(Received for publication, March 8, 1995; and in revised form, May 24, 1995)
From the
Polypeptide hormones and neuropeptides are initially synthesized
as precursors possessing one or several domains that constitute the
propeptide. Previous work from our laboratory demonstrated that
expression of anglerfish prosomatostatin-I (proSRIF-I) in rat anterior
pituitary GH
Neuropeptides and peptide hormones are synthesized as
polypeptide precursors or prohormones that undergo a series of
post-translational processing reactions during their transport through
the secretory pathway to generate the mature hormone. A common feature
of prohormones is that the bioactive peptide is usually flanked by
pairs of basic amino acids or less commonly, single basic residues.
Endoproteolytic cleavage at a defined set of basic residues, mediated
by prohormone convertase enzymes (Steiner et al., 1992; Seidah
and Chrtien, 1992; Zhou et al., 1993),
results in excision of the peptide, which may then be subject to
further covalent modifications. For several prohormones, proteolytic
processing is initiated in the trans Golgi network and
continues during targeting to and within nascent immature secretory
granules (Mains and May, 1988; Fisher and Scheller, 1988; Lepage-Lezin et al., 1991; Xu and Shields, 1993). The mature hormones are
then concentrated, resulting in the formation of dense core secretory
granules that fuse with the plasma membrane and discharge their
contents in response to external stimuli; this has been designated
``regulated secretion'' (Burgess and Kelly, 1987). For
most prohormones, removal of the propeptide occurs quite late in the
secretory pathway. Consequently, it is possible that propeptides may
function both in the proximal steps of secretion by promoting correct
prohormone folding, exit from the endoplasmic reticulum, and/or
transport through the Golgi cisternae and in more distal events by
targeting precursors to post-Golgi secretory vesicles. Indeed, several
laboratories including our own (Stoller and Shields, 1989; Sevarino et al., 1989) have demonstrated that the propeptides of some
hormone precursors mediate intracellular transport and processing.
Expression in pituitary GH Mutagenesis and gene
fusion studies have shown that the propeptide of a yeast vacuolar
hydrolase precursor, pro-proteinase A, contains a vacuolar targeting
signal and that this sequence is important for facilitating proper
folding of the enzyme and efficient transit through the secretory
pathway (Klionsky et al., 1988). Studies on the Saccharomyces cerevisiae vacuolar enzyme precusor,
procarboxypeptidase Y, have defined a four-amino-acid motif in its
propeptide that confers vacuolar sorting (Valls et al., 1990);
a receptor for this domain, the product of the vps10 gene, has
recently been identified (Marcusson et al., 1994). In
addition, several members of the subtilisin protease family from
diverse species of bacteria and yeast are synthesized as proenzymes in
which the proregion is thought to act as an intramolecular chaperone
facilitating correct folding and activation of the enzyme (Silen et
al., 1989; Zhu et al., 1989). Most significantly, trans-expression of the propeptides of several of these
enzymes from separate plasmids was shown to activate these proteases in vivo and in vitro (Silen and Agard 1989; Fabre et al., 1992; Zhu et al., 1989). In the case of the
yeast Yarrowia lipolytica extracellular alkaline protease,
deletion of the propeptide resulted in its intracellular accumulation
early in the secretory pathway, and trans-expression of the
propeptide partially restored secretion of the mature enzyme (Fabre et al., 1992). Similarly, expression of the TGF- To further understand the
function of prohormones in intracellular sorting and processing, our
laboratory has been investigating the trafficking of preproSRIF. The
prosomatostatins comprise a prohormone family and are among the
simplest of peptide hormone precursors. The mature 14-residue hormone,
SRIF-14, is located at the carboxyl terminus of the propeptide and is
preceded by an Arg-Lys pair at the processing site; these are the only
dibasic amino acids in the precursor. Mammals possess a single gene
that encodes a common precursor to both SRIF-14 and SRIF-28, the latter
being a 14-residue NH
Figure 1:
Construction of chimeric cDNA encoding
the signal and propeptide of anglerfish SRIF-I fused to anglerfish
proSRIF-II. A PCR using oligonucleotides A and B was used to generate a
cDNA fragment corresponding to the SRIF-I signal and propeptide
terminating at residue Leu-66 (L). Oligonucleotides C and D
corresponding to residue 1 of the anglerfish SRIF-II propeptide and the
carboxyl terminus of SRIF-28, respectively, were used to prime a second
PCR. The two cDNAs were fused in a third PCR using oligonucleotides A
and D, which generated the full-length chimera. In each precursor, the wavyline corresponds to the signal peptide, and the adjacentarrow corresponds to its cleavage site. The arrow at position RK (Arg-Lys) in preproSRIF-I
corresponds to the prohormone converting enzyme 2 cleavage site, which
yields SRIF-14; the arrowhead at DL indicates the
carboxyl-terminal residues fused to proSRIF-II. The arrow adjacent to the Glu-Arg (ER) residues in preproSRIF-II
indicates the cleavage site that generates SRIF-28. To eliminate a
potential processing site in proSRIF-I (Glu-67-Arg-68), the
carboxyl-terminal 16 amino acids of the SRIF-I propeptide were excluded
from the chimera (see ``Experimental
Procedures'').
