Proprotein Processing within Secretory Dense Core Granules of Tetrahymena thermophila*

Niels R. Bradshaw, N. Doane Chilcoat, John W. Verbsky, and Aaron P. TurkewitzDagger

From the Department of Molecular Genetics and Cell Biology, the University of Chicago, Chicago, Illinois 60637

Received for publication, July 19, 2002, and in revised form, November 13, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the ciliate Tetrahymena thermophila, the polypeptides stored in secretory dense core granules (DCGs) are generated by proteolytic processing of precursors, and the mature products assemble as a crystal. Previous observations suggested that this maturation involves precise cleavage at distinct motifs by a small number of enzymes. To test these inferences, we analyzed the determinants for site-specific processing of pro-Grl1p (Granule lattice protein 1) by complete gene replacement with modified alleles. Contrary to the predictions of previous models, none of the component amino acids in a putative processing motif was necessary for targeted cleavage. Indeed, cleavage at a range of alternative positions near the native site was consistent with normal DCG assembly. Furthermore, substitution of other classes of processing site motifs did not perturb DCG structure or function. These results suggest that processing can be catalyzed by multiple proteases, for which substrate accessibility may be the prime determinant of site specificity. Consistent with this, inhibition of either subtilisin or cathepsin family proteases resulted in delayed processing of pro-Grl1p.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In some eukaryotic cells, a subset of proteins destined for exocytosis can segregate, condense, and then crystallize within vesicles called dense core granules (DCGs).1 Core assembly occurs during granule maturation, in which the best characterized feature is site-specific proteolytic processing of DCG cargo proteins (1). In pancreatic beta -cells, pro-insulin is cleaved by subtilisin-related enzymes called prohormone convertases (PCs), to generate mature active insulin (see Ref. 2 and reviewed in Ref. 3). This is also linked to structural transitions; whereas proinsulin will dimerize in the trans-Golgi network and immature granule compartments, only processed insulin forms the crystal core of mature DCGs (4, 5). The PCs recognize and cleave substrates following paired basic residues, which may be presented in the context of defined local structures (6-8). The enzyme activities are sensitive to both pH and calcium concentration, with the result that processing occurs in specific sub-compartments of the secretory pathway (9). PCs are not the only class of enzymes involved in proprotein processing in secretory granules. A variety of less well characterized enzymes appear to play roles in metazoans (10, 11). Similarly, recent results (12-14) from studies of protozoa indicate that both serine and cysteine proteases may be involved in regulated secretory vesicle maturation.

The ciliate Tetrahymena thermophila has DCGs, termed mucocysts, whose cargo includes a family of polypeptides derived proteolytically from Grl (Granule lattice protein) precursors (15-17). An orthologous set of granule cargo proteins in Paramecium tetraurelia are the trichocyst matrix proteins (tmps) (18-21). In both organisms, the granule cores consist of highly organized protein crystals that undergo spring-like expansion upon exocytosis. This mechanism propels the granule cargo into the environs of the cells. Each T. thermophila cell contains several thousand DCGs, virtually all of which are docked at the plasma membrane, so cargo release upon exocytic stimulation is massive and synchronous (22). Proprotein processing within immature DCGs in ciliates appears closely linked with organization and function of the granule cores. First, only processed proteins can be detected within the crystallizing cores (23, 24). Second, defects in proprotein processing and core assembly are strictly correlated in mutants (25-28). Third, mutants defective in core assembly are also defective in rapid extrusion of DCG cargo upon exocytosis (29). Finally, while the precise structural basis for the spring mechanism is unknown, a conformational transition in processed Grl1p is correlated with expansion but does not occur in the proprotein (17). These observations suggest that, as in neuroendocrine cells, proprotein processing is an essential regulatory mechanism for assembly of functional DCGs in ciliates. However, neither the processing enzymes themselves nor the processing determinants within the proproteins are known. Candidates for the latter have been deduced by comparing Grl and tmp sequences.

