©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Propeptide of Anglerfish Preprosomatostatin-I Rescues Prosomatostatin-II from Intracellular Degradation (*)

(Received for publication, March 8, 1995; and in revised form, May 24, 1995)

Ye-Guang Chen (1)(§) Ann Danoff (3) Dennis Shields (1) (2)(¶)

From the  (1)Departments of Developmental and Molecular Biology and (2)Anatomy and Structural Biology and the (3)Division of Endocrinology, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(3) 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(3) 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(3) 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.


INTRODUCTION

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(3) cells of a chimera consisting of the signal sequence and propeptide of anglerfish preproSRIF-I fused to ape alpha-globin resulted in targeting of alpha-globin to the regulated secretory pathway; furthermore, the chimera was correctly processed to yield alpha-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-beta1 (TGF-beta1) (^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).

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-beta1 propeptide in trans together with TGF-beta1 lacking its propeptide facilitated a low level of secretion of biologically active TGF-beta1 (Gray and Mason, 1990).

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(2)-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(3) 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(3) 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.


EXPERIMENTAL PROCEDURES

Materials

[^3H]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(3) 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).


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(3) 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(3).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(3).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(3).A107 cells.

Pulse-Chase, Immunoprecipitation and HPLC Analysis

Confluent GH(3).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).

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 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(4) 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(3)CN; 4-6 min 5-16% CH(3)CN; 6-20 min 16-35% CH(3)CN; 20-34 min 35-45% CH(3)CN; 34-37 min 45-46% CH(3)CN; 37-39 min 46-80% CH(3)CN, all containing 0.1% trifluoroacetic acid. One-minute fractions were collected, and the radioactivity was determined by liquid scintillation counting.

For peptide sequencing, a 100-mm dish of confluent GH(3).C28.7 cells was labeled with 500 µCi/ml [S]Cys, or 1 mCi/ml [^3H]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.


RESULTS

Expression of the preproSRIF-I-proSRIF-II Chimera in GH(3)Cells

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 GH(3) cells, and approximately 60% of the mature hormone is sorted to the regulated secretory pathway in rat pituitary GH(3) 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(3) 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(3) 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.

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(3) 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(3).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(r) 24,000) was evident in GH(3).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(3).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.


Figure 2: The SRIF-I propeptide rescues proSRIF-II from intracellular degradation. GH(3).C28.7 cells (lanes1-10) and GH(3).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(3).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.


Figure 3: Processing and secretion of the chimera. A, HPLC resolution of SRIF-28 related polypeptides. GH(3).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 (bullet), 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.



To test this directly, cells were labeled with [^3H]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 [^3H]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.


Figure 4: Partial amino-terminal amino acid sequencing of the processed SRIF-28 polypeptide. Two separate dishes of GH(3).C28.7 cells were radiolabeled continuously with [^3H]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 ^3H- 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).



To determine the processing efficiency and secretion kinetics of the chimera, GH(3).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(3) 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.


Figure 5: Processing of the chimera requires an intracellular acidic pH. GH(3).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 (bullet); 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(3).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(3)C28.7 and GH(3)A.107 cells. In GH(3)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(3).A107 cells was similar to that shown in panelD. As observed previously (Danoff et al., 1991), immunoreactive SRIF-28 in GH(3)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.


Figure 6: Immunolocalization of the SRIF-chimera. GH(3).C28.7 and GH(3).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(r) 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(3).C28.7 cells, panelsA, C, and E; GH(3)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(3).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(3) cells (Elgort and Shields, 1994). Similarly, both the anglerfish SRIF-I and human SRIF propeptides were efficiently secreted from the GH(3)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(3).A107 cells, as expected proSRIF-II was also degraded (lanes11 and 12).


Figure 7: The SRIF-I propeptide functions only in cis. GH(3)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.




DISCUSSION

Previous work from our laboratory (Danoff et al., 1991) demonstrated that anglerfish proSRIF-II is degraded intracellularly in GH(3) 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).

It is unclear why the processing, sorting, and secretion of proSRIF-II is atypical; originally we had hypothesized that GH(3) 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. (^2)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.

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, alpha-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-beta1, 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(3) 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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health (NIH) Grant DK21860 and in part by grants from the Lucille Markey Charitable Trust and the Juvenile Diabetes Foundation (to D. S.) and a Clinical Investigator Award DK 18994 (to A. D.). Core support was provided by an NIH Cancer Center grant P30CA13330. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by NIH training grant CA09475.

To whom correspondence should be addressed. Tel.: 718-430-3306; Fax: 718-823-5877; shields{at}aecom.yu.edu.

^1
The abbreviations used are: TGF-beta1, transforming growth factor-beta1; TGN, trans Golgi network; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; SRIF, somatostatin.

^2
A. Danoff, unpublished results.


ACKNOWLEDGEMENTS

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.


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