The chimeric cDNA was transfected into GH
For HPLC analysis, the protein A-Sepharose beads
were suspended in 100 ml of TEU (0.5 M Tris-HCl, pH 8.8, 20
mM EDTA, 8 M urea) containing 80 mM dithiothreitol and incubated at 65 °C for 20 min, centrifuged
at 15,000 For peptide sequencing,
a 100-mm dish of confluent GH
To test this idea, we
constructed a hybrid precursor containing the signal peptide and
propeptide of anglerfish SRIF-I fused to proSRIF-II (Fig. 1) and
expressed this chimera in GH
Figure 2:
The SRIF-I propeptide rescues proSRIF-II
from intracellular degradation. GH
Figure 3:
Processing and secretion of the chimera. A, HPLC resolution of SRIF-28 related polypeptides.
GH
To
test this directly, cells were labeled with
[
Figure 4:
Partial
amino-terminal amino acid sequencing of the processed SRIF-28
polypeptide. Two separate dishes of GH
To determine the processing efficiency and
secretion kinetics of the chimera, GH
Figure 5:
Processing of the chimera requires an
intracellular acidic pH. GH
Figure 6:
Immunolocalization of the SRIF-chimera.
GH
Figure 7:
The SRIF-I propeptide functions only in cis. GH
Previous work from our laboratory (Danoff et al.,
1991) demonstrated that anglerfish proSRIF-II is degraded
intracellularly in GH It is unclear why the processing, sorting, and secretion of
proSRIF-II is atypical; originally we had hypothesized that GH The
propeptides of several precursors have been implicated in mediating
intracellular transport as well as protein folding and enzyme
activation (for review, see Baker et al.(1993)). Studies on
the yeast vacuolar procarboxypeptidase Y (Valls et al., 1987;
Johnson et al., 1987; Valls et al., 1990)
demonstrated that a four-amino-acid motif present in the propeptide
acted as a positive sorting signal on proteins not normally targeted to
the vacuole. Recently, a receptor, the product of the vps10 gene has been identified that specifically recognizes this motif.
In bacteria,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
cells resulted in efficient and accurate
cleavage of the prohormone to generate the mature 14-amino acid
peptide, SRIF-I. We also implicated the propeptide in mediating
intracellular sorting to the trans Golgi network where
proteolytic processing is initiated. In contrast, expression of a
second form of the precursor, proSRIF-II in GH
cells
resulted in its intracellular degradation in an acidic, post-trans Golgi network compartment, most probably lysosomes. To further
investigate the positive sorting signal present in proSRIF-I, we
constructed a chimera comprising the signal peptide and proregion of
SRIF-I fused to proSRIF-II and expressed the cDNA in GH
cells. Here we demonstrate that the propeptide of SRIF-I rescued
proSRIF-II from intracellular degradation quantitatively and diverted
it to secretory vesicles. Furthermore, the chimera was processed to
SRIF-28, an amino-terminally extended form of the hormone that is the
physiological cleavage product of proSRIF-II processing in
vivo. Most significantly, the SRIF-I propeptide functioned only in cis as part of the fusion protein and not in trans when expressed as a separate polypeptide. These data suggest that
the SRIF-I propeptide may possess a sorting signal for sequestration
into the secretory pathway rather than functioning as an intramolecular
chaperone to promote protein folding.
cells of a chimera consisting of
the signal sequence and propeptide of anglerfish preproSRIF-I fused to
ape
-globin resulted in targeting of
-globin to the regulated
secretory pathway; furthermore, the chimera was correctly processed to
yield
-globin (Stoller and Shields, 1989). Analogous results were
obtained by Sevarino et al.(1989). Similarly, the
amino-terminal domain of proenkephalin targeted frog dermophin to the
regulated secretory pathway (Seethaler et al., 1991). Deletion
of the propeptide from rat serum albumin, a protein secreted via the
constitutive pathway, inhibited its exit rate from the endoplasmic
reticulum, implying that the propeptide might be involved in protein
folding in this organelle (McCracken and Kruse, 1989). Likewise, the
propeptide of transforming growth factor-
1 (TGF-
1) (
)promotes its correct folding and secretion and may
regulate the biological function of the mature growth factor (Gentry
and Nash, 1990; Brunner et al., 1989).
1
propeptide in trans together with TGF-
1 lacking its
propeptide facilitated a low level of secretion of biologically active
TGF-
1 (Gray and Mason, 1990).
-terminally extended form of the
tetradecapeptide (Noe and Speiss, 1983). In several species of fish,
however, two distinct genes expressed in different populations of islet
D-cells encode separate precursors, preproSRIF-I and II, from which are
released the 14- and the 28-amino-acid peptides, respectively (Noe and
Spiess, 1983; Noe et al., 1987; McDonald et al.,
1987). Our previous experiments showed that these two precursors had
significantly different fates when their cDNAs were stably expressed in
rat anterior pituitary GH
cells. PreproSRIF-I, which
displays substantial homology to mammalian SRIF precursors, was
processed efficiently and accurately to the mature 14-amino-acid
peptide and targeted to the regulated secretory pathway (Stoller and
Shields, 1988). In contrast, proSRIF-II, which has much less homology
to mammalian proSRIFs, was quantitatively degraded intracellularly in a
chloroquine-sensitive post-TGN compartment (Danoff et al.,
1991). Since the SRIF-I propeptide possesses positive sorting
information, we hypothesized that it might divert proSRIF-II from the
``degradation pathway'' to secretory vesicles. To test this
idea, we constructed a chimera comprising the SRIF-I signal and
propeptide fused to proSRIF-II and introduced this cDNA into GH
cells. Here we demonstrate that when expressed in cis but not in trans the SRIF-I propeptide rescued proSRIF-II
from intracellular degradation. Furthermore, the chimera was processed
to mature SRIF-28, the physiological cleavage product of proSRIF-II,
and targeted to secretory vesicles.
