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(Received for publication, September 29,
1995; and in revised form, November 29, 1995) From the
The specific pathway of tropoelastin secretion was investigated
in fetal calf ligamentum nuchae (FCL) cells using brefeldin A (BFA) to
disrupt the secretory pathway. Electron microscopic studies of
BFA-treated FCL cells showed ultrastructural changes consistent with
the reported effects of BFA on intracellular organelles. When FCL cells
were labeled with [ The formation of elastic fibers in the extracellular matrix
involves the secretion and subsequent alignment of tropoelastin
monomers onto a microfibrillar scaffold where they then become
cross-linked to form an insoluble elastin matrix. Ultrastructural
studies have suggested that the secretion of tropoelastin occurs at
specific sites on the cell surface(1) . This targeted secretion
is thought to be mediated by an elastin receptor or chaperone complex
that not only directs the secretion of tropoelastin but facilitates the
assembly of the protein onto the developing elastic fibers at the cell
surface(2, 3, 4) . The definitive function of
the elastin chaperone complex, however, has yet to be established. This
is due, in large part, to the fact that very little is known concerning
the intracellular events required for the trafficking and secretion of
the tropoelastin monomer. Cell-free translation studies using
tropoelastin mRNA have shown that the 70-kDa tropoelastin monomer
contains a signal sequence of 24-26 residues(5) , which
is cleaved as the completed polypeptide chain enters the
ER( One approach
to study the intracellular pathway of a secreted protein is to use
drugs that affect protein trafficking at distinct sites along the
secretory pathway. Since tropoelastin does not require any
modifications within the Golgi, it was of interest to study the effect
of brefeldin A (BFA) on the secretion of tropoelastin. Over the past
several years, this fungal metabolite has been used extensively to
block protein transport from the ER to the Golgi(11) .
Morphological studies have shown that in the presence of BFA, the Golgi
apparatus disassembles and the ER becomes extensively
dilated(12, 13) . These morphological attributes
reflect a retrograde fusion of the cis-, medial-, and trans-Golgi cisternae into the
ER(12, 14, 15, 16) . Since the
effect of BFA on protein synthesis is minimal, secretory proteins tend
to accumulate within the mixed ER/Golgi
compartment(12, 17) . Remarkably, the effects of BFA
have been shown to be completely reversible upon removal of the drug,
with reassembly of the Golgi apparatus and resumed
secretion(18, 19) . In the present study, the
secretion of tropoelastin was investigated in fetal calf ligamentum
nuchae (FCL) cells. In fetal tissues, cells that are committed to
elastogenesis can devote as much as 40% of their total protein
synthesis to tropoelastin. Here we demonstrate that BFA treatment of
FCL cells results in complete inhibition of tropoelastin secretion. It
was expected that an intracellular accumulation of the protein would
therefore occur; however, tropoelastin was found to be rapidly and
selectively degraded in the ER. Although not well characterized, there
is increasing evidence to support the existence of a pre-Golgi
degradation pathway that is independent of lysosomes(20) . Such
a degradative system is required to dispose of proteins that are
retained in the ER due to misfolding or failure to assemble into
oligomeric complexes. In addition to abnormal proteins, several normal
proteins also undergo ER degradation in a regulated manner, such as the
enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (21, 22) and apolipoprotein B(23) . The
results from this study provide the first evidence of a BFA-induced
degradation event mediated by a cysteine protease and provide
supportive evidence for a selective process of ``quality
control'' in the ER. The implications of these results, with
respect to the intracellular events required for tropoelastin secretion
and the general process of ER degradation, are discussed.
For metabolic labeling, L-[4,5- Reagents used during metabolic labeling included
ammonium chloride (NH
To determine total
protein synthesis and secretion, trichloroacetic acid-precipitable
radioactivity was determined from lysates and medium of cells
metabolically labeled as indicated in the text. For each sample, 10
µl of lysate or medium was mixed with an equal volume of 10 mg/ml
bovine serum albumin, and 15 µl of this solution was spotted on dry
glass microfiber filters (Whatman, Hillsboro, OR). After 30 min at 4
°C in dishes flooded with cold 10% trichloroacetic acid, the
filters were washed 3 For immunoprecipitation, medium and lysates
were precleared by incubation with 10 µg/ml normal mouse IgG for 2
h and an additional 1 h with 25 µl of Staphylococcus aureus added to each tube. The Staphylococcus aureus was
pelleted by centrifugation, and the supernatants were transferred to
clean tubes containing 100 µl of 50 mg/ml bovine serum albumin in
lysis buffer and 10 µg of BA-4 antibody. Medium and lysates were
incubated overnight at 4 °C with gentle agitation. The following
day, 40 µl of Staphylococcus aureus was added to each tube
and incubated for 1 h at 4 °C with gentle agitation. The immune
complexes were pelleted, and the pellets were washed two times with
lysis buffer and one time with nondetergent buffer (10 mM Tris-HCl (pH7.5), 5 mM EDTA (pH 7.5)). After the final
wash, each pellet was resuspended in 35 µl of Laemmli sample buffer
containing dithiothreitol and incubated at 100 °C for 6 min. The
samples were electrophoresed on SDS-polyacrylamide gels, fixed for 20
min, and treated with EN All immunoprecipitation experiments were conducted a minimum of
three times to ensure reproducibility of results. Furthermore, in each
experiment, one well of cells was always a control, with no treatment,
in order to access the quality of labeling for that particular
experiment and provide a direct comparison for the treated cells.