As a family the Grls/tmps share less than 20% amino acid identity, but regions of limited sequence similarity flank the known proteolytic processing sites and have therefore been tentatively identified as targets for processing proteases (17, 28). At least some of the Grls/tmps are cleaved twice to yield two mature peptides, and a single proprotein can contain a different motif at each of two processing sites. The simplest model is that, as for proinsulin, each of the motifs is recognized by a distinct endopeptidase whose activity is regulated by substrate availability and compartmental conditions. Alternatively, a single enzyme may recognize multiple motifs but with different affinities (30).

We have now characterized the activities responsible for Grl1p processing in vivo by generating Grl1p variants with substitutions at residues surrounding the known cleavage site. By using complete replacement of the wild type gene with these modified alleles, we could test the relevance of the putative recognition motifs for processing site specificity and granule assembly. To extend this analysis we asked whether the cleavage of different putative motifs was dependent on their position within the polypeptide. We demonstrate that cleavage does not depend on any residues in the putative processing motifs. The precise locus of cleavage can be subtly shifted in these variants, but this does not compromise the assembly or function of the resulting cores. These results, supported by protease inhibition experiments, strongly suggest that this system depends on multiple proteases that can collectively cleave at a wide variety of peptide bonds.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

All reagents were from Sigma unless otherwise indicated.

Cell Culture-- Cell cultures were gently swirled at 30 °C in SPP (2% proteose peptone, 0.2% yeast extract (both from Difco) with 0.009% ferric EDTA). T. thermophila strain CU428.1 (mpr1-1/mpr1-1 (mp-s, VII)) was provided by Peter Bruns (Cornell University, Ithaca, NY). The strain is wild type with respect to regulated exocytosis. Genetic nomenclature for T. thermophila can be found in Ref. 31.

Site-directed Mutagenesis-- In general, variants of Grl1p were generated by inverse PCR using as template pBluescript SK+ (Stratagene, La Jolla, CA) containing the KpnI-BglII fragment of the GRL1 genomic sequence. Expand High Fidelity Polymerase was used (Roche Molecular Biochemicals) followed by a 5-min polish with T4 DNA polymerase (New England Biolabs, Beverly, MA) to remove 3' As. Several variants (details on primers accessible at home.uchicago.edu/~nelselde) were generated using the Quick Change Mutagenesis kit (Stratagene). Clones were sequenced across the entire insert using M13.F and M13.R primers and the Big Dye sequencing kit (Amersham Biosciences). Inserts were then shuttled into a vector containing GRL1 cDNA sequence carrying a silent mutation (C249G,T250C,A252G) that introduces a PstI site and 1100 bases of 3'- and 500 bases of 5'-genomic flank. The NEO2 cassette, which confers resistance to paromomycin sulfate in T. thermophila, was inserted in the XhoI site 500 bases downstream. In cases where novel sequences were inserted, we chose the codons used most frequently in highly expressed T. thermophila genes (32). In the case of the exchange of the GRL1 sequence with those from GRL3, GRL5, and GRL7, we used the exact coding sequence from the respective genes.

Transformation and Gene Replacement-- Particle-mediated transformation of T. thermophila was as described (33). Cells were bombarded with 15 µg of DNA digested with BamHI and SacI and were recovered in SPP containing 100 µg/ml paromomycin sulfate. Cell lines were transferred every 2nd day to media of increasing drug concentration until the mutant construct had replaced all macronuclear copies of wild type Grl1p. Complete replacement was confirmed by Southern blotting of genomic DNA digested with PstI and HindIII, using a probe corresponding to the GRL1 gene.

Western Blotting-- SDS-PAGE (15% polyacrylamide) was performed according to Ref. 34. Antibody blotting on nitrocellulose membranes was according to Ref. 35, and antibodies were visualized using signal substrate (Pierce). The rabbit antiserum specific for Grl1p (called anti-p40) has been described (26). Isolation of DCG cargo was performed by dibucaine stimulation (see below) followed by differential centrifugation, as described in Ref. 36.