Materials
[H]Leucine was
purchased from Amersham Corp. [
S]Cysteine was
purchased from DuPont NEN. Chloroquine was purchased from Sigma.
Lipofectin was purchased from Life Technologies, Inc. The mammalian
expression vector pCEP4 containing a hygromycin selectable marker was
purchased from InVitrogen. Reverse phase HPLC columns were purchased
from Vydac (Hesperia, CA). Rabbit antisera specific for the propeptides
of human SRIF and anglerfish SRIF-I and -II were prepared against
recombinant antigens isolated from bacteria (Elgort and Shields 1994);
the serum specific for the SRIF-II propeptide was designated R60.
Generation of Chimeric DNA Encoding the Signal Peptide
and Proregion of Anglerfish SRIF-I Fused to proSRIF-II
The
starting cDNAs for this construction were plasmids pDS5/pLAS I (Danoff et al., 1991) and pLJ/pLAS-II (Danoff et al., 1991)
encoding anglerfish preproSRIF-I and preproSRIF-II, respectively. A
cDNA fragment I encoding the signal peptide and most of the propeptide
of anglerfish preproSRIF-I (Fig. 1) was generated by PCR using
oligonucleotides A (5`-CCCGGGATCCGCAGACGCCGGCAGA-3`) and B
(5`-CTCTCTGTCGAGCTGTAGGTCGGCGTGGGC-3`) (reverse).
Oligonucleotide B was a 30-mer containing 15 nucleotides from the
carboxyl terminus of the SRIF-I propeptide (italics) and 15 nucleotides
from the amino terminus of the preproSRIF-II propeptide. Fragment II
encoding the pro- and mature peptides of anglerfish proSRIF-II was also
generated by PCR using oligonucleotides C
(5`-GCCCACGCCGACCTACAGCTCGACAGAGAG-3`) and D
(5`-CCCGGGATCCGTTGGTCCGGTGACG-3`) (reverse). Oligonucleotide C was a
30-mer, containing 15 nucleotides from the SRIF-I propeptide (italics)
and 15 from the amino terminus of the proSRIF-II propeptide. The
chimeric cDNA was constructed by hybridizing fragment I and fragment II
at their cohesive ends and amplified by PCR using oligonucleotide
fragments A and D (Fig. 1). The resulting cDNA was digested with BamHI (both oligonucleotides A and D have the BamHI
restriction site) and subcloned into vectors pTZ19R (U. S. Biochemical
Corp.) for DNA sequencing and pcDNA/neo (Invitrogen) for expression in
GH cells. The fidelity of the chimeric cDNA was confirmed
by DNA sequencing as well as by in vitro transcription and
translation; the translation product was immunoprecipitable with
antiserum R33, which is specific for SRIF-28 (Danoff et al.,
1991).
cells
using the Lipofectin procedure (Life Technologies, Inc.) as described
previously (Danoff et al., 1991). Cells were selected for
growth in the presence of 1 mg/ml G418 (Sigma) for 11 days. Several
G418-resistant colonies were established; these were assayed for the
expression of chimeric protein by pulse labeling followed by
immunoprecipitation with anti-SRIF-28 serum. One such colony designated
GH
.C28.7 was chosen for further analysis; it should be
noted that identical results were obtained with several of the other
clonal lines (data not shown).
Co-expression of SRIF Propeptides with Anglerfish
proSRIF-II
cDNA encoding the signal peptide and proregion of
human SRIF (Elgort and Shields 1994) was subcloned directly into the BamHI site of the mammalian expression vector pCEP4. A cDNA
fragment encoding the signal peptide and proregion of anglerfish SRIF-I
was generated by PCR techniques such that a stop codon was introduced
after Lys-107 (Fig. 1) and the fragment ligated into the pCEP4
expression vector. For selection of cells expressing the human or
anglerfish SRIF-I propeptide, GH.A107 cells expressing
native proSRIF-II were transfected with the appropriate DNA and grown
in the presence of 125 µg/ml of hygromycin for 10-14 days.
Hygromycin-resistant cells were analyzed for the expression of the SRIF
propeptide by pulse-chase followed by immunoprecipitation using
antibodies specific for each individual propeptide (Elgort and Shields,
1994). Experiments were performed on mixed populations of
hygromycin-resistant GH
.A107 cells.
