to an approximate
concentration of 1 × 106 cell ml
1. The
cell suspension (800 µl) was placed in a 4-mm electroporation cuvette
and mixed 1 min prior to the pulse with 30 µg of
pCIneoIII
1 linearized with BcgI or 30 µg of
pIRES-hyg-HA-trans linearized with SspI. Cells were pulsed
at 1650 microfarads/250 V using the EasyJect electroporator (Flowgen)
and immediately transferred from the cuvettes into a
T162-cm2 flask containing complete medium. Cells were
incubated for 48 h and subsequently selected by incubation in
media containing G418 sulfate or hygromycin at a concentration of 500 µg ml
1. The resulting G418 or hygromycin-resistant
colonies were transferred to a fresh T162-cm2 flask and
grown to confluence to give a pooled population of transfectants. A
random sample of cells from the pooled population of transfectants was
serially diluted in complete medium containing 200 µg
ml
1 G418 sulfate or hygromycin to a concentration of 25 cells/ml
1 and 200 µl aliquoted per well of a 96-well
plate. Wells containing a single colony of cells were identified after
several days of growth and the clones grown to confluence before being
transferred to 6-well plates. To identify clones expressing type
III
1 procollagen, proteins present in the medium of the clones were
precipitated by the addition of PEG-3000 to a concentration of 5%
(w/v), harvested by centrifugation at 12,000 × g,
separated by SDS-PAGE, and transferred to nitrocellulose. The membrane
was probed with a 1/1000 dilution of an antibody against type III
procollagen and developed using a chemiluminescense substrate. The
expression level of pro-
1(III)
1 was quantitated by metabolic
labeling, and B12 was identified as the cell line with the highest
expression level. Similarly, indirect immunofluorescence staining using
anti-Myc antibody identified clones expressing HA-trans-Myc at the cell
surface and F2 was identified as a high expressing cell line.
Metabolic Labeling and Pulse-Chase--
All the following
procedures were performed at 37 °C. B12 cells were plated to
50-80% confluence in 100-mm2 culture dishes and incubated
for 4 h in complete medium. Cells were rinsed twice with 5 ml of
PBS, once with 5 ml of methionine- and cysteine-free minimal Eagle's
medium supplemented with 0.3 mg ml
1 glutamine (starve
medium), and incubated for 45 min in 5 ml of the starve medium. Cells
were incubated with 3 ml of starve medium containing
[35S]methionine and [35S]cysteine (labeling
medium) at a total concentration of 100 µCi ml
1 for the
indicated times.
For pulse-chase experiments, cells were radiolabeled as above, and the
chase was initiated by the addition to the labeling medium of 7 ml of
pre-warmed Dulbecco's modified Eagle's medium (with 1.5 mg
ml
1 glucose) supplemented with 0.3 mg ml
1
glutamine and containing 7.1 mM cycloheximide and
L-methionine, respectively. When required, exogenous
reagents were present during the starve, pulse, and chase periods at
the following concentrations: ascorbate at 50 µg ml
1,
,
'-dipyridyl at 0.3 mM, and azetidine-2-carboxylic
acid at 10 mM. When required, ferrous ions were present
during the chase period at a final concentration of 5 mM by
the addition of solid Fe2(SO4)3 to
the chase medium.
All the following procedures were performed at 4 °C. After the
completion of the labeling or chase periods, the cells were immediately
lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1% (v/v) Triton X-100, 150 mM NaCl,
0.02% (w/v) NaN3, 0.1 mg ml
1 soybean trypsin
inhibitor (SBTI), 0.5 mM phenylmethylsulfonyl fluoride
(PMSF)). When required, N-ethylmaleimide (NEM) was present in the PBS and lysis buffer at a concentration of 20 mM.
Cells were lysed for 30 min, and the lysate was centrifuged at
12,000 × g for 15 min to remove nuclei and cell
debris. Proteins present in the chase medium were precipitated by the
addition of PEG as described above.
Cross-linking with DSP--
B12 cells were radiolabeled for
1 h as described above and pelleted by centrifugation. Harvested
cells were washed in PBS and resuspended in 100 µl of PBS. The cell
suspension was mixed with 10 µl of freshly prepared 20 mM
DSP in Me2SO and incubated at 4 °C for 30 min. Cells
were washed with 2 mM glycine in PBS to quench the
cross-linker, washed once with PBS, and resuspended in 200 µl of
Nonidet P-40 lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% (v/v) Nonidet P-40, 5 mM EDTA, 1 mM PMSF, 0.1 mg ml
1 SBTI, 1 mM
NEM). Cells were lysed at 4 °C for 30 min, and the lysate was
centrifuged at 12,000 × g for 15 min.