Exocytosis Assays-- Two different secretagogues induce massive regulated exocytosis in T. thermophila. Exposure to dibucaine triggers rapid secretion of granule contents, which can subsequently be isolated in a highly purified form (16). Exposure to Alcian blue similarly triggers global fusion of DCGs, but this dye also precipitates the DCG contents as they are being secreted, with the result that cells become instantaneously encapsulated (37). Such capsules are easily detected by light microscopy (×100-400). The first assay can therefore be used to assess the exocytic competence of a culture, whereas the second is useful for judging the competence of individual cells.

Cells were grown to stationary phase (~106/ml), washed once, and then starved overnight in DMC, a 1:10 dilution of Dryl's medium (1.7 mM sodium citrate, 1 mM NaH2PO4, 1 mM Na2HPO4, and 1.5 mM CaCl2) with additional 100 µM MgCl2 and 0.5 mM CaCl2 (38). Cells were stimulated by vigorous addition of Alcian blue 8GX (1%) to 0.025%. Following light agitation for 30 s, the cells were diluted with at least 10 volumes of 0.25% proteose peptone, 0.5 mM CaCl2. Cells were pelleted by centrifugation for 45 s in a clinical centrifuge and fixed with formalin, and the fraction of encapsulated cells was determined.

N-terminal Sequencing-- Cells grown to stationary phase in 2% proteose peptone, 0.2% yeast extract, 0.003% iron EDTA were stimulated to secrete with 2.5 mM dibucaine, and the secreted material was purified. In the final step, the secreted protein was centrifuged at 17,000 × g for 20 min to yield a flocculent pellet whose volume was estimated. beta -Mercaptoethanol was added to 10%, and the sample was heated at 55 °C for 30 min. Under these conditions, virtually all mature Grl1p remains soluble, whereas most other polypeptides form aggregates.2 Samples were centrifuged at 17,000 × g for 10 min, and the supernatants were subjected to SDS-PAGE (15% polyacrylamide, 0.09% bisacrylamide) and transferred to polyvinylidene difluoride (Osmonics, Westboro, MA). The strong band(s) corresponding to mature Grl1p were excised and subjected to 5 cycles of N-terminal sequencing by automated Edman degradation (Carol Beech, Macromolecular Structure Analysis Facility, University of Kentucky).

Biosynthetic Labeling-- Cells grown to stationary phase were pelleted and starved for 2 h in DMC. They were stimulated with Alcian blue, as above, and then resuspended for 1 h in DMC. 2 × 106 cells/ml were labeled for 15 min with 0.1 mCi/ml [3H]lysine (PerkinElmer Life Sciences) in DMC. Cells were then separated from the labeling medium by pelleting through a pad of 5% Ficoll in DMC with 2 mg/ml lysine and resuspended in DMC with 2 mg/ml lysine at 106 cells/ml. At each time point, 106 cells were withdrawn and lysed by addition of 1/4th volume of 5× NDET (NDET is 1% Nonidet P-40, 0.4% sodium deoxycholate, 66 mM EDTA, 10 mM Tris-HCl, pH 7.0) with protease inhibitors (0.5 µg/ml leupeptin, 12.5 µg/ml antipain, 10 µg/ml E-64, 10 µg/ml chymostatin). Samples were incubated for 1 h at 4 °C, after which the insoluble fraction was pelleted at 17,000 × g for 10 min. Supernatants were pre-cleared with Sepharose CL4B and then incubated with protein A-conjugated Sepharose that had been preincubated with anti-p40 antibody. For experiments done in the presence of protease inhibitors, cells were treated as above with the following variations. Following stimulation cells were allowed to settle to remove the small fraction of cells trapped in capsules. Cells were incubated for 1 h following stimulation in DMC supplemented with protease inhibitors as follows: 20 µM subtilisin inhibitor III (Calbiochem), 10 µM cathepsin inhibitor III(Calbiochem), 50 µM benzyloxycarbonyl-Phe-Ala-CH2F (gift of Jim Miller, University of Chicago), and all subsequent steps prior to detergent lysis were done in the presence of inhibitors.