Pulse-Chase, Immunoprecipitation and HPLC
Analysis
Confluent GH.C28.7 cells grown in 35-mm
tissue culture dishes, were washed twice with PBS, pulse-labeled in
RPMI medium (Life Technologies, Inc.) with 500 µCi/ml
[
S]Cys for 15 min at 37 °C, and chased for
various times in growth medium containing 15% horse serum and 2.5%
fetal calf serum. The cell lysate and medium was adjusted to a final
concentration of 156 mM NaCl, 40 mM Tris-HCl, pH 8.3,
5 mM EDTA, 2% Triton X-100, 100 units/ml Trasylol, 5 mM cysteine, 1 mg/ml bovine serum albumin. Appropriate antibodies
were added to each sample, and immunoprecipitation was performed
overnight at 4 °C followed by the addition of protein A-Sepharose
(Zymed, CA) as described previously (Stoller and Shields, 1988). Prior
to gel electrophoresis, the beads were washed twice with 150 mM NaCl, 10 mM Tris-HCl, pH 8.3, 5 mM EDTA, 0.1%
Triton X-100, and twice with PBS, followed by boiling for 3 min in
SDS-PAGE loading buffer (8.5% sucrose, 2 mM EDTA, 200 mM Tris, pH 8.8, 5% SDS, 200 mM dithiothreitol, 0.005%
bromphenol blue).
g for 10 min, and the supernatant was
treated with iodoacetic acid (3.2 mg/ml) for 10 min at room
temperature. Acetonitrile containing 0.1% trifluoroacetic acid was
added to the samples to give a final concentration of 8%. Samples were
applied to a Vydac C
reverse-phase column using a
Waters-Millipore HPLC system. The column was eluted at a flow rate of
1.5 ml/min with a gradient of 0-4 min 5% CH
CN;
4-6 min 5-16% CH
CN; 6-20 min 16-35%
CH
CN; 20-34 min 35-45% CH
CN;
34-37 min 45-46% CH
CN; 37-39 min
46-80% CH
CN, all containing 0.1% trifluoroacetic
acid. One-minute fractions were collected, and the radioactivity was
determined by liquid scintillation counting.
.C28.7 cells was labeled with
500 µCi/ml [
S]Cys, or 1 mCi/ml
[
H]Leu for 2 h. The media and cell lysates were
collected and incubated with SRIF-28 antiserum, and the
immunoprecipitates combined. The immunoprecipitated peptides were
resolved by HPLC, and the fractions corresponding to mature SRIF-28
were pooled, lyophilized, and subjected to 30 cycles of automated Edman
degradation using an Applied Biosystems model 301A sequencer.
Immunofluorescence
Immunofluorescence was
performed as described previously (Danoff et al., 1991).
Briefly, cells were grown on polylysine-coated glass coverslips, fixed
in 3% paraformaldehyde, permeabilized with 0.2% saponin in PBS
containing 1% fetal calf serum as blocking agent, and incubated for 60
min with either a rabbit antisera directed against the SRIF-II
propeptide (R60) or SRIF-28 (R33) together with a mouse monoclonal
antibody 53FC3 against Golgi mannosidase II (Burke and Warren, 1984).
Samples were double labeled by incubation with fluorescein
isothiocyanate-conjugated goat anti-mouse serum and
rhodamine-conjugated goat anti-rabbit serum and examined using a
Bio-Rad MRC 600 laser scanning confocal microscope.
Expression of the preproSRIF-I-proSRIF-II Chimera in
GH
Previous work from our laboratory
has established that anglerfish preproSRIF-I and human proSRIF are
processed efficiently to SRIF-14 with about 75% efficiency in GHCells
cells, and approximately 60% of the mature hormone is sorted to
the regulated secretory pathway in rat pituitary GH
cells
(Stoller and Shields, 1988; Elgort and Shields, 1994). In contrast,
anglerfish proSRIF-II is degraded in an acidic post-Golgi compartment,
and no mature SRIF-28 is processed from this precursor (Danoff et
al., 1991). We hypothesized that proSRIF-II degradation occurred
because either GH
cells lack a prohormone processing enzyme
that cleaves at the monobasic residue, to generate SRIF-28, or that the
SRIF-II propeptide might be incorrectly folded and therefore would be
functionally incompetent in GH
cells. To distinguish
between these two possibilities and since earlier studies (Stoller and
Shields, 1989) had demonstrated that anglerfish proSRIF-I and rat
proSRIF contain positive sorting signals (Sevarino et al.,
1989), we hypothesized that the SRIF-I propeptide might rescue
proSRIF-II from intracellular degradation.
cells. To exclude the
possibility of prohormone processing occurring at sites present in the
SRIF-I propeptide, the chimera possessed a truncated SRIF-I proregion
comprising the first 66 residues of the propeptide, which lacks both
the monobasic and dibasic cleavage sites (Fig. 1) fused to the
entire proregion (74 amino acids) and mature hormone (28 amino acids)
of preproSRIF-II. The sequence of the chimera was confirmed by DNA
sequencing and by in vitro transcription and translation
followed by immunoprecipitation with anti-SRIF-28 serum (data not
shown).
proSRIF-II Degradation Is Rescued by the SRIF-I
Propeptide
We had previously shown that proSRIF-II degradation
occurred in a compartment distal to the 20 °C temperature block, i.e. in a post-TGN compartment (Danoff et al., 1991).
