(Received for publication, February 15, 1995; and in revised form, May 16, 1995)
From the
Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic
reticulum (ER). Both of these proteins are closely associated with
polysome-associated
Type I procollagen is a trimeric molecule composed of two
pro An important feature of native triple
helical structure of procollagen is that this structure can include
only trans-peptide bonds. However, it has been estimated that
16% of the X-Pro bonds and 8% of the X-Hyp bonds in
nascent type I collagen are in cis- rather than trans-confirmation(9) . Since, the temperature for
folding kinetics of collagen is consistent with the normal Arrhenius
activation energy for cis/trans-isomerization of peptide
bonds, cis/trans-isomerization is considered to be a
significant factor in collagen
folding(10, 11, 12) . Consequently, when some
of these enzymes are inhibited by the addition of cyclosporin A (CsA),
the rate of in vivo folding of collagen is
retarded(13) . The cyclophilin proteins (CyPs) are a highly
conserved family of PP A number of ER resident proteins, designated
as molecular chaperones, have been associated with polysome-associated
pro-
For Western blots, proteins run on SDS-PAGE
were immediately electrotransferred to nitrocellulose paper. The paper
was blocked with 10% non-fat dry milk in 10 mM Tris-HCl, pH
7.4, 0.9 M NaCl (TBS) for 1 h and then in TBS/non-fat dry milk
with 2% normal goat serum (Life Technologies, Inc.). Antiserum or
preimmune serum was diluted 1:2000 in the same buffer and incubated
with gentle shaking overnight. The nitrocellulose was then rinsed three
times for 5 min in TBS/Tween. The secondary antibody, affinity-purified
biotinylated goat-anti-rabbit IgG (Fc) (Kirkegaard and Perry Labs,
Gaithersburg, MD) was diluted to 0.9 µg/ml and incubated with the
paper for 2 h. Washing between steps was performed three times for 30
min with 50 mM Tris-HCl, 0.9 M NaCl, 0.05% Tween, pH
7.4. The blot was visualized using ECL Western blot protocol (Amersham
Corp.). Hsp47 rabbit polyclonal antibodies were prepared against a
22-mer peptide corresponding to the N-terminal sequence of mouse Hsp47
that was conjugated to Keyhole limpet hemocyanin and was cross-reactive
with mouse 3T6 Hsp47(38) . Polyclonal rabbit anti-mouse
To study procollagen processing, 3T6 mouse fibroblasts were
labeled with [
Figure 1:
Q-Sepharose fractions of
[
Previous studies had also shown that microsomes may
possess a high PP To further characterize this protein, we
sought to determine if this protein possessed an N-terminal signal
sequence that directs the protein to the ER and a C-terminal
decapeptide extension distinct from cytosolic and mitochondrial forms
of cyclophilin(43) . To address these questions, total RNA was
isolated from Affinity-purified rabbit
antibodies prepared against a synthetic peptide corresponding to the
C-terminal decapeptide of CyPB in Western blots showed reactivity
against the 20.6-kDa protein in immunoprecipitates obtained with
anti-collagen or anti-Hsp47 antibodies (Fig. 1). Also, when the
dense polysome Q-Sepharose-retained fraction was immunoprecipitated
with anti-CyPB antibodies, a number of proteins were noted to
coimmunoprecipitate with CyPB. Some of these proteins were identified
by susceptibility to digestion by bacterial collagenase and/or Western
blot analysis as procollagen, PDI, Hsp47, and the translocon component
Sec61p (Fig. 2).
Figure 2:
Immunoprecipitate obtained with anti-CyPB
antibodies of dithiobis(succinimidyl propionate) cross-linked dense
polysomes derived from 3T6 cells. Dense polysomes prepared and
fractionated as described in Fig. 1were immunoprecipitated with
anti-CyPB antibodies. LaneA represents the
immunoprecipitate digested with bacterial collagenase (28) prior to SDS-PAGE. LaneB depicts the
immunoprecipitate obtained with anti-CyPB antibodies. The proteins
labeled in the figure were confirmed by Western blot. The proteins
identified were laneC, Sec61p; laneD, Hsp47; laneE, CyPB; laneF, GRP78; laneG,
GRP94.