Microscopy-- For electron microscopy, cells were fixed in 1.5% glutaraldehyde, 1% osmium tetroxide, as described (26). Indirect immunofluorescence images of regranulating cells were obtained by stimulating 50 ml of a 2-h starved culture with Alcian blue, as described above. Cells were pelleted and resuspended in 0.25% proteose peptone, 0.5 mM CaCl2. The cells were allowed to settle for ~10 min until a clear separation was visible between the settled blue-stained secreted material and free swimming cells that concentrated at the meniscus. These were collected and diluted to 20 ml with DMC. At each time point, 4 ml were pelleted and fixed in ice-cold 50% EtOH, 0.2% Triton X-100 for 10 min, pelleted, and resuspended in 0.5% bovine serum albumin in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl. DCGs were detected using monoclonal antibody 4D11 (39), a gift of Marlo Nelsen and Joe Frankel, University of Iowa.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

From earlier studies, pro-Grl1p is known to be cleaved following Lys188, and the surrounding region can be aligned with sequences flanking cleavage sites in two other Grl proproteins (17, 29). First, each cleavage site lies downstream of a small zone of basic residues (schematic in Table II). Short positively charged regions upstream of cleavage sites are conspicuous in proteins that are otherwise highly acidic. Close upstream of each cleavage site lies a cluster of hydrophobic residues at moderately conserved positions (Table I). The P'3 position (the residue 3 positions downstream of the cleaved peptide bond) is generally occupied by a hydrophobic residue, and there is always at least one hydrophobic residue in the first three positions following the cleavage site. Most significantly, the P1 position (that immediately preceding the cleaved peptide bond) is always occupied by a basic residue. Some or all of these shared features are potentially important for cleavage site specificity and are therefore indirect determinants of lattice assembly during granule biosynthesis. Alternatively, the role played by these features may be secondary to specificity based on accessibility in an otherwise tightly folded polypeptide. Only in the first instance would modifying these putative motifs be likely to interfere with pro-Grl1p processing and DCG formation.

                              
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Table I
Conserved motifs in proGrlps and tmps
The N-terminal residue of the mature processed peptides, as determined by Edman degradation, are shown in boldface in A-C. Amino acids that are relatively conserved are boxed. A, alignment of sequences in which cleavage occurs following a single basic residue. B, alignment of sequences at which cleavage occurs following a glycine residue. C, alignment of sequences in which cleavage occurs following a single asparagine residue.

To test the physiological importance of these features, we replaced all expressed (macronuclear) copies of GRL1 with variants altered in sequence at or around the cleavage site. In general, these variants of Grl1p, shown in Table II, fall into four classes as follows: 1) deletion of large regions around the cleavage site; 2) replacement of Lys188; 3) alteration of the hydrophobic residues flanking the cleavage site; and 4) up- or downstream translocation of residues immediately flanking the cleavage site. The products of such gene replacements are cells in which the variant genes are expressed at the endogenous locus, with the bacterial gene (NEO2) that confers neomycin resistance inserted in the downstream flank. The insertion of NEO2 allowed us to drive complete gene replacement (40). To confirm that the insertion of the heterologous gene did not itself interfere with Grl1p processing or granule assembly, we first replaced the wild type gene with an identical copy flanked by NEO2. The resulting drug-resistant transformants were indistinguishable from wild type in all DCG-related phenotypes. Complete replacement by the novel alleles could be distinguished by virtue of an engineered silent mutation that introduced a novel restriction site, as described under "Experimental Procedures."

                              
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Table II
GRL1 mutant alleles
Modified sequences are aligned vertically with respect to the wild type sequence shown at the bottom. The arrow indicates the position of the peptide bond between Lys188 and Glu189 that is cleaved in the wild type. Sequenced N termini of the mutant proteins are shown in boldface. In cases where alternative cleavages generated two mature polypeptides, the sequence determined from the smaller product is underlined. N termini that were inferred based on gel mobility shifts are indicated in italics. The single exception is construct 10 (Delta Lys188,Glu189), for which we concluded that the cleavages occur somewhere within the 4 downstream residues following Glu187, based on the mobility of the products. Names of constructs are based on the single letter code for amino acids.