Sucrose Gradient Centrifugation--
All sucrose solutions were
prepared in gradient buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1%(v/v) Nonidet P-40, 1 mM PMSF). Equal volumes of 25 and 5% (w/v) sucrose were differentially mixed using a gradient maker to create a 10-ml 5-25% gradient that was gradually layered onto 0.5 ml of 50% (w/v) sucrose in a Beckman SW-40
tube. All the following procedures were performed at 4 °C unless
otherwise stated. Cells were radiolabeled as described above and lysed
for 30 min in gradient buffer containing 0.1 mg ml
1 SBTI.
The lysate was centrifuged at 12,000 × g for 15 min,
and the supernatant was slowly layered onto a 9.5-ml 5-25% sucrose gradient. Proteins were separated through the gradient by
centrifugation at 40,000 rpm (Beckmann SW-40 rotor) for 16 h.
Immunoprecipitation--
For the immunoprecipitation of proteins
under denaturing conditions, 10% (w/v) SDS was added to the cell
lysate supernatant or to PEG precipitates from medium to a final
concentration of 0.5%, and the sample was boiled for 5 min. The sample
was then diluted 10-fold with immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1% (v/v)
Triton X-100, 150 mM NaCl, 0.02% (w/v) NaN3)
and centrifuged for 15 min to remove non-solubilized material. Samples
immunoprecipitated under native conditions were diluted directly into
immunoprecipitation buffer.
After the above preparations, 10% (w/v) protein A-Sepharose in PBS was
added to the supernatants to a concentration of 0.5%, and the samples
were incubated with gentle agitation for 1 h. The protein
A-Sepharose was pelleted from the sample by centrifugation for 15 min,
and the supernatant was incubated with gentle agitation overnight with
0.5% protein A-Sepharose and the appropriate antibody at the following
concentrations: 1 µl of antibody/1-ml sample for SPA-470 and
anti-calreticulin; 2 µl of antibody/1-ml sample for anti-PDI and
anti-p57; and 5 µl of antibody/1-ml sample for anti-calnexin and
anti-type III procollagen. For the anti-BiP and anti-GRP94 antibodies,
protein G-Sepharose was used instead of protein A-Sepharose, and the
antibodies were used at a concentration of 2 µl of antibody/1-ml
sample. The immunocomplexes were pelleted by centrifugation for 30 s and washed twice with immunoprecipitation buffer, once with high salt
buffer (50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1%
(v/v) Triton X-100, 500 mM NaCl, 0.02% (w/v)
NaN3), and once again with immunoprecipitation buffer. The
immunoprecipitates were then prepared for analysis by SDS- PAGE.
Immunofluorescence--
F2 cells were plated to 80% confluence
on 13-mm coverslips and cultured overnight in complete medium
supplemented with 50 µg/ml ascorbate if required. The following
procedures were performed at room temperature. Cells were washed twice
in PBS and then fixed with 4% (w/v) paraformaldehyde in PBS for 15 min. Three washes with PBS were followed by three with
NaBH4 solution (0.5 mg/ml in PBS) to reduce
autofluorescence, and where appropriate the cells were permeabilized
with 0.1% (v/v) Triton X-100, 0.5% (w/v) SDS in PBS for 4 min. The
coverslips were rinsed in PBS before incubation with a 1/100 dilution
of the anti-Myc mouse monoclonal antibody (9E10) for 1 h. After
washing, incubation with a 1/40 dilution of a donkey anti-mouse Cy2
conjugate (Amersham Pharmacia Biotech, Buckinghamshire, UK) was
performed for 1 h.
Poly(L-Proline) Precipitation--
Fractions from
sucrose gradients were incubated with gentle agitation for 60 min with
10% (w/v) poly(L-proline)-Sepharose. The
poly(L-proline) precipitate was pelleted by centrifugation at 12,000 × g for 15 min and washed four times with
P4-H buffer (10 mM Tris, pH 7.8, 0.1 M NaCl,
100 mM glycine, 0.1% Triton X-100). Proteins were eluted
from the poly(L-proline)-Sepharose by incubating the
precipitate with gentle agitation for 5 min in P4-H buffer containing 3 mg ml
1 poly(L-proline) (3000 kDa). Proteins
were precipitated from the eluate by the addition of trichloroacetic
acid to 15% (w/v) and acetone to 25% (v/v), and the sample was
incubated with gentle agitation for 60 min. Precipitates were harvested
by centrifugation at 12,000 × g for 15 min, washed
with acetone, and prepared for SDS-PAGE analysis.
SDS-PAGE--
Immunoprecipitates were resuspended in SDS-PAGE
buffer (62.5 mM Tris-HCl, pH 6.8, 1% (w/v) SDS, 10% (v/v)
glycerol, bromphenol blue) and boiled for 5 min in either the absence
(non-reducing) or presence (reducing) of 50 mM dithiothreitol.
Samples were subjected to electrophoresis through a polyacrylamide gel,
and the dried gels were visualized by autoradiography using Kodak
BIOMAX film.
 |
RESULTS |
The primary purpose of this study was to investigate the molecular
mechanism leading to the retention of partially folded procollagen
chains within the ER. To facilitate this study we first constructed a
cell line expressing a procollagen "mini-chain," designated
pro-
1(III)
1, that we have previously studied using a
semi-permeabilized cell expression system (31). These experiments have
shown that procollagen "mini-chains" translated in the presence of
SP cells are efficiently translocated, modified, and assembled into a
correctly aligned triple helix (25). Here we used HT1080 as the host
cell line because these cells do not express any fibril-forming procollagens and have been shown to support the folding and assembly of
types I and II procollagen (32, 33).