Figure 1:
Time course of secretion
of (A) total trichloroacetic acid-precipitable proteins and (B) immunoprecipitated tropoelastin (TE) in FCL cell
lysates and medium following a 10-min pulse with
[
Figure 2:
Fibronectin is immunoprecipitated with Staphylococcus aureus independent of fibronectin primary
antibody. FCL cells were radiolabeled for 2 h with
[
Figure 3:
BFA treatment of FCL cells results in
blocked secretion of tropoelastin with no concurrent intracellular
accumulation. FCL cells were pulsed with
[
Figure 4:
Electron micrographs of cultured FCL cells
showing the Golgi region of control cells treated with 1 µl/ml
Me
Figure 5:
Electron micrographs of cultured FCL cells
showing the peripheral region of control cells treated with 1 µl/ml
Me
In
addition to FCL cell ultrastructure, the general condition of the cells
with respect to protein synthesis and secretion was investigated by
measuring total trichloroacetic acid-precipitable counts from lysates
and medium of cells treated with 1 µl/ml Me
Figure 6:
Effect of BFA treatment on total protein
synthesis and secretion. FCL cells were metabolically labeled in
complete medium with [
Figure 7:
Effect of BFA treatment on mRNA levels of
tropoelastin in FCL cells. FCL cells were incubated in the presence of
10 µg/ml BFA for 15 min, 1 h, 2 h, and 4 h in complete medium (lanes 3-6). As a control, one plate of cells was
incubated in leucine-free medium alone for 1.25 h (lane 1),
and a second plate was incubated with 1 µl/ml Me
Figure 8:
FCL cells pulsed and chased in the
presence of BFA result in rapid degradation of the tropoelastin trapped
within the cells, while fibronectin remains unchanged. FCL cells were
pulsed for 1 h with [
Figure 9:
The cysteine protease inhibitor, ALLN,
inhibits the BFA-induced degradation of tropoelastin. A, FCL
cells were pulsed for 1 h with [
Studies on the ER degradation
of HMG-CoA reductase and T-cell receptor Although it is well documented
that BFA treatment results in the retention of secreted proteins in the
fused ER/Golgi compartment, the possibility that tropoelastin is
transported to some degradation compartment following BFA treatment
could not be ruled out. However, nocodazole had no effect on
tropoelastin degradation when the drug was added into the chase medium (Fig. 9A). This observation suggests that movement of
tropoelastin via microtubules is not a prerequisite for the degradation
to occur. The inclusion of nocodazole in the chase, however, does not
preclude a degradative compartment from fusing with the ER during BFA
treatment. Thus, FCL cells were pretreated with nocodazole before being
metabolically labeled and chased, with both BFA and nocodazole being
present in the pulse and chase. Since the retrograde transport of Golgi
elements into the ER is a microtubule-dependent event(35) , the
preincubation of the cells with nocodazole prior to being pulsed in the
presence of BFA would block, or at least severely reduce, the
redistribution of Golgi and other vesicular compartments into the ER.
As shown in Fig. 9B, the addition of nocodazole prior
to BFA treatment did not increase the stability of tropoelastin during
the chase. In contrast, the degradation of tropoelastin in these cells
appeared to occur even more rapidly. These results confirm that the
degradation of tropoelastin takes place in the ER as a result of its
retention in that compartment.
Figure 10:
In the presence of BFA, tropoelastin
accumulates in the ER to a threshold level before degradation occurs.
FCL cells were labeled with [
Over the past several years, a number of studies have
provided good evidence for the existence of a proteolytic system
contained within the ER that functions as a ``quality
control'' mechanism. Although not well characterized, this system
is thought to be responsible for the degradation of unassembled or
misfolded proteins as well as excess subunits of proteins that undergo
oligomerization in the ER, such as the T-cell antigen
receptor(36) . In addition to unassembled and misfolded
proteins, normal proteins are also degraded in the ER, indicating that
this system may play a role in regulated proteolytic degradation.