Remarkably, each of the Grl1p variants in the four classes was processed to yield a product whose mobility approximated that of the wild type mature peptide (Fig. 1). The samples in Fig. 1 represent protein released within seconds following stimulation of cell cultures with the secretagogue dibucaine, indicating that both wild type and variant proteins were stored in functional DCGs. Under these conditions, protein release from one mutant with a large upstream deletion, Delta 161-187, was markedly less than in wild type (not shown). The processing also appeared to be roughly as precise as that of the wild type protein, since the products appeared as a single band or a closely spaced doublet. However, the gel mobilities also indicated that alternative cleavage sites were being used in some variants. To understand how these were specified and thereby gain insight into site selection in the wild type protein, we analyzed each of these four classes in detail. An underlying question was, why are residues surrounding the cleavage site conserved if their substitution does not affect pro-Grl1p processing?


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Fig. 1.   Processed Grl1p from wild type (wt) and mutant strains. Purified DCG cargo was resolved by SDS-PAGE and transferred to nitrocellulose. The processed proteins were visualized using the anti-Grl1p antiserum. The numbers above each lane correspond to the particular mutant strain designated in Table II. 1, Delta 161-187; 2, Delta 189-200; 3, K188R; 4, K188A; 5, K188N; 6, K188P; 7, K188D; 8, K188W; 9, K188E; 10, Delta Lys188, Glu189; 11, Y190A,V191A; 12, Y190D,V191E; 13, L185A,L186A; 14, Delta Phe158, Leu159, Glu160; 15, -4:Glu187, Lys188, Glu189; 16, +6:Glu187, Lys188, Glu189; 17, +6:Leu185, Leu186, Glu187, Lys188, Glu189. In all cases the processed products are of similar mobility. The position of a molecular weight standard (ovalbumin, 43 kDa) is shown by a bar at the right of the 1st panel. B, heat extracts of DCG cargo were resolved by SDS-PAGE and stained with Coomassie Blue. Processed Grl1p from several mutant lines are revealed to migrate as closely spaced doublets. The resolution of doublets in B but not A is due to differences in gel dimensions and running conditions.

The Role of the P1 Residue in Site Selection-- Precise target recognition may be determined in large part by the P1 residue, as a result of the physical restrictions of the substrate-binding pocket. The potential importance of Lys188 as a determinant was also reinforced by preliminary analysis of one of the variants. N-terminal sequencing of proteins isolated from stimulated cell supernatants allowed precise determination of the cleavage sites in the altered proteins. In Delta 161-187 the 27 amino acids upstream of Lys188 were deleted, which includes the stretch of basic amino acids (Table II, line 1). Nonetheless the N-terminal residue of the mature polypeptide remained identical to that in the wild type, Glu189. This implied that processing takes place following Lys188, the identical residue targeted in the wild type protein. This result suggested that target site selection is not designated in any straightforward manner by the amino acid sequence upstream of the P1 residue and, in particular, that the basic region is not an essential determinant.

We therefore focused on the potential role of the single basic residue at the P1 position, by replacing Lys188 with a variety of other amino acids (Table II, lines 3-9). The conservative substitution of Lys188 with Arg had no effect, but replacement with either negatively charged or uncharged residues led to small shifts in the processing site. Substitutions by Ala (K188A) or Pro (K188P) shifted the processing site one residue downstream, to Glu189. Substitution with either Asp (K188D), Glu (K188E), or Trp (K188W) led to cleavage following Glu187. A basic residue in the wild type protein may therefore be part of the preferred substrate for the processing protease, but the locus of cleavage, as defined within the range of a single residue, must be determined by other features. A striking outcome of these results was the implication that processing, which takes place after a basic residue in the wild type protein, can also occur following negatively charged residues. In addition, a hydrophobic residue at the P'1 position is acceptable.

One possibility was that charge density rather than the particular charges was important for site determination, since the wild type sequence is Glu187-Lys188-Glu189 and the variants above maintain at least two charged residues. Accordingly, we deleted both Lys188 and Glu189 (Delta Lys188, Glu+) (Table II, line 10). Processing was also seen in this variant, producing a doublet of products whose mobility indicated that the cleavages had almost certainly occurred within the first three or four residues, none of which is charged. While the precise N termini were not determined for these products, results below directly demonstrate that a variety of downstream residues can be accommodated and confirm the premise that no flanking charged residues are required.