Our previous work with this procollagen polypeptide demonstrated that
assembly occurred in distinct stages. Initially a trimer is formed that
is stabilized by inter-chain disulfides within the C-propeptide. Once
sufficient proline residues within the triple helical domain are
hydroxylated, the triple helix folds in a C to N direction resulting in
the N-propeptide associating and forming interchain disulfide bonds.
Thus the formation of interchain disulfides within the N-propeptide
does not occur until the triple helical domain has folded. These two
disulfide-bonded intermediates can be separated by non-reducing
SDS-PAGE allowing an evaluation of the folding status of the expressed
procollagen chains (25).
Cell lines expressing pro-
1(III)
1 were isolated as individual
clones, and one of these cell lines, designated B12, was used for this
study. To determine the folding status of the pro-
1(III)
1 chains
synthesized in this cell line, we pulse-labeled newly synthesized proteins and specifically immunoprecipitated the pro-
1(III)
1 chains from cell lystates. The resulting immunoprecipitates were then
separated under reducing and non-reducing conditions (Fig. 1). The cells were radiolabeled in the
presence or absence of ascorbate (vitamin C), an essential co-factor of
P4H. In the absence of added ascorbate P4H activity is reduced leading
to the synthesis of underhydroxylated procollagen chains that do not
form a triple helix.

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Fig. 1.
Time course of disulfide bond formation
during the synthesis of
pro- 1(III) 1 in the
presence and absence of added ascorbate. A stable cell line (B12)
expressing pro- 1(III) 1 chains was pulse-labeled for 20 min with
[35S]Met/Cys and chased in the presence of cycloheximide
and L-methionine. Ascorbate was either absent or present in
the pulse and chase medium as indicated. After the times indicated, the
cells were rinsed in PBS containing NEM and lysed in a buffer also
containing NEM. Pro- 1(III) 1 chains were immunoprecipitated from
cell lysates and separated either under reducing (A) or
non-reducing (B) conditions and visualized by
autoradiography.
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In the absence of added ascorbate, radiolabeled pro-
1(III)
1
chains were visualized under non-reducing conditions as two forms with
apparent molecular masses of 292 and 210 kDa (Fig. 1B).
These forms correspond to a trimer and dimer respectively stabilized by
the formation of inter-chain disulfide bonds within the C-propeptide.
In the presence of ascorbate the chains migrated predominantly with an
apparent molecular mass of 260 kDa. We have shown previously that this
form represents a trimer that is stabilized by the formation of
inter-chain disulfide bonds within the C- and N-propeptide (25). The
higher mobility of this trimer relative to the trimer stabilized by
inter-chain disulfide bonds within the C-propeptide only is a result of
a decrease in the hydrodynamic volume (34). These results demonstrate
that the pro-
1(III)
1 synthesized in this cell line folds to form
a triple helical molecule when expressed in the presence of co-factors
for P4H and that we can distinguish between two disulfide-bonded
intermediates by carrying out electrophoresis under non-reducing
conditions. For simplicity these intermediates will subsequently be
referred to as a non-helical or helical trimer.
Effect of Ascorbate on Procollagen Secretion--
Incubating the
B12 cell line in the absence of ascorbate clearly prevents folding of
the procollagen molecule leading to a build-up of non-helical trimer.
To investigate whether the cells could recognize this intermediate and
retain it within the cell, we examined the effect of ascorbate
depletion on the secretion of pro-
1(III)
1 chains. Proteins
synthesized by B12 cells were radiolabeled and chased over a 180-min
period (Fig. 2). Pro-
1(III)
1 chains
were immunoprecipitated from the cell lysates (Fig. 2A) and
from the medium (Fig. 2B). Immunoprecipitated proteins were subjected to SDS-PAGE under reducing conditions. In the presence of
ascorbate, pro-
1(III)
1 chains were secreted into the medium after
40 min of chase with a maximal level of secretion of radiolabeled protein at 80 min of chase (Fig. 2B). In the absence of
added ascorbate, very little radiolabeled material was secreted, the small amount present in the medium after 80 min of chase probably representing a minimal amount of protein that folds even in the absence
of added ascorbate (see Fig. 1B, 40 min). Thus, it would appear that in the absence of added ascorbate the non-helical trimers
formed are retained within the cell.

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Fig. 2.
Time course of secretion of
pro- 1(III) 1
synthesized in the presence or absence of ascorbate. B12 cells
were pulse-labeled for 20 min and chased in the presence of
cycloheximide as described in Fig. 1. Ascorbate was either present or
absent from the medium during the time course as indicated.
Pro- 1(III) 1 chains were precipitated from the medium
(B) or cell lysates (A) and separated by SDS
carried out under reducing conditions.
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This retention could be a consequence of the ascorbate having a general
effect on protein secretion rather than its role as a co-factor of P4H.