To determine if the SRIF-I propeptide could rescue proSRIF-II from
degradation, GH.C28.7 cells expressing the chimera were
pulse-labeled for 15 min with [
S]Cys, an amino
acid present only in the carboxyl-terminal mature SRIF peptide, and
chased for 2 h either at 37 or 20 °C followed by subsequent
incubation at 37 °C for up to 3 h. At each time point, the cell
lysate and medium were treated with anti-SRIF-28 serum, followed by
SDS-PAGE (Fig. 2). A major SRIF-28 immunoreactive polypeptide (M
24,000) was evident in GH
.C28.7
cells after the initial pulse labeling and 2 h of chase at 20 °C (lanes1 and 3). Following the temperature
shift from 20 to 37 °C, this 24-kDa chimera disappeared from the
cells (lanes4 and 5) and was secreted into
the medium (lanes9 and 10). Similarly, the
SRIF-28-immunoreactive chimera was also secreted when the cells were
chased only at 37 °C (lanes2 and 7).
Consistent with our previous data (Danoff et al., 1991),
incubation at 20 °C inhibited secretion, and the chimera was
retained in the cells (lanes3 and 8). In
contrast to the behavior of the chimeric precursor, when
GH
.A107 cells expressing native proSRIFII were chased at 37
°C for 2 h, the level of intracellular proSRIF-II was diminished,
but no SRIF-immunoreactive material was recovered in the medium (lanes12 and 17). As previously observed
(Danoff et al., 1991), incubation at 20 °C prevented
proSRIF-II degradation (lane13); however, subsequent
chase at 37 °C (lanes14 and 15)
resulted in its loss from the cells, and no SRIF-28 material was
recovered in the medium (lanes19 and 20)
due to its intracellular turnover.
.C28.7 cells (lanes1-10) and GH
.A107 cells (lanes 11-20) were pulse-labeled for 15 min
with [
S]Cys at 37 °C and chased for 2 h
either at 37 °C (lanes2, 7, 12, and 17) or 20 °C followed by subsequent
incubation at 37 °C for the indicated times (lanes4 and 5; 9 and 10; 14 and 15; 19 and 20). At each time point, the cell
lysate and media were treated with anti-SRIF-28 serum (R33), and the
immunoprecipitable material was analyzed by SDS-PAGE using a 15%
gel.
The SRIF Chimera Is Processed to SRIF-28
The
preceding data showed that the SRIF-I propeptide rescued proSRIF-II
from intracellular degradation. However, we were unable to determine if
the chimera was processed correctly to SRIF-28 using SDS-PAGE because
it was too small to resolve accurately; we therefore used HPLC methods.
GH.C28.7 cells were pulse-labeled for 15 min with
[
S]Cys and chased for 2 h at 37 °C followed
by immunoprecipitation of the intracellular and secreted material with
anti SRIF-28 antibodies, and the peptides were analyzed by HPLC (Fig. 3A). Following the initial pulse labeling, a
major intracellular polypeptide corresponding to unprocessed chimera (panelA, fraction 34) was evident. During
the 2-h chase, the chimera was processed to a polypeptide with the
identical retention time as mature SRIF-28 suggesting that it was
processed correctly (panelsC and D,
fraction 13) and interestingly, no SRIF-14 was evident. Since
the processed form of the chimera had an HPLC retention time that was
identical to SRIF-28 (Fig. 3A) and migrated on SDS-PAGE
at about 3-4 kDa, the expected size of SRIF-28 (data not shown),
this suggested that it was correctly processed to mature SRIF-28.
.C28.7 cells were pulse labeled for 15 min with
[
S]Cys (panelsA and B) chased for 2 h at 37 °C (panelsC and D), the intracellular and secreted material incubated with
anti-SRIF-28 serum and the immunoprecipitable polypeptides resolved by
reverse phase HPLC (see ``Experimental Procedures''). The
radioactivity in each fraction was determined by liquid scintillation
counting. The elution positions of mature SRIF-28 (fraction 13) and the chimera (fraction 34) are indicated.
Intracellular SRIF-28 material (
), secreted SRIF-28 polypeptides
(
). B, processing and secretion kinetics of the proSRIF
chimera. Cells were pulse-labeled for 15 min with
[
S]Cys and chased for up to 6 h; at each time
point, the intracellular and secreted SRIF-28 immunoreactive
polypeptides were resolved by HPLC, and the areas under each peak were
used to calculate the processing and secretion efficiencies. Percent
secretion = (secreted (total SRIF-28 immunoreactive
material))/(total SRIF-28 immunoreactive material (intracellular +
secreted))
100. Percent processing = (mature SRIF-28
(intracellular + secreted))/(total SRIF-28 immunoreactive material
(intracellular + secreted))
100.
H]Leu or [
S]Cys, the cell
lysate and media were incubated with anti-SRIF-28, the
immunoprecipitated material was combined, and the SRIF-28 polypeptide
was separated by HPLC. The HPLC-purified SRIF-28 peptide (fraction 13) was subjected to 30 cycles of Edman degradation (Fig. 4). If cleavage of the chimera occurred correctly at the
single Arg-processing site of proSRIF-II (Fig. 1), then
[
H]Leu radioactivity should be detected at cycle
8 and [
S]Cys radioactivity should be present at
cycles 17 and 28 of Edman degradation. The data of Fig. 4demonstrate that this was correct; as expected, Leu was
present at residue 8, and cysteine was detected at 17. Due to loss of
material from the sequencer at each cycle and decreased repetitive
yield, we were unable to detect significant levels of
[
S]Cys radioactivity above background levels at
residue 28. Nevertheless, these data demonstrate that the chimera was
cleaved correctly to yield SRIF-28, the physiological processing
product of proSRIF-II.