Replacement of Adjacent Hydrophobic Residues Leads to a Change in Site Selection-- Since the residues at, or immediately adjoining, the cleavage site are apparently not essential for specificity, we focused on potential structural cues. In particular, we asked whether flanking residues somewhat more distal to the cleavage site were involved in creating a 2° element, such as a flexible exposed loop, which could be targeted by a protease. As noted above, the cleavage site is flanked by nearby hydrophobic residues that could anchor and thereby define a short loop. We therefore replaced these residues both up- and downstream of Lys188.

We substituted the downstream residues Tyr190 and Val191 with acidic residues to generate Y190D,V191E (Table II, line 12). This resulted in a minor change in processing specificity. We observed two products, which were generated by alternative cleavage following Glu187 and Lys188. To be certain that this change did not result from the presence of charged residues as opposed to the absence of hydrophobic residues, we replaced Tyr191, Val192 with alanines (Y191A,V192A) (Table II, line 11). Similar cleavage products were obtained. These results suggest that if local 2° structure orients cleavage specificity, it does not depend on these downstream residues. However, the presence of two alternatively processed products hinted that these residues might stabilize a local structure.

The upstream hydrophobic residues Leu185 and Leu186 were similarly replaced with Ala (L185A,L186A) (Table II, line 13). In this variant, processing occurred following Ala184. Cleavage after an uncharged amino acid confirms that the range of acceptable P1 residues is quite broad. That the cleavage site was shifted four positions upstream suggests a role for these upstream hydrophobic residues in site selection. This is also consistent with the fact that cleavage after Lys188 was maintained in Delta 161-187, as described above. In this protein, Lys188 follows immediately after Phe158-Leu159-Gln160. As a result, hydrophobic residues occupy the P3 and P4 positions. This is equivalent to the wild type but unlike the L185A,L186A construct. In summary, these results indicated that most of the conserved features are not critical for processing at or near the wild type site. The largest deviation seen was a shift 4 residues upstream from the wild type site, resulting from substitutions at Leu185 and Leu186.

In light of the finding that local modifications had little or no effect on site specificity, we considered the possibility that the signal for processing is distal in the primary sequence. Such a determinant could still lie nearby in the folded polypeptide. A signal of this type might be detected as an additional feature shared among the Grls/tmps. The most obvious candidate was a 3-residue motif, FLQ, that is found immediately upstream of the basic regions discussed above and that is the most highly conserved sequence element in these proteins (17). We therefore expressed Delta Phe158, Leu159, Glu160, in which these three residues were deleted (Table II, line 14). However, cells expressing this variant were indistinguishable from wild type with regard to DCG functions, and Grl1p processing occurred after Lys188 as in wild type.

The Processing Site Can Be Shifted by Translocation of Lys188 and Flanking Residues-- An alternative strategy for asking whether the residues flanking Lys188 specify the cleavage site, or whether the site is determined largely by the position within the polypeptide, was to determine whether the site could be translocated to another position within the 1° sequence (Table II, lines 15-17). For this we deleted Glu187-Lys188-Glu189 and reinserted the tripeptide both upstream and downstream. Guided by the hint that the downstream hydrophobic residue might help to stabilize a cleavage site structure, we translocated Glu187-Lys188-Glu189, either four positions upstream (-4:Glu187, Lys188, Lys189) or six positions downstream (+6:Glu187, Lys188, Glu189), so that a hydrophobic amino acid was present as the next residue. In these variants the sequence of amino acids remaining at the original cleavage zone is Leu-Ala-Leu-Leu-Tyr-Val, very dissimilar to the wild type.

In cells expressing either -4:Glu187, Lys188, Glu189 or +6:Glu187, Lys188, Glu189, the mature polypeptide appeared as a doublet (Fig. 1B). In the former, N-terminal sequencing revealed that the predominant product resulted from cleavage following new amino acid 189. The absolute position of the cleaved bond within the polypeptide was therefore reasonably well conserved with respect to wild type. This conservation was striking in light of the fact that residue 189 in the variant is Leu, indicating that processing can also occur following a large hydrophobic residue. We conclude that the processing of Glu-Lys-Glu, with a downstream hydrophobic residue, is sensitive to the position of this peptide within the 1° sequence. It is clear that Glu-Lys-Glu is not necessary to specify the site of processing.

