(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 [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.
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()(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.
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-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).
Reagents used during metabolic labeling included
ammonium chloride (NHCl), N-acetyl-Leu-Leu-norleucinal (ALLN), BFA, cycloheximide,
leupeptin, nocodazole, and pepstatin A (Sigma). NH
Cl 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.
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 5 min with room temperature 10%
trichloroacetic acid and 1
5 min with 95% ethanol and left to
dry prior to counting.
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 ENHANCE (DuPont NEN) for 1 h. Gels
were then dried and exposed to XAR-5 x-ray film (Eastman Kodak Co.).
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
[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).
Figure 2:
Fibronectin is immunoprecipitated with Staphylococcus aureus independent of fibronectin primary
antibody. FCL cells were radiolabeled for 2 h with
[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).
Figure 3:
BFA treatment of FCL cells results in
blocked secretion of tropoelastin with no concurrent intracellular
accumulation. FCL cells were pulsed with
[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 EN
HANCE, 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.
Figure 4:
Electron micrographs of cultured FCL cells
showing the Golgi region of control cells treated with 1 µl/ml
MeSO 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.
Figure 5:
Electron micrographs of cultured FCL cells
showing the peripheral region of control cells treated with 1 µl/ml
MeSO 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.
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 MeSO 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.
Figure 6:
Effect of BFA treatment on total protein
synthesis and secretion. FCL cells were metabolically labeled in
complete medium with [H]leucine for up to 4 h in
the presence of 1 µl/ml Me
SO 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.
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 MeSO 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).
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 [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.
Figure 9:
The cysteine protease inhibitor, ALLN,
inhibits the BFA-induced degradation of tropoelastin. A, FCL
cells were pulsed for 1 h with [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,
NH
Cl, 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.
Studies on the ER degradation
of HMG-CoA reductase and T-cell receptor 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.
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 [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.
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, 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.
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.