Examples of such proteins include HMG-CoA reductase (21) and
apolipoprotein B-100(23, 37, 38) . In many
of the studies of proteins that undergo intracellular degradation, BFA
has been used to characterize the nature of the degradative
compartment. It has been shown, for example, that the regulated
degradation of HMG-CoA reductase and apolipoprotein B-100 is unaffected
by BFA treatment, indicating that the proteolytic event must take place
in a pre-Golgi compartment. In contrast, the degradation of secretory
immunoglobulin M in B lymphocytes has been reported to occur in a
post-ER compartment, since the degradation of this protein is strongly
inhibited by BFA treatment(39) . In the present study,
treatment of FCL cells with BFA induced in the degradation of
tropoelastin as a consequence of the protein being retained in the
fused ER/Golgi compartment. Retention alone, however, is not sufficient
to cause the degradation of proteins, since fibronectin, another
protein normally secreted by these cells, readily accumulated in the ER
of BFA-treated FCL cells without any apparent degradation. The
selective degradation of tropoelastin raises important questions
concerning the specificity and regulation of the ER proteolytic system.
Of particular interest from the present study was the observation that
tropoelastin initially accumulated in the fused ER/Golgi compartment
prior to its rapid degradation. This observation appears to be a
relatively consistent feature of ER degradation, in that a lag period
with little degradation often precedes the rapid phase of degradation (20, 40) . Although it is unclear as to why a lag
period exists, it has been suggested that the site of degradation may
be in a specific subcompartment of the ER, and thus a certain degree of
time is required to allow for sorting and delivery to this
area(20) . Another reason for the lag period may be that,
within the ER, tropoelastin normally associates with a chaperone
protein that protects the protein from degradation. Thus, with BFA
treatment, the continued synthesis of tropoelastin into the fused
ER/Golgi compartment exhausts the available chaperone and leads to
either misfolded or simply unfolded protein that is susceptible to
proteolytic degradation. This hypothesis is supported by the fact that
half-lives of chaperone proteins are often quite long, for example
greater than 30 h for calnexin(41) , while the secretion rate
of tropoelastin is on the order of 40 min. During a 4-h pulse in the
presence of BFA, therefore, the production of a chaperone protein could
be relatively minimal as compared with the amount of tropoelastin
synthesized and retained in the ER. Furthermore, studies of procollagen
I and its association with a collagen-binding glycoprotein localized to
the ER, termed colligin or hsp47(42, 43) , have set a
precedence for such a protective function of an ER chaperone. It has
been demonstrated that procollagen I can bind to colligin and that this
interaction can protect the procollagen I from degradation by a serine
protease that is present in microsomal preparations(44) . Since
the expression of colligin has only been detected in collagen-secreting
cells(45) , the possibility exists that a specific
tropoelastin-binding chaperone is present in elastogenic cells. One
interesting property of tropoelastin is its ability to undergo a phase
transition and form a coacervate under physiological conditions (46) . Because this process is concentration-dependent, it is
also possible that retention of tropoelastin in the fused ER/Golgi
compartment during BFA treatment leads to coacervation of the protein
and thus its subsequent recognition and targeting for degradation as a
misfolded protein or aggregate. Support for a concentration-dependent
requirement for tropoelastin degradation is provided by the observation
that, in the presence of BFA, tropoelastin degradation was more rapid
in FCL cells when the cells were pretreated with nocodazole. Since this
pretreatment restricts the retrograde fusion of the Golgi saccules with
the ER, the concentration of tropoelastin in the ER would increase
faster due to the smaller size of the compartment. The tendency of
tropoelastin to coacervate provides further evidence that the transport
of tropoelastin through the cell may be mediated by a chaperone
protein, since coacervation is clearly an undesirable feature of a
protein that must traverse the secretory pathway. The accumulation
and lag period observed prior to the BFA-induced degradation of
tropoelastin may also represent the time required for the protein to
reach a threshold level critical for activation of the enzyme
responsible for its degradation. Since the nature of the proteolytic
enzymes contained within the ER remains largely unknown, the control of
the active states of these enzymes has yet to be determined. In the
present study, the degradation of tropoelastin was found to be
independent of lysosomal function and strongly inhibited by the
cysteine protease inhibitor, ALLN. Similarly, a cysteine protease that
is inhibited by ALLN has been implicated in the degradation of HMG-CoA
reductase(47) , apolipoprotein B-100 (48) and T-cell
receptor chains(49) . The degradation of these proteins in the
ER, however, appears to be considerably more complex than the
involvement of a single cysteine protease. For example, while HMG-CoA
reductase degradation is sensitive to depletion of intracellular
Ca Recently, ER-60 protease, an
ER-resident protein with cysteine protease
activity(53, 54) , has been characterized and related
to ER degradation in vivo for the first time by its
association with misfolded human lysozyme prior to its
degradation(52) . In addition to ER-60, a second protease,
termed ER-72 protease, has also been identified in the ER and is
inhibited by cysteine protease inhibitors(55) . Although both
of these proteases localize to the ER and can be inhibited by ALLN,
their specific role in ER degradation of proteins in vivo remains to be determined. It is important to note that FCL
cells treated with ALLN alone during the pulse did not result in an
obvious increase in tropoelastin secretion. This observation suggests
that the intracellular degradation and turnover of tropoelastin
observed in the present study is not a significant event during normal
secretion of the protein. The ability of tropoelastin to be selectively
degraded in the ER, however, may be of extreme importance in the event
of an aberrant accumulation of tropoelastin in the ER. One situation
where this may occur is in disease states where the elastin gene is
disrupted, such as supravalvular aortic
stenosis(56, 57) . The production of the abnormal gene
product from the defective allele could result in a tropoelastin
protein that is incompetent for transport from the ER and thus be
lethal for the cell if not disposed of early in the secretory pathway.