We investigated this possibility by using an alternative approach to
inhibit P4H activity. Along with ascorbate and oxygen, iron is an
essential co-factor of this enzyme (10). Chemicals that chelate iron
such as
,
'-dipyridyl are inhibitors of this enzyme and have been
shown to be effective when added to cells grown in culture leading to
the under-hydroxylation of procollagen (35). In the present study, a
pulse labeling and chase experiment was carried out using B12 cells
incubated in the presence of
,
'-dipyridyl (Fig.
3). Here no pro-
1(III)
1 chains were
secreted even after 180 min of chase (Fig. 3B, lanes 1-5)
with all the radiolabeled chains remaining within the cell (Fig.
3A, lanes 1-5). When these samples were separated under non-reducing conditions the pro-
1(III)
1 chains separated as non-helical trimers (results not shown). When B12 cells were labeled in
the presence of
,
'-dipyridyl and then incubated in medium containing iron and protein synthesis inhibitor, a reversal of this
block in secretion was observed (Fig. 3B, lanes 18-20).
After 40 min of chase in the presence of iron, the radiolabeled protein in the cell lysate decreased in electrophoretic mobility when separated
under reducing conditions (Fig. 3A, compare lanes
6 and 7). This decrease in mobility is characteristic
of proline hydroxylation which is thought to affect SDS binding (36).
Pro-
1(III)
1 chains were secreted into the medium after 80 min of
chase (Fig. 3B, lane 18), indicating that the secretion
block is reversible and that the time taken for the non-helical trimer
to be hydroxylated, folded, and secreted is between 40 and 80 min. This
experiment also demonstrates that the retention of the
pro-
1(III)
1 chains within the cell is due to a lack of
hydroxylation or folding rather than a general effect of ascorbate
on the secretory pathway.

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Fig. 3.
Time course of secretion from cells of
pro- 1(III) 1 labeled
in the presence of
, '-dipyridyl and chased in
the presence and absence of iron. Proteins synthesized by B12
cells were pulse-labeled for 20 min and then incubated in the presence
of cycloheximide. The iron chelator , '-dipyridyl was present
during the labeling period but was either present (A, lanes
1-5, and B, lanes 11-15) or absent (A, lanes
6-10, and B, lanes 16-20) during the chase medium.
Ferrous ions were added to the chase medium to reverse the inhibitory
effect of , '-dipyridyl (A, lanes 6-10, B lanes
16-20). Ascorbate was added to the medium throughout the labeling
and chase periods. At the chase times indicated the pro- 1(III) 1
chains were isolated from the cell lysates or medium and separated by
carrying out SDS-PAGE under reducing condition.
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Localization of the Block in Secretion of Non-helical Procollagen
Trimer--
Having established that cells can recognize and retain
non-helical procollagen chains, we next addressed the question of where the block in secretion occurs. For this study we constructed a procollagen chain where the C-propeptide was replaced with the transmembrane domain from influenza virus hemagglutinin. We have shown
that this protein can fold correctly to form a triple helical molecule
when expressed in SP cells (7). We also included a Myc epitope at the N
terminus of the mature polypeptide to allow visualization by indirect
immunofluorescence. As the synthesized polypeptide remains
membrane-bound, we could also monitor secretion by visualizing
cell-surface expression. A stable cell line expressing this construct
was isolated, cloned, and designated F2. Expression of the
transmembrane procollagen was visualized in F2 cells using an antibody
against the Myc epitope (Fig. 4). When
cells were incubated in the absence of ascorbate, no fluorescence was
detected unless the cells were permeabilized prior to addition of the
primary antibody (Fig. 4,
Triton). When cells were
permeabilized a clear reticular pattern of staining was seen
characteristic of the ER network (Fig. 4, +Triton). However,
incubation of F2 cells in the presence of ascorbate led to the
appearance of cell-surface staining before and after permeabilization.
These results clearly demonstrate the tight regulation of procollagen
secretion as no cell-surface staining was seen in the absence of added
ascorbate, thus confirming the conclusions drawn from the
pulse-labeling experiments. The pattern of staining in the absence of
added ascorbate is consistent with a block in secretion at the level of
the ER.

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Fig. 4.
Localization of procollagen chains retained
within the cell. A stable cell line (F2) expressing a Myc-tagged
membrane-bound form of procollagen was grown on coverslips incubated
either in the presence or absence of ascorbate (ASC) as
indicated. Cells were fixed and either permeabilized with Triton X-100
or not permeabilized before addition of anti-Myc antibody as indicated.
The cells were then incubated in the presence of secondary antibody,
and immunofluorescence was visualized.
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Effect of Azetidine-2-carboxylic Acid Incorporation on Procollagen
Hydroxylation and Secretion--
The retention of procollagen within
the cell could be a result of the lack of hydroxylation or a lack of
folding of the collagen triple helix. To address this point we created
conditions that prevented folding of the triple helical domain but did
not affect hydroxylation of proline residues. The formation of the
triple helix of procollagen can be prevented by the incorporation of proline analogues, such as azetidine-2-carboxylic acid into the polypeptide chain (11). To examine the effect of incorporation of this
proline analogue on procollagen secretion, B12 cells were pulse-labeled
for 20 min and chased over a 180-min period in the presence of
azetidine-2-carboxylic acid and in either the absence (Fig.