.C28.7 cells were
radiolabeled continuously with [
H]Leu (panelA) or [
S]Cys (panelB) for 120 min at 37 °C. Following incubation, the
intracellular and secreted material was treated with anti SRIF-28 serum
and the
H- and
S-labeled polypeptides were
combined and resolved by HPLC. Fraction 13 corresponding to SRIF-28 was
collected and applied to an Applied Biosystems sequencer and subjected
to 30 cycles Edman degradation. The radioactivity in each cycle was
determined by liquid scintillation counting. Letters correspond to the
predicted amino acid sequence of anglerfish proSRIF-II (Hobart et
al., 1980).
.C28.7 cells were
labeled for 15 min with [
S]Cys and chased for
various times up to 6 h, and the intracellular and secreted SRIF-28
immunoreactive material was quantitated after HPLC analysis (Fig. 3B). Following a lag of
15 min, the
unprocessed chimera was secreted linearly for up to 90 min. After 3 h
of chase, approximately 75-80% of the pulse labeled precursor was
secreted. In addition, processing of the chimera, which was
approximately 40-45% efficient, was evident only following a lag
time of 30-60 min, presumably the time taken to reach the
intracellular site where the processing enzyme is located, and was
linear for approximately 3 h, after which time it reached a plateau (Fig. 3B); these processing kinetics were virtually
identical to those of native proSRIF-I in GH
cells (Stoller
and Shields, 1988).
Processing of the Chimera Requires an Acidic pH
We
have previously shown that proSRIF processing, which can be initiated
in the TGN, requires an acidic pH that is generated by a
bafilomycin-sensitive vacuolar H-ATPase, and recently
we demonstrated that proSRIF processing in the TGN occurs at pH 6.1
(Stoller and Shields, 1989; Xu and Shields 1993, 1994). Since an
aspartyl protease, with an acidic optimum, possibly the mammalian
homologue of the S. cerevisiae Yap3 enzyme, has been
implicated in proSRIF-II processing (Cawley et al., 1993;
Bourbonnais et al., 1993), we hypothesized that processing of
the chimera would also be dependent on an acidic pH. Cells were
pretreated with or without 40 µM chloroquine and
pulse-labeled with [
S]Cys for 15 min followed by
a 2-h chase in the absence or presence of chloroquine and the SRIF-28
immunoreactive polypeptides resolved by HPLC (Fig. 5). In
agreement with our previous data (Danoff et al., 1991),
chloroquine almost quantitatively inhibited processing; only
6% of
the chimera was processed to SRIF-28 compared with
35% for the
control untreated cells (compare panelsE and F). These data indicate that, like native proSRIF-I,
processing of the chimera to the mature hormone also occurred in an
intracellular compartment possessing an acidic pH.
.C28.7 cells were preincubated
for 30 min in the absence (panelsA, C, and E) or presence (panelsB, D, and F) of 40 µM chloroquine followed by
pulse-labeling with [
S]Cys for 15 min and chased
for 0 min (panelsA and B) or 120 min (panelsC-F) in the absence (panelsA, C, and E) or presence (panelsB, D, and F) of chloroquine. At each
time point, the cell lysates and media were immunoprecipitated with
antiserum R33 and analyzed by HPLC. No detectable SRIF-28 material was
secreted following the 15-min pulse (not shown). Intracellular material
(
); secreted material (
).
Immunolocalization of SRIF Chimera
Our earlier
studies (Danoff et al., 1991) using scanning confocal
immunofluorescence microscopy, had demonstrated a low level of diffuse
reticular staining of SRIF-28 material in the endoplasmic reticulum and
in the perinuclear Golgi region of GH.A107 cells; however,
no staining was evident in secretory granules, presumably because the
peptide was mis-targeted to lysosomes and degraded. To confirm that a
consequence of proSRIF-II rescue was correct targetting to the
secretory pathway, immunolocalization studies were performed on both
GH
C28.7 and GH
A.107 cells. In
GH
C28.7 cells treated with anti SRIF-28 antisera, most of
the SRIF-immunoreactive material displayed a perinuclear punctate
staining characteristic of the Golgi apparatus and secretory vesicles,
respectively (Fig. 6, panelA). Also there was
significant colocalization with the Golgi marker mannosidase II (panelC). An identical staining pattern was evident
when the cells were incubated with an antibody that recognizes only the
propeptide of SRIF-II (panelE). The slight
difference in staining by the mannosidase II monoclonal antibody in panelsC and D was due to the confocal
optical section rather than to an intrinsic difference in Golgi
morphology between these clonal lines. In earlier studies (Danoff et al., 1991), which showed a different optical section, the
appearance of the Golgi apparatus in GH
.A107 cells was
similar to that shown in panelD. As observed
previously (Danoff et al., 1991), immunoreactive SRIF-28 in
GH
A107 cells exhibited faint reticular staining
characteristic of the endoplasmic reticulum (panelB), which was confirmed with anti-propeptide antibody (panelF). Some staining of the Golgi apparatus was
also detected. However, in contrast to the chimera, no
SRIF-immunoreactive material was present in secretory vesicles,
presumably because it was degraded in lysosomes. These results further
demonstrated that the SRIF-I propeptide could target proSRIF-II away
from the degradation pathway and into post-Golgi apparatus secretory
vesicles.