To determine whether CyPB was retained
within the ER as previously suggested (45) or implied further
in the secretory pathway, cells were pulse labeled with
[
Figure 3:
Kinetics of total cellular procollagen,
Hsp47-bound procollagen, and CyPB-bound procollagen. Pulse-chase
experiments were performed by labeling cells with 100 µCi/ml
[
To limit procollagen secretion to certain
compartments within the secretory pathway, cells were treated with
agents that inhibited diacylglycerol/phorbol ester-binding proteins (41) or multiple GTP-binding proteins including heterotrimeric
G protein(s)(40) . Calphostin C, a specific inhibitor of the
highly conserved cysteine-rich C
Figure 4:
Comparison of kinetics of Hsp47 bound
procollagen in control, mastoparan, calphostin C, and GTP
Figure 5:
Comparison of kinetics of CyPB-bound
procollagen in control, mastoparan, calphostin C, and GTP
Figure 6:
A,
distribution of Hsp47, CyPB, and procollagen I in 3T6 cells after
modulation of ER to Golgi transport. Cells were incubated for 40 min in
the absence or presence of mastoparan, calphostin C, or GTP
In
previous immunocytochemical studies, we noted that Hsp47 was
colocalized with procollagen in what appeared to be pre-Golgi vesicular
structures during tooth development(46) . To verify this
relationship, cells were treated with GTP
Figure 7:
Kinetics
of total cellular procollagen, Hsp47-bound procollagen, and CyPB-bound
procollagen following treatment of cells with cyclosporin A. Cells were
treated with 1 µM cyclosporin A for 1 h. Pulse-chase
experiments were then performed by labeling cells with 100 µCi/ml
[
Although previous studies have
demonstrated that Hsp47 and other molecular chaperones remain bound to
altered procollagen in Currently, evidence is accruing
that suggests that proteins are sorted and concentrated during export
from the ER. One paradigm that has been advocated presumes that the
protein to be transported is insensitive and relies on the passive
movement of exported proteins into designated regions of the ER
specialized in export(51) . However, to ensure net
concentration of the protein, it appears that a mechanism must exist to
prevent backflow. To account for this, two additional models that hinge
on the interaction of the transported protein with transport machinery
have been proposed(51) . One of the schemes is analogous to the
process of receptor-mediated endocytosis to concentrate-transported
protein (52) . The other suggests that signals also participate
in the active recruitment of coat components, leading to the formation
of nascent budding sites(40, 51, 53) . In
both instances, release from a chaperone-mediated retention system is
suggested as the first step in a more regulated pathway involving
multiple transport components that initiate, facilitate, or enhance the
efficiency of transport(51) . We suggest that there is an
additional alternative for procollagen. In this system, Hsp47 is seen
to possess both chaperone and anti-chaperone properties similar to that
recently demonstrated for PDI(54) . Thus, during translation
the high levels of Hsp47 function as a chaperones and limit protein
aggregation and facilitate chain registration and/or posttranslational
modification of procollagen(28) . Later, as procollagen
concentrations increase and are released from GRP78 and GRP94, Hsp47
and/or in consort with CyPB and PDI act as anti-chaperones and provide
the basis for the concentration of procollagen destined for export.
However, at present it is still not clear whether the Hsp47 and CyPB
that escorts procollagen into pre-Golgi intermediate vesicles initiate
and/or coordinate the dynamics of coat assembly on the lipid bilayer.
It will be important now to examine the transport of procollagen after
alteration of Hsp47 and CyPB to define the general need for sorting and
concentration and the nature of plausible signals involved in
conveyance control from the ER.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1(I) procollagen chains. Hsp47 possesses
chaperone properties early during the translation of procollagen while
the cis/trans-isomerase properties of CyPB facilitate
procollagen folding. In this report, we further investigate the
interaction of these proteins with procollagen I during export from the
ER. To inhibit vesicular budding and retain procollagen within the ER,
cells were treated with the heterotrimeric G protein inhibitor
mastoparan or calphostin C, a specific inhibitor of
diacylglycerol/phorbol ester binding proteins. To arrest procollagen in
pre-Golgi intermediate vesicles, cells were treated with guanosine
5`-3-O-(thio)triphosphate. Pulse-chase experiments of cells
labeled with [
S]methionine followed by
immunoprecipitation during the chase period with anti-procollagen,
anti-Hsp47, and anti-CyPB antibodies were performed to reveal the
relationship between Hsp47/CyPB/procollagen I. The distribution of
procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was
verified by immunofluorescence. Hsp47 and CyPB remained associated with
procollagen retained within the ER. Hsp47 and CyPB were also associated
with procollagen exported from the ER into pre-Golgi intermediate
vesicles. Treatment of cells with cyclosporin A diminished the levels
of CyPB bound to procollagen and diminished the rate of Hsp47 released
from procollagen and the rate of procollagen secretion, suggesting that
Hsp47 release from procollagen may be driven by helix formation. Also,
these studies suggest that Hsp47 may resemble protein disulfide
isomerase and possess both chaperone and anti-chaperone properties.