5, A-C, lanes 1-5) or
presence (lanes 6-10) of
,
'-dipyridyl. In addition, a
pulse-chase was performed in the absence of the analogue and presence
of
,
'-dipyridyl in order to ascertain the secretion kinetics of
the under-hydroxylated non-helical trimer (lane 11).
Pro-
1(III)
1 chains were immunoprecipitated from the cell lysates
(Fig. 5, A and B) and the media (Fig.
5C). When separated under reducing conditions (Fig.
5A), the pro-
1(III)
1 chains synthesized in the
presence of the analogue and
,
'-dipyridyl (lanes
6-10) had a faster mobility than the chains synthesized in the
presence of the analogue but in the absence of
,
'-dipyridyl (lanes 1-5). This indicates that in the presence of the
proline analogue, hydroxylation of proline residues occurs. This
probably reflects the fact that only a small percentage of the proline residues in the polypeptide will be replaced with the proline analogue.
When the folding status of the protein synthesized in the presence of
the analogue was analyzed, most of the material migrated with the
mobility of the non-helical trimer even when synthesized in the absence
of
,
'-dipyridyl (Fig. 5B, lanes 1-5). This contrasts
with the almost complete folding and assembly of a helical trimer in
the absence of the analogue (Fig. 1B). This demonstrates
that although hydroxylation occurred the triple helical domain could
not fold correctly. However, the incorporation of the proline analogue
did not dramatically affect the folding and association of the
C-propeptide as folding of this domain is critical for the formation of
an inter-chain disulfide-bonded trimer (29).

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Fig. 5.
Retention of hydroxylated non-helical trimers
of pro- 1(III) 1.
B12 cells were pulse-labeled for 20 min as described previously in the
presence (lanes 1-10) or absence (lane 11) of
the proline analogue azetidine-2-carboxylic acid (AZE).
Cells were then incubated for the indicated times in the presence
(lanes 6-11) or absence (lanes 1-5) of
, '-dipyridyl (DP). Ascorbate was present in the medium
throughout the experiment. The pro- 1(III) 1 synthesized were
isolated from cell lysates (A and B) or the
medium (C) and separated by SDS-PAGE under reducing
(A and C) or non-reducing (B)
conditions. hyd, hydroxylated; unhyd,
unhydroxylated.
|
|
Analysis of the medium from cells incubated in the presence of the
proline analogue revealed that very little pro-
1(III)
1 was
detected (Fig. 5C). This low level of secretion was also
seen in the presence of
,
'-dipyridyl and could reflect release of intracellular material due to prolonged incubation with the proline analogue. This contrasts with the rapid secretion of pro-
1(III)
1 chains from B12 cells in the absence of analogue (Fig. 2B).
These results indicate that the retention of procollagen within the cell is due to a lack of folding of the collagen triple helix rather
than a lack of proline hydroxylation.
Cross-linking of Procollagen to Proteins within the ER--
The
retention of non-native proteins within the cell has been hypothesized
to be mediated by ER resident chaperones that confer their ER residency
to such proteins by selectively binding to them (1). Assuming that such
a process was responsible for the retention of procollagen chains
within the cell, a cross-linking approach was adopted in an attempt to
identify ER resident proteins that selectively bound to non-helical
pro-
1(III)
1 chains. Proteins synthesized by B12 cells were
radiolabeled for 1 h and subsequently incubated in either the
absence or presence of DSP, a thiol-cleavable homo-bifunctional reagent
that is specific for primary amine groups. Ascorbate was omitted in
order to minimize the conversion of the non-helical trimer to the
helical trimer. Cross-linked complexes containing the following well
characterized ER resident proteins PDI, ERp57, calreticulin, calnexin,
BiP, GRP94, and Hsp47 were separately immunoprecipitated from the cell
lysates before or after prior denaturation of the lysate by boiling in SDS.
The immunoprecipitated complexes were subjected to SDS-PAGE analysis
under reducing conditions in order to cleave the cross-linker and allow
the identification of the individual components of the cross-linked
complexes. Most of the antibodies employed effectively immunoprecipitated their corresponding target proteins from the cross-linked lysates under both native and denaturing conditions with
the exception of the BiP antibody which worked best after denaturation
of the sample (Fig. 6, lanes 6 and 14). The antibody to the pro-
1(III)
1 chain also
reacted poorly with its antigen under native conditions (Fig. 6,
lane 1).

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Fig. 6.
Cross-linking of non-helical trimer of
pro- 1(III) 1 to ER
resident proteins. Proteins synthesized by B12 cells were labeled
for 1 h and proteins cross-linked with DSP as described under
"Material and Methods." Cells were lysed and immunoprecipitations
carried out directly (lanes 1-8) or after denaturation of
the proteins (lanes 8-15) with the antibodies indicated.