.C28.7 and GH
.A107cells were grown on
polylysine-coated coverslips, fixed with 3% paraformaldehyde, and
permeabilized with 0.2% saponin and incubated with rabbit anti-SRIF-28 (panelsA and B), mouse monoclonal antibody,
53FC3, directed against a M
135,000 Golgi membrane
protein mannosidase II (panelsC and D)
(Burke and Warren, 1984), rabbit antiserum R60 directed against the
propeptide of SRIF-II (panelsE and F).
Then, both rhodamine-conjugated goat anti-rabbit IgG and fluorescein
isothiocyanate-conjugated goat anti-mouse IgG were incubated with the
samples in panelsA and C and panels D and F, respectively, to double label the cells. Cells in panelsB and E were treated only with
rhodamine-conjugated goat anti-rabbit IgG. Samples were examined using
a confocal laser scanning microscope. The bars correspond to
10 µm. GH
.C28.7 cells, panelsA, C, and E; GH
A.107 cells, panelsB, D, and F.
The SRIF-I Propeptide Functions Only in Cis
For
several bacterial and yeast protease precursors that are synthesized as
proenzymes, the proregion has been shown to function both in mediating
correct folding of the protease and in inhibiting its activity (Baker et al., 1993). Furthermore, in several cases, the proregion
could confer correct protein folding and hence proteolytic activity in trans, i.e. as a separate polypeptide (Fabre et
al., 1992; Silen and Agard 1989; Zhu et al., 1989). It
was therefore of interest to determine if the SRIF-I propeptide could
also rescue proSRIF-II from intracellular degradation when expressed in trans. To test this idea, the original neomycin-resistant
GH.A107 cells synthesizing proSRIF-II were transfected with
a second expression plasmid that encoded either the anglerfish SRIF-I
propeptide or the human SRIF propeptide and also conferred resistance
to the antibiotic hygromycin (see ``Experimental
Procedures''). Hygromycin-resistant cells were selected, and the
secretion of proSRIF-II and each propeptide was determined by
sequential immunoprecipitation using species specific antibodies to
each respective propeptide (Fig. 7). We have recently
demonstrated that the propeptides of SRIF-I and human proSRIFs are
secreted as intact polypeptides from GH
cells (Elgort and
Shields, 1994). Similarly, both the anglerfish SRIF-I and human SRIF
propeptides were efficiently secreted from the GH
A.107
cells following 2 h of chase (lanes1-4).
However, when expressed in trans as a separate polypeptide,
neither propeptide was able to rescue proSRIF-II from intracellular
degradation (lanes7 and 8 with 9 and 10). These results contrast markedly with expression
of the chimera (Fig. 2), which was efficiently secreted. In
control GH
.A107 cells, as expected proSRIF-II was also
degraded (lanes11 and 12).
A.107 cells were co-transfected with cDNAs
encoding the anglerfish SRIF-I propeptide (lanes1 and 2 and lanes 7 and 8) or the human
SRIF propeptide (lanes3 and 4 and lanes 9 and 10) or control cells received no additional
cDNAs (lanes5 and 6 and lanes 11 and 12). Stable transfectants expressing the respective
propeptides were selected by their resistance to the antibiotic
hygromycin (see ``Experimental Procedures''). Cells were
pulse-labeled with [
S]Cys for 15 min and chased
for 120 min at 37 °C. The cell lysates (C) and media (M) were then incubated sequentially with different
antibodies: first with anti-SRIF-I propeptide antisera (lanes1, 2, 5, and 6) followed by
anti-SRIF-II propeptide antisera, R60 (lanes7, 8, 11, and 12) or first with anti human
SRIF propeptide antisera (lanes3 and 4)
followed by anti SRIF-II propeptide antisera, R60 (lanes9 and 10). After immunoprecipitation with the respective
antisera, samples were analyzed by SDS-PAGE and
fluorography.
cells in a post 20 °C, acidic
compartment, most likely lysosomes. This observation is in marked
contrast to that of proSRIF-I or the human precursor expression where
processing and targeting to the regulated secretory pathway is quite
efficient (Stoller and Shields, 1988; Elgort and Shields, 1994). Indeed
the expected phenotype for expression of a prohormone in cells lacking
prohormone converting enzymes would be secretion of the unprocessed
precursor. Expression of proSRIF-II in mouse anterior pituitary AtT-20
cells, which express prohormone converting enzymes 1/3 and low levels
of prohormone converting enzyme 2 (Zhou et al., 1993), did not
result in degradation, instead proSRIF-II was aberrantly cleaved to
yield a nonphysiological peptide (Sevarino et al., 1989),
suggesting that this precursor is also poorly recognized in these
cells. However, the sorting efficiency of a chimera comprising the rat
SRIF propeptide fused to anglerfish proSRIF-II was improved relative
to the low efficiency of proSRIF-II sorting in these cells. The goal
of our present studies was to understand how proSRIF-II was mistargeted
in different pituitary cell lines. As a first step to answering this
question, we utilized our previous observation that proSRIF-I possesses
positive sorting information and constructed a chimera consisting of
the SRIF-I propeptide fused to proSRIF-II. Here we have demonstrated ( Fig. 2and Fig. 3) that the SRIF-I propeptide rescued
proSRIF-II from intracellular degradation quantitatively, and
approximately 40% of the chimera was processed to SRIF-28, the
physiological product of native proSRIF-II processing (Noe and Spiess,
1983).