During translation, high levels of Hsp47 are seen to limit protein
aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB
and protein disulfide isomerase act as anti-chaperones and provide the
basis for concentration of procollagen for ER export.
1(I) chains and one pro
2(I) chain. Like other secretory
proteins(1, 2, 3, 4) , procollagen
-chain synthesis begins in the cytosol with translation of a
signal peptide. Subsequently, the signal sequence binds to a
recognition particle that docks the complex with a membrane receptor on
the endoplasmic reticulum (ER). (
)Coincident with membrane
association, the nascent protein is shifted into the membrane and
traverses into the cisternal space(5, 6, 7) .
Subsequently, the association of
-chain carboxyl propeptides
provides the appropriate alignment and orientation for ensuing triple
helix formation(8) .
ases expressed ubiquitously in
prokaryotes and eukaryotes(14, 15, 16) .
These proteins act as intracellular receptors for the immunosuppressant
CsA(17, 18) . The cyclophilin family, like heat shock
proteins, possesses a conserved core domain flanked by variable domains
at the N and C termini(15, 19) . It has been suggested
that the variable domains encode subcellular targeting information.
Consequently, CyPs have been classified into several isoforms; CyPA is
cytosolic, while CyPB-C and the Drosophila cyclophilin, ninaA,
possess ER signal sequences directed to the secretory pathway (19) . In addition to their rotamase activity (cis/trans-isomerase)(20, 21, 22, 23) ,
CyPs also appear to possess chaperone properties for protein
trafficking and macromolecular
assembly(14, 16, 24, 25, 26, 27) .
For example, the Drosophila ninaA gene encodes a
photoreceptor-specific CyP. Furthermore, ninaA and rhodopsin Rh 1
colocalize to secretory vesicles, suggesting the Rh 1 requires ninaA as
it travels through the distal compartments of the secretory
pathway(19) .
1(I) chains(28, 29) . These have included the
GRP78/Bip, GRP94, protein disulfide isomerase (PDI), and Hsp47, which
appear to function in a series of coupled or successive reactions
during procollagen production and assembly(30) . While
investigating the relationship between various molecular chaperones and
evolving procollagen chains associated with dense polysomes, a 20.6-kDa
protein was observed to be closely associated with procollagen, Hsp47,
PDI, GRP78, and GRP94. Here, we report this 20.6-kDa protein to be a
CyPB that associates early in the translation/translocation of
procollagen and may function in consort with other ER resident proteins
to prevent aggregation of nascent chains, mediate proline
isomerization(3) , and possibly improve the catalytic effect of
protein disulfide isomerase(31) . Also, CyPB and Hsp47 were
localized to the ER and to pre-Golgi intermediate vesicles associated
with procollagen, suggesting that Hsp47 and CyPB have a role in
procollagen sorting and transport.
Cell Culture and Metabolic
Labeling
Mouse 3T6 cells obtained from the American Type
Tissue Collection were used in all experiments. The cells were grown
and maintained in plastic flasks using Dulbecco's modified
Eagle's medium, 1.16 g/liter glutamine, 10% fetal bovine serum,
10 µg/ml ascorbate, 100 units of penicillin, and 100 µg/ml
streptomycin at 37 °C. In those instances when it was necessary to
label proteins, the medium was removed and replaced with fresh
methionine-free Dulbecco's modified Eagle's medium
containing [S]methionine (100 µCi/ml, DuPont
NEN) for varying periods (see figure legends).