It remains to be determined if the degradative pathway identified in
the present study plays a role in ``quality control'' of
elastin gene products in diseases such as supravalvular aortic
stenosis.
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3787-3794
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A POSSIBLE CLEARANCE PATHWAY FOR ABNORMAL TROPOELASTIN (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H]leucine in the presence of
BFA, radiolabeled tropoelastin was not secreted, nor was there an
intracellular accumulation of the protein. In contrast, fibronectin
accumulated within the cells in the presence of BFA. Northern analysis
of mRNA levels in FCL cells showed that the message for tropoelastin
was unaffected by BFA treatment. Pulse chase experiments conducted in
the presence of BFA demonstrated that the tropoelastin retained within
the cells was rapidly degraded. Ammonium chloride, nocodazole, and
cycloheximide had no effect on the degradation of tropoelastin,
indicating that the degradation did not involve the endosome/lysosome
pathway, movement via microtubules, or a short-lived protein,
respectively. Incubation of FCL cells with BFA in the presence of N-acetyl-Leu-Leu-norleucinal, however, allowed tropoelastin to
steadily accumulate in the cells. Cells pulsed in the presence of BFA
alone showed that tropoelastin initially accumulates within the cells
for approximately 1 h prior to being degraded, thus indicating that a
critical threshold of tropoelastin must be reached before degradation
can occur. Results from this study provide evidence for selective
degradation of a soluble secreted protein by a cysteine protease
following retention of the protein in the endoplasmic reticulum.
)(6, 7) . From the ER to the cell
surface, tropoelastin remains unchanged with no glycosylation and
little, if any, other post-translational modifications. Early attempts
to explore the synthesis and secretion of tropoelastin, using
morphological and cytochemical techniques, showed the presence of small
vesicles in the vicinity of the Golgi and cell periphery that contained
amorphous material with staining properties identical to that of
elastin(8) . Similar material was also identified within the ER
cisternae and Golgi saccules. Later studies, using immunoelectron
microscopy, confirmed the presence of tropoelastin in elastogenic cells (9, 10) ; however, in many cases, the specific
identity of the immunolabeled compartments was unclear.
Cells and Reagents
Bovine ligamentum nuchae were
obtained from late gestation fetuses at a local slaughterhouse. FCL
fibroblasts were obtained from tissue explants and cultured as
described previously (24) . All experiments described in this
report were with first passage cells from 230-270-day fetal
nuchal ligaments. For all experiments, cells were grown to confluency
in Dulbecco's modified Eagle's medium supplemented with L-glutamine, nonessential amino acids, antibiotics, and 10%
fortified bovine calf serum (Hyclone Laboratories, Inc., Logan, UT)
prior to metabolic labeling.H]leucine (1 mCi/ml) was
purchased from ICN Pharmaceuticals, Inc. (Irvine, CA), and dialyzed
fetal bovine serum was purchased from Hyclone Laboratories, Inc.
Protease inhibitors, -amino-n-caproic acid,
phenylmethylsulfonyl fluoride, and N-ethylmaleimide were
purchased from Sigma and used in the lysis buffer at final
concentrations of 10, 5, and 5 mM, respectively. For
immunoprecipitation experiments, a monoclonal tropoelastin antibody
BA-4, raised to bovine
-elastin(25) , and a polyclonal
fibronectin antibody (Chemicon, Temecula, CA) were used. Immune
complexes were precipitated using heat-killed Staphylococcus aureus (Pansorbin cells, Calbiochem) or protein A immobilized on
Trisacryl (Pierce).