cells might lack the aspartyl protease that has been implicated
in proSRIF-II processing in islet D cells (Mackin et al.,
1991; Cawley et al., 1993), however this was not correct since
a significant fraction of the chimera was accurately processed to
SRIF-28 ( Fig. 3and Fig. 4). This suggests that native
proSRIFII was diverted from a processing to a degradation pathway prior
to interaction with the cleavage enzyme. We propose that proSRIF-II was
degraded because its propeptide was poorly recognized by the sorting
apparatus of mammalian cells. This hypothesis is based on comparison of
the amino acid sequences of seven proSRIFs, which revealed that
proSRIF-II deviated significantly from other members of this prohormone
family. Mammalian preproSRIFs exhibit remarkable amino acid
conservation and share
96% overall sequence identity, which
includes their signal peptides; this high degree of sequence identity
may indicate functional conservation. It is noteworthy that the
anglerfish SRIF-I propeptide has
38.5% identity with mammalian
precursors, and it is this polypeptide that is efficiently processed
and sorted, whereas anglerfish proSRIF-II shares only
24% identity
with the mammalian precursors. A highly conserved polar hexapeptide
motif (Ala-Pro-Arg-Glu-Arg-Lys) that includes the Arg-Lys processing
site is present in all proSRIFs except proSRIF-II. In proSRIF-II, a
single amino acid change alters this region to Pro-Pro-Arg-Glu-Arg-Lys.
We hypothesized that this substitution might cause proSRIF-II to be a
poor substrate for the prohormone converting enzyme 2, thereby causing
its missorting and subsequent degradation. This was not the case,
however, because mutagenesis of the first Pro to Ala had no effect on
the intracellular degradation of proSRIF-II. (
)We
therefore speculate that the structure of the SRIF-II propeptide may be
significantly different from its mammalian counterpart and is poorly
recognized by a putative receptor or ``quality control sorting
machinery'' located in the TGN or post-TGN vesicles.
-lytic protease of Lysobacter enzymogenes or
subtilisin from Bacillus subtilis are synthesized as
precursors with 166- and 77-residue propeptides, respectively (Baker et al., 1993). Folding of these proteases to generate an
active enzyme requires the propeptide domain. However, not only does
the propeptide function in cis as a component of the
proenzyme, but it can also function in trans as a separate
polypeptide. It has been suggested that these propeptides enhance the
rate of folding to the native state in contrast to chaperone-type
molecules, which prevent protein aggregation or misfolding (Baker et al., 1993). In addition, several mammalian propeptides,
including the proregion of TGF-
1, which is necessary for its
secretion, can function in cis or in trans. Recently,
Fabre et al.(1992) demonstrated that the intracellular
transport of a yeast alkaline extracellular protease was rescued by trans-complementation using its propeptide. We have recently
demonstrated that the human SRIF propeptide itself, lacking any mature
hormone sequence, can be transported through the secretory pathway,
implying that it possesses the structural features necessary for
correct folding, exit from the endoplasmic reticulum, and sorting in
the TGN (Elgort and Shields, 1994). In light of these observations, we
investigated the possibility that the anglerfish SRIF-I propeptide
might function in trans to rescue proSRIF-II from
intracellular degradation. However, in contrast to the above mentioned
studies, the SRIF-I propeptide functioned only in cis when
covalently linked to proSRIF-II (Fig. 7). Indeed when the human
or anglerfish SRIF-I propeptides were co-expressed with proSRIF-II,
both of these propeptides were efficiently secreted, whereas the
proSRIF-II was degraded intracellularly. We suggest that because the
propeptide of anglerfish SRIF-I more closely resembles its mammalian
counterpart than does proSRIF-II, its conformation facilitates
interaction with a putative receptor apparatus in the TGN of GH
cells, thereby ensuring sorting into secretory granules (Chung et al., 1989); however, the existence of a prohormone sorting
receptor still remains to be demonstrated, definitively. If such a
model were correct, the folded SRIF-I propeptide would bind a putative
receptor and facilitate sorting to secretory granules. The propeptide
could not effect sorting in trans because although it might be
recognized by the receptor machinery, proteins not directly bound to it
or lacking the functional equivalent of a sorting domain would be
mistargeted. Currently, we are using several native and mutated SRIF
propeptides to identify components of the putative sorting apparatus.
Finally, studies in which specific regions of the rat SRIF propeptide
were deleted suggested that the conformation of the proregion is not
important for recognition of the SRIF processing site (Sevarino and
Stork, 1991). Consequently, it will be of interest to determine the
effect of such deletions on the sorting of the chimeric precursor to
determine if specific domains of the SRIF-I propeptide effect rescue of
proSRIF-II from intracellular degradation; these experiments are
currently in progress.
1, transforming growth factor-
1;
TGN, trans Golgi network; HPLC, high performance liquid
chromatography; PCR, polymerase chain reaction; PAGE, polyacrylamide
gel electrophoresis; SRIF, somatostatin.
We thank Arkady Elgort for generating and generously
supplying the various anti-propeptide antisera, Dr. Ruth Angeletti for
performing the protein sequencing, and Michael Cammer for help with the
confocal microscopy experiments. We particularly thank Cary Austin and
Duncan Wilson for helpful suggestions and discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.