Isolation of Intact Polysomes and Nascent
Procollagen
Dense polysomes were prepared after a modified
protocol of Kirk et al.(32) . In essence, Mouse 3T6
cells were grown to near confluence as described above, and protein
synthesis was blocked by the addition of cycloheximide (100 µg/ml)
for 10 min. In some instances, cross-linking with
dithiobis(succinimidyl propionate) was used to determine the near
neighbors of polysome-associated proteins(28) . The cells were
suspended in buffer A (0.2 M Tris-HCl, pH 7.4, 0.24 M KCl, 0.0075 M MgCl, 0.1 mg/ml cycloheximide,
0.2 mg/ml heparin, 2 mM dithiothreitol, 0.05% sodium
deoxycholate, 0.16 mg/ml phenylmethylsulfonyl fluoride, and 0.78 mg/ml
benzamidine), and Triton X-100 was then added to a final concentration
of 2%. The cells were homogenized and centrifuged at 10,000
g for 30 min to remove nuclei and cellular debris. The
resulting supernatant was collected and the volume adjusted to 5 ml
with buffer A. The supernatant (2.5 ml) was then layered on top of 1 ml
of 1 M sucrose layered over 1.5 ml of 2 M sucrose.
The samples were centrifuged for 12 h in a Beckmann SW 55 Ti rotor at
100,000
g. Polysomes were collected, washed with
water, suspended in Q-Sepharose buffer (0.02 M Tris-HCl, pH
7.4, 0.24 M KCl, 0.0075 M MgCl
), and
applied to a 3-ml Q-Sepharose Fast Flow column (Pharmacia Biotech Inc.)
as described by Bergman and Kuehl (33) and modified by Kirk et al.(32) . The resulting flow-through fraction
contained tRNA-free nascent chains. The retained tRNA-bound material
was eluted with 1.0 M NaCl.
N-terminal Sequencing
Proteins separated
by SDS-PAGE were electroblotted onto Problot membranes at 50 mA for 3
h. The Problot was then soaked in blotting buffer (25 mM Tris,
192 mM glycine, 20% methanol). The Problot was stained with
Coomassie Blue G for 30 s and then destained using acetic
acid/methanol/water (1:1:18, v/v) for 12 h. The membranes were dried,
and the PP band was cut for N-terminal sequencing.
Reverse Transcriptase Polymerase Chain
Reaction
Total RNA was isolated from 10
3T6
mouse cells using a Total RNA Isolation System (Promega). The first
strand cDNA synthesis reaction was catalyzed by superscript II RNase
H-reverse transcriptase (RT) (Life Technologies, Inc.) with either of
two 5`-end-specific primers and one 3`-end-specific primer for mouse
CyPB(34) . The resulting products were analyzed by SDS-PAGE
using
-actin primers and product as a control(35) . The
polymerase chain reaction-amplified fragments were ligated into the
Invitrogen TA cloning system and subsequently sequenced to verify the
polymerase chain reaction product. Sequencing was achieved using the
Sequenase kit 2.0 (U. S. Biochemical Corp.).
PAGE and Immunoblotting
Samples for gel
electrophoresis were suspended in Laemmli SDS-PAGE buffer and boiled
for 5 min before loading onto 4-20% polyacrylamide gradient slab
gels after the method of Laemmli(36) . The gels were fixed,
dried, and autoradiographed using the method of Bonner and
Laskey(37) .1(I) procollagen antibodies were made from acid-soluble
procollagen derived from lathyritic mouse skin(28) . Polyclonal
rabbit antibodies were made against a peptide corresponding to the
C-terminal decapeptide (VEKPFAIAKE) of CyPB (34) and affinity
purified on a peptide column. The antibodies were further characterized
by Western blot to CyPB that was expressed in Escherichia coli containing the CyPB expression plasmid(34) . Notably,
these antibodies failed to show any reactivity to purified CyPA (Sigma)
in Western blots.
Immunoprecipitation and Analysis by Gel
Electrophoresis
Cells were freed from the support medium
with trypsin (0.025% EDTA (0.02%) and treated with bacterial
collagenase (0.01%) and 10% fetal calf serum for 3 min to block trypsin
activity and remove extracellular collagen. The samples were suspended
in a equal volume of 2 immunoprecipitation buffer (0.2 M Tris-HCl, 0.3 M NaCl, 2% Triton X-100, 2% deoxycholate,
0.2% SDS, pH 7.2 containing apyrase (Sigma) to deplete ATP). The
samples were centrifuged for 5 min at 10,000
g in an
Eppendorf centrifuge, and a 50-µl sample of the radiolabeled
supernatant was added to a mixture of protein A-Sepharose and antibody
in PBS-azide. The samples were then incubated at 4 °C with constant
shaking and then centrifuged at 10,000
g for 10 min.