Cl), N-acetyl-Leu-Leu-norleucinal (ALLN), BFA, cycloheximide,
leupeptin, nocodazole, and pepstatin A (Sigma). NHCl was
prepared fresh as a 2 M stock in distilled water and used at a
final concentration of 20 mM. ALLN was stored at -20
°C as a 10 mg/ml stock in ethanol and used at final concentration
of 10 µg/ml. BFA and nocodazole were stored at -20 °C as
10 mg/ml stocks in Me
SO and used at final concentrations of
10 and 20 µg/ml, respectively. Cycloheximide and leupeptin were
prepared fresh in distilled water as a 0.1 M stock, used at a
final concentration of 0.5 mM, and as a 10 mM stock,
used at a final concentration of 200 µM, respectively.
Pepstatin A was prepared fresh as a 1 mg/ml stock in methanol and used
at a final concentration of 10 µM.Metabolic Labeling and
Immunoprecipitation
Confluent monolayers of FCL cells, grown in
six-well tissue culture plates (Falcon number 3046, Baxter, McGaw Park,
IL), were washed with leucine-free medium containing 5% dialyzed fetal
bovine serum and incubated in this medium for 1 h. Any pretreatment of
cells with ALLN or nocodazole is as indicated in the text and figure
legends. Each well of cells was metabolically labeled with 50 µCi
of [H]leucine in 1 ml of leucine-free medium
containing 5% dialyzed fetal bovine serum for various lengths of time
as indicated. For pulse chase experiments, cells were rinsed twice with
complete medium and chased in 1 ml of complete medium containing 5%
dialyzed fetal bovine serum. Following metabolic labeling, medium was
collected, and cell layers were washed twice with cold
phosphate-buffered saline. To each well, 1 ml of cold lysis buffer (25
mM Tris-HCl (pH 7.5), 5 mM EDTA (pH 7.5), 250 mM NaCl, 0.1% Triton X-100) with protease inhibitors was added. The
culture dishes were placed on a platform shaker at 4 °C, and cell
lysates together with the detached cells were collected after 30 min.
Cellular debris was pelleted by centrifugation, and the cell lysates
were transferred to clean microfuge tubes.
5 min with room temperature 10%
trichloroacetic acid and 1 5 min with 95% ethanol and left to
dry prior to counting.HANCE (DuPont NEN) for 1 h. Gels
were then dried and exposed to XAR-5 x-ray film (Eastman Kodak Co.). Electron Microscopy
Confluent cultures of FCL
cells, plated in four-well Lab-Tek tissue culture chamber slides
(Thomas Scientific, Swedesboro, NJ), were incubated with 10 µg/ml
BFA or 1 µl/ml MeSO in complete medium for 4 h. Cell
layers were then washed in phosphate-buffered saline and fixed in
situ with 3% glutaraldehyde in 0.1 M sodium cacodylate
buffer (pH 7.4) for 30 min. After being washed several times with
cacodylate buffer, the cell layers were treated sequentially with 1%
osmium tetroxide in buffer, 2% tannic acid in buffer, and 2% uranyl
acetate in distilled water. Cell layers were then dehydrated in a
graded series of methanol to propylene oxide, infiltrated and embedded
with Epon (SPI Supplies, West Chester, PA). En face thin
tissue sections were cut on a Reichert ultracut ultramicrotome and
counterstained with methanolic uranyl acetate (26) followed by
lead citrate(27) . Sections were examined in a Ziess 902
transmission electron microscope at an accelerating voltage of 80 kV.RNA Isolation and Northern Analysis
Confluent
cultures of FCL cells in 100-mm dishes were incubated for 15 min, 1 h,
2 h, or 4 h with 10 µg/ml BFA in complete medium. Control cultures
were incubated for 1 h with an equal volume of MeSO alone
(1 µl/ml). Following incubation, total RNA was isolated from the
cell layers by guanidinium isothiocyanate/phenol
extraction(28) . RNA concentration and purity was determined by
spectrophotometric analysis. Total RNA (5 µg) was separated by
electrophoresis through a 1% agarose gel containing 1 M formaldehyde, transferred to Hybond-N nylon
membrane (Amersham Corp.), and hybridized and washed under stringent
conditions as described previously(29) . Probes for this study
were labeled with [
-P]CTP (ICN, Irvine, CA)
by random primed labeling. The tropoelastin probe was prepared from
clone 12.1, a 2.2-kilobase bovine tropoelastin cDNA (30) . The
membrane was hybridized concurrently with the tropoelastin probe and a
cDNA probe for glyceraldehyde-3-phosphate dehydrogenase. Hybridized
complexes were detected by exposure of the membrane to X-OMAT AR film
(Kodak).