Samples were separated by SDS-PAGE under reducing conditions.
|
|
Several co-immunoprecipitating bands were seen with antibodies to ER
proteins when lysates were not denatured prior to immunoprecipitation (Fig. 6, lanes 2-5). These were not covalently cross-linked
as they were absent in the immunoprecipitates from denatured lysates (Fig. 6, lanes 10-13). After cross-linking, the
pro-
1(III)
1 chain was present in the immunoprecipitate of the
antibody against PDI, but only when the immunoprecipitation was
performed under native conditions (Fig. 6, lanes 2 and
10). The pro-
1(III)
1 chain was not
co-immunoprecipitated from uncross-linked cell lysates with the
antibody against PDI or other ER resident proteins (results not shown).
Pro-
1(III)
1 chains were not present in cross-linked lysates
immunoprecipitated with antibodies against ER resident proteins other
than PDI. Some cross-linking of Hsp47 to proteins other than
pro-
1(III)
1 was observed (Fig. 6, lanes 8 and
16). Furthermore, an association of Hsp47 with
pro-
1(III)
1 chains could not be demonstrated using additional
cross-linking reagents (results not shown). These results show that by
using this approach we were not able to cross-link the non-helical
trimer to any of the major ER proteins. However, we were able to
cross-link this molecule to a protein that associated with PDI and
could be co-immunoprecipitated with the antibody under native
conditions. Other proteins co-immunoprecipitating with PDI under native
conditions include polypeptides with relative molecular masses of
approximately 60, 62, 200, and 250 kDa (Fig. 6, lane 2). The
200-kDa polypeptide was sensitive to digestion with collagenase
(results not shown) and is probably type IV collagen, whereas the
polypeptide at 250 kDa remains unidentified. The polypeptides at 60 and
62 kDa were shown to co-migrate with the two glycoforms of the
-subunit of P4H (Fig. 7). Here in the
absence of cross-linker (lanes 1-3), neither PDI nor the
-subunit antibodies co-immunoprecipitate the pro-
1(III)
1
chain. However the
-subunit of P4H is co-immunoprecipitated with the
antibody to PDI (lane 2). In the presence of cross-linker (lanes 4-6), the
-subunit antibody clearly
immunoprecipitates the pro-
1(III)
1 chain (lane 6). As
PDI is the
-subunit of P4H, it seems likely that P4H binds to the
non-helical trimer and can be cross-linked via the
-subunit of P4H
and can consequently be immunoprecipitated with antibodies to PDI. This
suggests that P4H itself is regulating the intracellular retention of
procollagen.

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Fig. 7.
Cross-linking of
pro- 1(III) 1 chains to
the -subunit of prolyl 4-hydroxylase.
Proteins synthesized by B12 cells were labeled for 1 h and
proteins and immunoprecipitations either carried out directly
(lanes 1-3) or after cross-linking with DSP (lanes
4-6) with the antibodies indicated. Samples were separated by
SDS-PAGE under reducing conditions.
|
|
Demonstration of the Association of P4H with Procollagen by
Velocity Centrifugation--
The results obtained with cross-linking
suggest that non-helical procollagen chains form a complex with P4H. To
confirm these results using an alternative approach and to gain some
information on the components of the complex, we carried out velocity
centrifugation on cell lysates. B12 cells were radiolabeled for 1 h, and proteins present in the cell lysate were separated on a 5-25%
sucrose gradient. Ascorbate was omitted from the media in order to
minimize the rate of conversion of the non-helical trimer to the
helical trimer. After centrifugation the gradients were divided into 14 fractions with fraction 1 being at the bottom of the gradient and
fraction 14 being at the top (Fig. 8). To
determine the position within the gradient of individual molecules that
are not part of a complex, lysates were first denatured with SDS, and
proteins were precipitated with specific antibodies. Alternatively,
lysates were separated without denaturation and P4H isolated from
fractions by binding to prolyl-Sepharose, an affinity matrix that will
specifically bind to free P4H (37). The results show that P4H separated
in fractions 5, 6, and 7 (Fig. 8A), and PDI separated in
fractions 9-14 (Fig. 8B), whereas pro-
1(III)
1
separated in fractions 3, 4, and 5 (Fig. 8C). Thus there was
a clear separation of these three proteins by velocity
centrifugations.

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Fig. 8.
Sucrose gradient analysis of cell lysates
labeled in the absence of ascorbate. Proteins synthesized by B12
were labeled for 1 h prior to lysis with Nonidet P-40 lysis buffer
as described. Lysates were either separated directly (A, C,
and D) or denatured with SDS (B) prior to
separating onto a 5-25% sucrose gradient. Gradients were fractionated
and each fraction immunoprecipitated with antibodies to PDI or
pro- 1(III) 1 either directly (B and D) or
after denaturation with SDS (C). Free prolyl 4-hydroxylase
was also isolated from each fraction by binding to
poly-L-proline (A). Samples were separated by
SDS-PAGE under reducing conditions.
|
|
We then separated the cell lysate without prior denaturation and
immunoprecipitated the resulting fractions with antibodies to PDI (Fig.
8D). PDI itself was present as expected in fractions 9-14,
and co-precipitated P4H
-subunit was also, as expected, present in
fractions 5, 6, and 7. However, PDI and the
-subunit of P4H were
also seen in fractions 2, 3, and 4 along with pro-
1(III)
1 chains.