The resulting immunoprecipitates were then washed twice with PBS-azide.
The final pellets were suspended in 2
gel electrophoresis
sample buffer, heated for 10 min at 90 °C, and then centrifuged to
remove protein A-Sepharose. Samples of the supernatants were counted in
a scintillation counter, and another sample was analyzed by PAGE and
autoradiography as described above.
Modulation of ER to Golgi Transport
To
limit procollagen transport to pre-Golgi intermediate vesicles, cells
were permeabilized with digitonin (39) and incubated in the
presence of 25 µM GTPS for various periods up to 60
min (40) . To inhibit vesicular budding and retain procollagen
within the ER, cells were treated with the heterotrimeric G protein
inhibitor mastoparan (17 µm)(40) . Also, in some
experiments cells were treated with calphostin C, a specific inhibitor
of the highly conserved cysteine-rich C
H
motif
present in the regulatory domain of protein kinase C(41) . For
these studies, 120 nm of calphostin C was added from a stock solution
in dimethyl sulfoxide. To ensure that inhibition by calphostin was
specific, parallel control samples were run in the dark since this
inhibitor behaves as a caged substrate when activated by exposure to
light(41) . In some experiments, cells were treated with 100
µg/ml cycloheximide to inhibit further protein synthesis.
Indirect Immunofluorescence
Fixed cells
were blocked with 5% swine serum in PBS, and the Cell membranes were
permeabilized with 0.1% saponin before incubation with
antibody(39) . Before the addition of the second primary
reagent to detect Golgi marker proteins (-1,2-mannosidase II),
cells were permeabilized with 0.1% saponin for 20 min in PBS/swine.
Cells were subsequently washed and exposed to the primary antibody.
Rabbit primary antibodies were detected with either a fluorescein
isothiocyanate goat anti-rabbit IgG or Texas Red goat anti-rabbit IgG
(Molecular Probes). Coverslips were mounted in Moviol
(Calbiochem-Behring Corp.) and viewed in Axiovert microscope (Carl,
Zeiss, Oberochen, Germany).
PP
PPase Activity
activity was determined in a standard coupled chymotrypsin assay
utilizing N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide(42) .
S]methionine, and dense
procollagen polysomes were prepared after the method of Kirk et al.(32) . The resulting polysomes were then fractionated
using Q-Sepharose Fast Flow chromatography to yield two pools of
procollagen. One pool consisted of elongating procollagen bound to
peptidyl-tRNA that was initially retained (RT) and eluted with salt.
The flow-through fraction consisted of recently completed nascent
chains that were disrupted during column fractionation. The eluted RT
fraction was then immunoprecipitated with anti-collagen I antibodies.
Hsp47 was noted to coimmunoprecipitate with nascent procollagen I
chains. In addition to Hsp47, GRP78, and GRP94, an additional 20.6-kDa
protein was recovered in immunoprecipitates associated with peptidyl
tRNAs (Fig. 1). Measurement of the PP
ase activity of
the dense polysome preparation, Q-Sepharose fractions, and
immunoprecipitates revealed an enrichment in PP
ase activity (Table 1).
S]methionine-labeled dense polysomes derived
from 3T6 cells. The toppanel depicts the
fractionation of dense polysomes by Q-Sepharose chromatography. LaneA depicts the flow-through fraction consisting
of recently completed nascent chains disrupted during column
fractionation. LaneB depicts the elongating chains
bound to peptidyl tRNA that was initially retained (RT) and eluted with
high salt. LaneC represents the RT fraction that was
digested with bacterial collagenase subsequent to precipitation. The bottompanel depicts an immunoprecipitate of a
dithiobis(succinimidyl propionate) cross-linked RT fraction obtained
with anti-collagen I antibodies. The various proteins labeled in the
figure were identified by susceptibility to bacterial collagenase and
Western blot analysis. The proteins identified were as follows: lane1, Hsp47; lane2, PDI; lane3, CyPB; lane 4, GRP94; lane5, GRP78.
activity that is inhibited by CsA.