Time Course of Tropoelastin Secretion
To
characterize the normal secretion rate of tropoelastin in FCL cells,
cells were pulsed for 10 min and the fate of that pulse was followed
over the course of a 90-min chase. Counts of total trichloroacetic
acid-precipitated proteins from the cell lysate and medium indicated
that approximately 40 min passed before proteins began to be secreted (Fig. 1A). Consistent with this time course, a very faint
band of immunoprecipitated tropoelastin appeared in the medium after 30
min of chase with a steady increase in band density thereafter (Fig. 1B). Since fibronectin binds directly to Staphylococcus aureus independent of any primary antibody (31) , the use of Staphylococcus aureus to
immunoprecipitate tropoelastin resulted in the presence of radiolabeled
fibronectin on the autoradiographs as well as tropoelastin. The time
course of fibronectin secretion was similar to that observed for
tropoelastin. To verify that the bands at the top of the autoradiograph
were indeed fibronectin, the cell lysate and medium from cells labeled
for 4 h with [H]leucine were divided into three
equal aliquots and immunoprecipitated for fibronectin or tropoelastin
using their respective primary antibodies followed by protein A to
precipitate the immune complex or by using the tropoelastin antibody
followed by Staphylococcus aureus. Fig. 2shows that
the mobility and relative intensity of the radiolabeled bands on
autoradiographs from the samples immunoprecipitated with the specific
antibodies and recovered with protein A were identical to those
observed using the tropoelastin antibody and Staphylococcus
aureus.
H]leucine and a 90-min chase. Trichloroacetic
acid-precipitable radioactivity was from 7.5 µl of cell lysate or
medium. Values are shown in cpm ± S.E. of triplicate samples. In panel B, radiolabeled fibronectin (FN) is seen at the top of the gel due to direct binding of fibronectin to the Staphylococcus aureus used to immunoprecipitate tropoelastin
(see Fig. 2).
H]leucine, and the cell lysate and medium were
divided into three equal aliquots. From one aliquot, fibronectin was
immunoprecipitated with a specific primary antibody followed by protein
A (lanes 1 and 2). Tropoelastin was
immunoprecipitated from the other two aliquots with a tropoelastin
antibody followed by either protein A (lanes 3 and 4)
or Staphylococcus aureus (Staph A) (lanes 5 and 6).
BFA Prevents Tropoelastin Secretion, but No Intracellular
Accumulation Occurs
To investigate the intracellular pathway of
tropoelastin secretion, FCL cells were metabolically labeled for 4 h
with [H]leucine in the presence of 10 µg/ml
BFA, and the cell lysates and medium were immunoprecipitated for
tropoelastin (Fig. 3). In the absence of BFA, radiolabeled
tropoelastin was observed in both the cell lysate and medium. As
expected with this length of pulse, the proportion of tropoelastin in
the medium was greater than that present in the cell. Treatment of the
cells with BFA during the pulse resulted in a complete inhibition of
tropoelastin secretion. Although the tropoelastin was not secreted, no
accumulation of tropoelastin was observed within the cells. In
contrast, radiolabeled fibronectin accumulated in the cells following
the inhibition of its secretion by BFA treatment.
H]leucine for 4 h in the absence or presence of
10 µg/ml BFA. Tropoelastin (TE) was immunoprecipitated
from the cell lysates and medium using Staphylococcus aureus to collect the immune complexes. Samples were run on an 8.75%
SDS-polyacrylamide gel, fixed, treated with ENHANCE, and
exposed to x-ray film. Fibronectin (FN) binds directly to Staphylococcus aureus as shown in Fig. 2. Molecular
mass markers (kDa) are as indicated on the left.
Characterization of the Effect of BFA on FCL Cell
Ultrastructure and Total Protein Synthesis
Since the BFA
treatment did not result in an intracellular accumulation of
tropoelastin, it was important to verify that the BFA treatment of FCL
cells induced morphological changes that were characteristic of the
reported effects of BFA on cell ultrastructure. FCL cells treated for 4
h with 1 µl/ml MeSO alone showed no aberrant
morphology. At one pole of the nucleus, numerous stacks of Golgi
cisternae were observed surrounding the microtubule-organizing center (Fig. 4A). In contrast, cells treated with 10 µg/ml BFA
in MeSO for 4 h showed no recognizable Golgi structures (Fig. 4B). Instead, the Golgi region of these cells was
occupied by an extensive array of tubulovesicular elements situated in
the vicinity of the microtubule-organizing center. In addition to the
Golgi apparatus, the cisternae of ER in FCL cells treated with BFA were
also affected. In normal FCL cells, the ER is situated in the periphery
of the cell and consists of small, slightly dilated cisternae (Fig. 5A). In BFA-treated cells, the ER is still
peripherally located; however, the cisternae are grossly expanded and
highly irregular in shape (Fig. 5B). This morphology is
consistent with the reported accumulation of secreted proteins and
retrograde fusion of Golgi elements into the ER that occur in
BFA-treated cells(13, 15, 19) .