This clearly demonstrates that a complex between P4H and the
procollagen chain was present in the cell lysate and that this complex
could be precipitated with antibodies to PDI. The fact that we could
isolate such a complex in the absence of cross-linking after sucrose
gradient fractionation but not directly from cell lysates by
co-immunoprecipitation could be due to a stabilization of the
P4H-procollagen interaction in the presence of sucrose. These results
confirm the cross-linking experiments demonstrating that P4H interacts
and forms a stable complex with procollagen chains.
Dissociation of P4H from Procollagen Chains following
Folding--
If P4H regulates the secretion of procollagen by binding
to non-helical trimers then this interaction should persist until the
protein folds, whereupon the complex dissociates allowing secretion
from the cell. To determine whether procollagen binding to P4H is
regulated by the folding status of the pro-
1(III)
1 chains, B12
cells were pulsed-labeled in media containing
,
'-dipyridyl. The
labeled cells were subsequently chased for 40 min with protein synthesis inhibitors either in the presence of the chelator (Fig. 9A) or excess ferrous ions
(Fig. 9B). The 40-min chase period performed in the presence
of ferrous ions was shown to be sufficient for the complete conversion
of the under-hydroxylated non-helical to the hydroxylated helical
trimer (Fig. 3, and results not shown). The proteins present in the
cell lysate were separated on a 5-25% sucrose gradient, and complexes
containing PDI were immunoprecipitated from the fractions isolated. In
the presence of
,
'-dipyridyl a stable complex was formed as
evidenced by the co-immunoprecipitation of P4H and pro-
1(III)
1
chains (Fig. 9A, fractions 1-5). However, after
incubation in the presence of excess iron, no complex was detected
(Fig. 9B). This demonstrates that following addition of iron
P4H dissociates from the procollagen chain that has folded allowing
subsequent secretion from the cell (Fig. 3B).

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Fig. 9.
Dissociation of the complex between
pro- 1(III) 1 and P4H
after restoration of hydroxylation. Proteins synthesized by B12
cells were labeled for 1 h in the presence of , '-dipyridyl
and chased either in the presence of the iron chelator (A)
or in the presence of ferrous ions (B) for 40 min. After the
chase period, cells were lysed, and cell lysates were separated by
sucrose gradient fractionation as described. Each fraction was
immunoprecipitated directly with antibodies to PDI and separated by
SDS-PAGE carried out under reducing conditions.
|
|
The complex formed between P4H and procollagen could be regulated not
by the folding status but by the extent of hydroxylation of the proline
residues within the collagenous domain. Indeed in vitro
binding studies have shown that the affinity of P4H for the collagen
helical domain is higher for unhydroxylated chains than hydroxylated
chains (38). To address this point we incubated cells in the presence
of the proline analogue azetidine-2-carboxylic acid. As described in
Fig. 5, this does not prevent hydroxylation of the triple helical
domain but does prevent folding and subsequent secretion. Cells were
pulse-labeled in the presence of ascorbate, cross-linking agent was
added, and cell lysates were immunoprecipitated with antibodies to PDI.
In the absence of azetidine-2-carboxylic acid a complex between P4H and
the synthesized pro-
1(III)
1 chains was only seen when
,
'-dipyridyl was included during the labeling period (Fig.
10, lanes 2 and
4). However, when the proline analogue was present a complex
formed between P4H and pro-
1(III)
1 chains even in the absence of
,
'-dipyridyl. This demonstrates that non-helical but hydroxylated
procollagen chains can still associate with P4H indicating that the
folding status of the chains rather than hydroxylation of proline
residues regulate the binding of P4H to procollagen chains within the
cell.

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Fig. 10.
Cross-linking of P4H to hydroxylated and
non-hydroxylated non-helical trimers of
pro- 1(III) 1.
Cells were labeled for 1 h either in the presence (lanes
5-8) or absence (lanes 1-4) of azetidine-2-carboxylic
acid. Labeling was carried out in the presence of ascorbate either in
the presence (lanes 3, 4, 7, and 8) or absence
(lanes 1, 2, 5, and 6) of , '-dipyridyl
(DP). After the labeling period, cells were treated with DSP
as described, and proteins were immunoprecipitated from cell lysates
with antibodies to PDI. Immunoprecipitated proteins were separated by
SDS-PAGE under reducing conditions.
|
|
 |
DISCUSSION |
It has been known for some time that the secretion of procollagen
from mammalian cells is regulated by the extent of hydroxylation of
this molecule (9, 22). Under normal physiological conditions, the level
of expression of procollagen in the extracellular matrix can be
affected dramatically by the availability of co-factors for P4H such as
ascorbate, oxygen, and iron. This has led to the hypothesis that there
is a mechanism by which the cell can recognize unhydroxylated
non-helical molecules and selectively retain them within the cell. The
molecular mechanism leading to this retention has been postulated to be
mediated either by P4H itself (22, 39) or by the collagen-binding
protein Hsp47 (15), which has been shown to bind to procollagen within
the ER. Support for a role of P4H in the retention process comes from
in vitro binding experiments which demonstrate that
formation of a complex between P4H and unhydroxylated procollagen
chains occurs and that the affinity of the enzyme for its substrate
diminishes upon hydroxylation of the triple helix (38). Indeed
unhydroxylated procollagen chains that are allowed to fold to form a
triple helix at low temperatures are no longer a substrate for the
enzyme (35). Hsp47 on the other hand shows no ability to discriminate
between hydroxylated and unhydroxylated forms of procollagen (19), but it is clear that the binding can be regulated by subtle changes in pH
(40). Thus, Hsp47 may associate with procollagen within the relatively
neutral pH of the ER and then dissociate upon transport to more acidic
compartments in the secretory pathway.