Furthermore, this activity was attributed to a protein belonging to the
CyPB family(43) . To ascertain if the 20.6-kDa protein
precipitating with procollagen nascent chains was a member of the CyPB
family, the RT fraction was separated by PAGE electroblotted onto
Problot membranes, and the 20.6-kDa band was cut out of the membrane
for N-ternimal sequencing. The resulting sequence
NDKKKGPKVTVKVYFDLQIG was identical to that previously reported for
mouse CyPB (34) being rich in lysine and thus distinct from
mitochondrial cyclophilins that possess a serine-rich N-terminal
extension(44) .
10
3T6 mouse cells using a total RNA
isolation system (Promega). The first strand cDNA synthesis reaction
was then catalyzed by SuperScript II RNase H-RT (Life Technologies,
Inc.). Next, the protein-coding region was amplified by the polymerase
chain reaction (45) with either of two 5`-end-specific primers
and one 3`-end-specific primer (see ``Materials and
Methods''). The polymerase chain reaction-amplified fragments were
ligated into the Invitrogen TA cloning system and subsequently
sequenced. These results verified that the amplified products from
mouse 3T6 cells were identical to CHP2 (CyPB)(34) . As such,
this sequence contained 25 amino acids (MKVLFAAALIVGSVVFLLLPGPSVA) that
resembled a signal sequence and a predicted cleavage site between
Ala-25 and Asn-26, in which the resultant protein would result in a
20.6-kDa final product. In addition, the protein concluded with a
C-terminal VEKPFAIAKE, distinct from cytosolic and mitochondrial
cyclophilins(43, 44) .
S]methionine chased in cold medium in excess of
the labeled amino acid and immunoprecipitated with various antibodies.
These data revealed that Hsp47 and CyPB were both closely associated
with procollagen during the first 10 min of the chase period. However,
subsequently both proteins progressively dissociated from procollagen
and were unassociated with secreted procollagen in the medium (Fig. 3).
S]methionine for 20 min; the cells were then
chased in medium containing an excess of unlabeled methionine. Cells
were harvested and lysed, and samples were immunoprecipitated with
anti-procollagen antibodies (total procollagen), anti-Hsp47 antibodies
(Hsp47-procollagen), and CyPB antibodies (CyPB-procollagen) and
separated by SDS-PAGE and autoradiograms prepared (see ``Materials
and Methods''). The bands representing procollagen I
-chains
were scanned using a densitometer. The value of the relative density
before chasing was designated as 1 unit. The mean values and the
standard deviations from at least three independent experiments are
plotted.
H
motif present
in the regulatory domain of protein kinase C, is known to be a potent
inhibitor of vesicular budding from the ER (41) and was
utilized to retain procollagen in the ER without directly modifying the
procollagen post-translational modification. Export from the ER was
also inhibited by mastoparan, a peptide that mimics G protein-binding
regions of seven transmembrane-spanning receptors activating and
uncoupling heterotrimeric G proteins from their cognate
receptors(40) . In both instances, immunoprecipitation of the
procollagen revealed that Hsp47 and CyPB remained bound to procollagen
compared to untreated cells ( Fig. 4and Fig. 5). These
results are consistent with the hypothesis that multiple GTP-binding
proteins (40) and a novel protein containing a
C
H
motif serve as a link in a signaling pathway
regulating vesicle budding from the ER(41) . To ensure that
both inhibitors were effective, the media were collected, and proteins
were precipitated and subjected to SDS-PAGE. In both instances, no
radiolabeled procollagen was detected in the medium. In addition, cells
were also monitored by indirect immunofluorescence to verify that
procollagen was retained within the ER. These studies confirmed that
cells treated with either calphostin C and mastoparan retained
procollagen, Hsp47, and CyPB to the ER region (Fig. 6).
S-treated
3T6 cells. Pulse-chase experiments were performed as described above,
and the samples were immunoprecipitated with anti-procollagen
antibodies separated by SDS-PAGE and autoradiograms prepared (see
``Materials and Methods''). The bands representing Hsp47 were
scanned using a densitometer. The value of the relative density before
chasing was designated as 1 unit. The mean values and the standard
deviations from at least three independent experiments are
plotted.