SO alone (A) and cells treated for 4 h with 10
µg/ml BFA (B). A, in control cells, numerous
stacks of Golgi (g) are observed at one pole of the nucleus (n) centered around the microtubule-organizing center (mtoc). This region also contains a multitude of small
vesicles and a few small cisternae of rough endoplasmic reticulum (rer). B, no visible stacks of Golgi are evident in
BFA-treated cells. Instead, a multitude of tubulovesicular structures (tv) are observed surrounding the microtubule-organizing
center. mt, microtubule. Bar, 0.5
µm.
SO alone (A) and cells treated for 4 h with 10
µg/ml BFA (B). A, the cisternae of endoplasmic
reticulum (rer) in control cells show normal ER morphology and
are interspersed with cables of actin filaments (a). B, in contrast, the cisternae of rough endoplasmic reticulum (rer) in cells treated with BFA appear swollen, diffuse, and
irregular in shape. Bar, 1.0
µm.
SO alone
(control) or 10 µg/ml BFA over a 4-h time course. As shown in Fig. 6, the synthesis of total Triton X-100-extractable
proteins, radiolabeled with [H]leucine, was found
to continue in both control and BFA-treated cells during the 4-h pulse.
BFA treatment does, however, lead to a 19% reduction over control cells
in the total trichloroacetic acid-precipitable counts after 4 h of
labeling. The treatment of FCL cells with 10 µg/ml BFA was
sufficient to block total protein secretion for at least 4 h, while in
control cells secretion of most radiolabeled proteins occurred after
approximately 1 h of pulse and steadily continued for the 4-h time
course.
H]leucine for up to 4 h in
the presence of 1 µl/ml MeSO alone (control) or 10
µg/ml BFA. Cell lysates and medium were collected and
trichloroacetic acid-precipitable radioactivity was determined from 7.5
µl of each sample. , control, lysate;
, control,
medium; , BFA, lysate;
, BFA,
medium.
Tropoelastin mRNA Levels Are Unaffected by BFA
It
has previously been reported that a time- and dose-dependent decrease
in tropoelastin gene expression occurs in smooth muscle cells treated
with monensin due to the repression of tropoelastin secretion (32) . In the present study, even though tropoelastin secretion
was inhibited, the amount of radiolabeled tropoelastin in the lysate of
BFA-treated FCL cells was actually less than that normally present in
the secretory pathway of untreated cells after 4 h. This raised the
possibility that a rapid down-regulation of tropoelastin gene
expression might result as a consequence of an accumulation of
tropoelastin in the ER during the initial stages of BFA treatment. To
investigate this possibility, the effect of BFA treatment on mRNA
levels of tropoelastin was investigated by Northern analysis. Fig. 7shows that treatment of FCL cells for up to 4 h with 10
µg/ml BFA has no effect on mRNA levels for tropoelastin.
SO only
for 1 h (lane 2). Ten µg of total RNA from each
experimental group was fractionated on a 1% agarose gel, transferred to
nylon membrane, and probed for tropoelastin (TE) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Selective Degradation of Tropoelastin Is Induced by BFA
Treatment
Since the synthesis of tropoelastin appears to
continue during BFA treatment, the lack of any accumulation of the
protein in the cell was hypothesized to be a result of intracellular
degradation. To study this possibility, FCL cells were pulsed for 1 h
in the presence of BFA and then chased in complete medium still
containing BFA in order to follow the fate of the radiolabeled
tropoelastin trapped within the cell. After 1.5 h of chase, the amount
of radiolabeled tropoelastin immunoprecipitated from the cells was
found to be less than half of the original amount, and by 3 h of chase,
little or no radiolabeled tropoelastin could be detected in the cell
lysate (Fig. 8). In contrast, the amount of fibronectin that was
initially radiolabeled during the 1 h pulse remained unchanged during
the 3-h chase. These results indicate that the tropoelastin trapped in
the fused ER/Golgi compartment is rapidly and selectively degraded.
H]leucine in medium
containing 10 µg/ml BFA. Cell lysates and media were then either
collected immediately or collected following a chase for a further 1.5
or 3 h in complete medium containing 10 µg/ml BFA. This protocol
allows the fate of the radiolabel tropoelastin to be studied in the
presence of BFA. Tropoelastin (TE) was immunoprecipitated from
the cell lysates as described in the legend to Fig. 3.
Fibronectin (FN) binds directly to Staphylococcus aureus as seen in Fig. 2.
Degradation of Tropoelastin Is Inhibited by ALLN but
Unaffected by Leupeptin, Ammonium Chloride, Pepstatin A, Cycloheximide,
and Nocodazole
To characterize the nature of the protease
responsible for the degradation of tropoelastin, several protease
inhibitors were tested for their ability to inhibit the BFA-induced
degradation. In a degradation assay similar to that shown in Fig. 8, the cysteine protease inhibitor, ALLN, was found to
completely inhibit the degradation of tropoelastin when included in the
chase together with BFA following the 1-h pulse (Fig. 9A).