To determine the molecular mechanism underlying the retention of
procollagen within the cell, we established cell lines expressing a
"mini-gene" coding for pro-
1(III) chains containing an "in frame" deletion within the collagen triple helix (29). We established conditions that generated an accumulation of non-helical trimeric molecules within the cell, and we looked for associated ER proteins. To
achieve this aim we cross-linked any potential associating proteins and
carried out immunoprecipitations with a number of well characterized ER
resident proteins. From the results we identified P4H as a potential
interacting partner, and this was confirmed by carrying out velocity
centrifugation to separate a complex between procollagen and P4H. No
binding to Hsp47 was detected even though binding to the full-length
pro-
1(III) chain has been reported (41). The reason for the lack of
binding of the mini-chain to Hsp47 could be due to the binding site for
Hsp47 being contained within the deleted sequence within the triple
helical domain. However, this lack of binding did allow us to study the
regulation of secretion of a collagenous protein in the absence of any
interaction with Hsp47. It is clear from our results that the retention
is still tightly regulated and is almost certainly due to the
association of P4H with the non-triple helical collagenous domain.
Thus, P4H fulfills all the characteristics of a protein required to
retain non-helical procollagen within the cell as follows: (i) it forms a stable association with non-helical molecules; (ii) this association is regulated by the folding status rather than hydroxylation; (iii) it
is localized in the ER due the -KDEL sequence at the C terminus of its
-subunit (PDI) (42); and (iv) it dissociates rapidly from the
protein upon folding of the triple helical domain.
The ability of P4H to interact also with hydroxylated but non-helical
procollagen chains also explains how the cell can recognize and retain
procollagen molecules containing naturally occurring mutations within
the triple helical domain. These mutations give rise to genetic
disorders such as osteogenesis imperfecta (14) and Ehlers-Danlos
syndrome type IV (43). In these cells, association of wild type and
mutant chains occurs normally, but folding of the triple helix is
prevented or slowed at the point of the mutation due to replacement of
glycine in the Gly-X-Y repeats with a more bulky amino acid
side chain. These molecules are retained within the cell resulting in a
dramatic reduction in the secretion of procollagen into the
extracellular matrix. Thus even though the mutant chains are
hydroxylated by P4H they are nevertheless retained due to lack of
folding. It is clear that P4H can recognize these chains as
non-helical, rather than unhydroxylated, and associates with them to
prevent secretion. The association of mutant chains with wild type
chains leads to retention of wild type chains within the cell
amplifying the severity of the mutation resulting in severe, often
lethal phenotypes.
This mechanism is distinct from two other mechanisms for retention of
procollagen chains within the cell. Procollagen chains with mutations
within the C-propeptide are retained within the cell due to their
interaction with BiP (21). Here the mutation prevents folding of the
C-propeptide with the resulting unfolded polypeptide interacting with
BiP and subsequently being degraded (44). As the mutant chains do not
interact with the wild type chains, these mutations generally give rise
to less severe phenotypes. Also, during the folding and assembly of
type I procollagen, which is composed of one pro-
2(I) and two
1(I) chains, the individual monomeric pro-
2(I) chains bind to
PDI. This binding retains them within the ER prior to assembly with
pro-
1(I) chains (23). Hence by three separate mechanisms procollagen
chains can be retained within the cell. This mechanism of retention
depends on whether they have a general folding defect and bind to BiP,
are folded into monomers but not formed trimers and bind to PDI, or
have formed non-helical trimers and bind to P4H.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Stephen High and Dr. David John
for comments on the manuscript.
 |
FOOTNOTES |
*
This work was supported by Wellcome Trust Grant 50600, The
Royal Society, and the Biotechnology and Biological Sciences Research Council.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.
Present address: School of Biochemistry and Molecular Biology, the
University of Leeds, Leeds, LS2 9JT, UK.
§
To whom correspondence should be addressed. Tel.: +44-61-275-5103;
Fax: 44-61-275-5082; E-mail: neil.bulleid{at}man.ac.uk.
 |
ABBREVIATIONS |
The abbreviations used are:
ER, endoplasmic
reticulum;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel
electrophoresis;
PMSF, phenylmethylsulfonyl fluoride;
DSP, dithiobis(succinimidyl propionate);
PDI, protein-disulfide isomerase;
SBTI, soybean trypsin inhibitor;
P4H, prolyl 4-hydroxylase.
 |
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