S-treated
3T6 cells. Pulse-chase experiments were performed as described above,
and samples were immunoprecipitated with anti-procollagen antibodies
separated by SDS-PAGE and autoradiograms prepared (see ``Materials
and Methods''). The bands representing CyPB were scanned using a
densitometer. The value of the relative density before chasing was
designated as 1 unit. The mean values and the standard deviations from
at least three independent experiments are
plotted.
S. The
distribution of proteins was monitored using the cis/medial
Golgi marker
-1,2-mannosidase II. Columna, row1 represents control cells labeled with
anti-Hsp47 antibodies. Columnb, row1 depicts Hsp47 limited to the ER in mastoparan-treated cells;
similar results were obtained with calphostin C. Columnc, row1 reveals the distribution of
Hsp47 in ER and punctate vesicles in cells treated with GTP
S. Columna, row2 depicts the
distribution of CyPB in control cells. Columnb, row2 depicts CyPB in the ER of cells treated with
calphostin C; similar results were obtained with mastoparan. Columnc, row3 demonstrates CyPB both in the
ER and in punctate pre-Golgi vesicles following GTP
S treatment. Columna, row3 depicts procollagen
in control cells. Columnb, row3 represents procollagen retained in the ER following treatment with
mastoparan; identical results were obtained with the use of calphostin
C. Columnc, row3 represents the
distribution of procollagen I in punctate pre-Golgi vesicles following
40 min of treatment with GTP
S. B, distribution of Hsp47
and Golgi markers in 3T6 cells. Following treatment of cells with
GTP
S, Hsp47 was localized to ER and punctate vesicles (panel a). In control cells (panelb), Hsp47 was
localized primarily to the ER with only a few vesicles apparent. Arrows indicate staining of Golgi with
-1,2-mannosidase
II.
S, a nonhydrolyzable
analog of GTP that blocks uncoating of both ER and Golgi
transport-derived vesicles(47, 48) . These studies
revealed accumulation of transported procollagen and Hsp47 in pre-Golgi
intermediate vesicles distributed throughout the cytoplasm of the cell
as well as the ER (Fig. 6). Immunoprecipitation of procollagen
in GTP
S-treated cells revealed that labeled Hsp47 and CyPB
coprecipitated with labeled procollagen ( Fig. 4and Fig. 5), in that CyPB trafficking through the secretory pathway
can be altered by CsA (27) . Mouse 3T6 cells were treated with
1 µM CsA, labeled, and chased as described above for 60
min (Fig. 7). These studies revealed that CsA treatment
diminished the levels of CyPB bound to procollagen and delayed the rate
of Hsp47 release from procollagen and the rate of procollagen
secretion. These data sustain previous studies that revealed that CyPB
traverses the secretory pathway rather than acting solely as a proline
isomerase functioning within the ER. Albeit CyPB has been shown to act
as a PP
ase for procollagen(13) , these results
suggest that the release of Hsp47 from procollagen is driven by helix
formation, thereby providing one mechanism by which CyPB plays a role
in procollagen export and secretion.
S]methionine for 20 min; the cells were then
chased in medium containing an excess of unlabeled methionine. Cells
were harvested and lysed, and samples were immunoprecipitated with
anti-procollagen antibodies (total procollagen), anti-Hsp47 antibodies
(Hsp47-procollagen), and CyPB antibodies (CyPB-procollagen) and
separated by SDS-PAGE and autoradiograms prepared (see ``Materials
and Methods''). The bands representing procollagen I
-chains
were scanned using a densitometer. The value of the relative density
before chasing was designated as 1 unit. The mean values and the
standard deviations from at least three independent experiments are
plotted.
,
-dipyridyl-treated cells and
procollagen in some forms of osteogenesis
imperfecta(29, 49, 50) . The studies reported
here indicate that Hsp47 association with procollagen is also
compartment dependent. In that Hsp47 has been shown not to undergo
Golgi processing of its N-linked oligosaccharides, we
anticipated that Hsp47 was involved early in procollagen
assembly(28) . However, the association of Hsp47 with
procollagen in the intermediate compartment residing between the ER and
Golgi suggests a further role for Hsp47 in mediating procollagen export
from the ER. This raises questions as to the role of Hsp47 during
transport from the ER to the Golgi.
S, guanosine
5`-3-O-(thio)triphosphate.
We thank JoAnn Walker for help in preparing the
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.