Since it has been demonstrated that ALLN inhibits the activity of the
lysosomal proteases, cathepsin L and cathepsin B(33) , the
ability of lysosomotropic agents to inhibit degradation was also
tested. NHCl, which raises intracellular pH and thus
inhibits acid proteases in endosome/lysosome degradative pathway, did
not inhibit the rapid degradation of tropoelastin in BFA-treated cells (Fig. 9A). Similarly, leupeptin, a reversible inhibitor
of trypsin-like serine proteases and most cysteine proteases, and
pepstatin A, an aspartic protease inhibitor, had no effect on
tropoelastin degradation (not shown).
H]leucine in
complete medium containing 10 µg/ml BFA. Following the pulse, the
cell lysates were collected immediately or after a 1.5 or 3 h chase in
complete medium containing BFA alone or together with either ALLN,
NHCl, cycloheximide (CHX) or nocodazole (NZ). B, FCL cells were pretreated for 2 h with
either ALLN or NZ and then pulsed for 1 h with
[H]leucine in the presence of BFA + ALLN or
BFA + NZ. Following the pulse, the cell lysates were collected
immediately or after a 1.5- or 3-h chase in complete medium containing
BFA + ALLN or BFA + NZ. Cell lysates were immunoprecipitated
for tropoelastin as described in the legend to Fig. 3.
subunit have shown that
the degradation of both proteins is inhibited by ALLN, but only HMG-CoA
reductase degradation is prevented by cycloheximide(34) . Thus,
the effect of inhibiting protein synthesis by cycloheximide on
tropoelastin stability was tested. Fig. 9A shows that
the presence of cycloheximide in the chase following the 1-h pulse had
no effect on the rate of tropoelastin degradation. This result suggests
that de novo protein synthesis is not needed for the
tropoelastin to be degraded, and thus a short-lived protein is not
required for the degradation event.Tropoelastin Accumulation in the ER Reaches a Threshold
Level before Degradation Occurs
A consistent observation made
during the course of these experiments was that the amount of
tropoelastin immunoprecipitated from cell lysates of FCL cells pulsed
for 1 h in the presence of BFA was always greater than that obtained
from cells pulsed for 4 h under similar conditions. To investigate the
kinetics of the BFA-induced degradation of tropoelastin, the effect of
BFA on intracellular accumulation and secretion of tropoelastin was
studied by metabolically labeling cells for increasing lengths of time
in the presence of BFA alone or together with the inhibitor of
degradation, ALLN. In control cells, with no drug added during the
pulse, or in cells with ALLN only, the amount of tropoelastin
immunoprecipitated from the cell lysates appeared to steadily increase
until approximately 1 h (Fig. 10). At this time point,
tropoelastin could also be immunoprecipitated from the medium. As
expected, BFA alone, or together with ALLN, completely inhibited
tropoelastin secretion. In the cell lysate of BFA-treated cells,
radiolabeled tropoelastin was observed to accumulate within the cells
for up to 1 h (Fig. 10). However, with increasing pulse times,
the amount of radiolabeled tropoelastin in the cell lysate steadily
decreased to a level lower than that observed for the 15-min pulse.
Although this appears to suggest that the synthesis of tropoelastin is
affected by the longer incubation times in BFA, the presence of ALLN in
the labeling medium together with BFA allowed a steady accumulation of
intracellular radiolabeled tropoelastin to occur (Fig. 10). This
observation provides conclusive evidence that tropoelastin continues to
be synthesized in the presence of BFA throughout the 4-h time period.
H]leucine in the
absence of BFA, with ALLN or BFA alone, or with BFA and ALLN together.
At time points up to 4 h of pulse, cell lysates and medium were
collected and immunoprecipitated for tropoelastin as described in the
legend to Fig. 3.
, the degradation of T-cell receptor
is
unaffected by Ca homeostasis(34) . On the
contrary, T-cell receptor
degradation is significantly inhibited
by N-tosyl-L-phenylalanine chloromethyl ketone,
whereas N-tosyl-L-phenylalanine chloromethyl ketone
has no effect on the degradation of HMG-CoA reductase. Furthermore,
increasing evidence suggests that resident ER chaperone proteins, such
as immunoglobulin heavy chain binding protein (BiP/GRP78) and protein
disulfide isomerase, associate with abnormal or excess proteins prior
to their degradation in a pre-Golgi compartment (50, 51, 52) . Overall, these results suggest
that the proteolytic system of the ER is extremely intricate and likely
involves multiple enzyme systems with cooperation from several
different resident ER chaperones.
)Cl, ammonium
chloride.
We thank Lisa Mecham and David Schettler for cell
culture assistance and Dr. R. Pierce for assistance with probe
preparation and Northern analysis.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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