Consistent with this conclusion, downstream translocation of the same motif (+6:Glu187, Lys188, Glu189) also generated processed products whose mobility was very similar to the wild type. Although we did not determine the precise N termini of the bands in this closely spaced doublet, it is clear that cleavage did not occur within the EKE motif. These results led us to conclude that Glu-Lys-Glu is also not sufficient to specify the cleavage site. We therefore considered whether Leu185 and Leu186 might be part of a minimal site-determining motif, since their replacement with Ala induced a large shift in the size of the processed product. To examine this, we included these two residues in the sequence that was translocated. The sequence Leu185-Leu186-Glu187-Lys188-Glu189 was deleted from its native locus and re-inserted at position +6 following Asp195. Processing of this protein generated a doublet of products that corresponded to cleavage following Tyr196 and Val198. In this case, therefore, translocation of the putative target sequence led to a downstream shift in the cleavage site by 8-9 residues. However, cleavage did not occur at the peptide bond between Lys and Glu that is targeted in the wild type, even though this was close to the peptide bond that was hydrolyzed in the variant. This is consistent with the evidence that Glu-Lys-Glu is not sufficient to determine the cleavage site.

Because cleavage can occur for all the variants discussed above, we conclude that the precise cleavage of the wild type protein following Lys188 is not accounted for either by its absolute position within the polypeptide backbone nor by a unique local 2° structure. Moreover, cleavage within the variants can occur with a wide range of residues at the P1 position. A single or small number of unusually promiscuous proteases or a larger family of more conventional enzymes could account for this last feature. Accessibility itself could be the major determinant of the zone of processing, if not of the cleavage site itself.

Cleavage in Heterologous Sequences: Dissimilar Processing Sites Show Surprising Interchangeability-- Identification of three classes of putative processing motifs suggested previously that the differential cleavage at these sites was a regulatory mechanism for core maturation. Because the results reported above, following extensive modifications at the Grl1p cleavage site, were not easily reconciled with this hypothesis, we tested the model directly by substituting the Lys188 motif in Grl1p with the other classes of motifs that are present within the Grl and tmp protein family. We replaced the region around the known cleavage site in Grl1p with the putative cleavage motifs from Grl7p(Ala46 to Val61), Grl3p(Val217 to Ser231), and Grl5p(Leu46 to Tyr60) (Table II, lines 18-20). In each case, the polypeptide transplanted from the donor corresponded to the sequence previously identified as conserved between at least two family members. The GRL1 gene was completely replaced by these constructs, and the chimeric proteins were stably expressed as determined by Western blotting. In all cases processing took place, yielding a single product that was packaged into DCGs (Fig. 2A). We conclude that such motifs were accessible to proteases when transplanted into a heterologous Grl1p context.


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Fig. 2.   Processing of Grl1p-Grl3p, Grl1p-Grl5p, and Grl1p-Grl7p chimeras. A, whole cell lysates were resolved by SDS-PAGE, and the mature polypeptides were detected by Western blotting with an antibody against Grl1p. The numbers above each lane correspond to a mutant strain as designated in Table II. In all cases, the chimeras were processed to products similar to the wild type (wt). B, newly synthesized proteins in wild type and mutant cells were pulse-labeled with [3H]lysine for 15 min, and Grl1p was immunoprecipitated from detergent cell extracts, resolved by SDS-PAGE, and visualized by autoradiography. Under these conditions, it is important to note that only the pre-processed form is soluble and therefore detectable. Time 0 corresponds to the beginning of the pulse. C, quantitative representation of the data shown in B. Pro-Grl1p disappears from mutant and wild type cells with indistinguishable kinetics.

The processed products of these chimeras were functionally indistinguishable from the wild type, in that neither DCG morphology (Fig. 4) nor exocytosis competence was affected. This strongly suggests that if the distinct sites present within the proproteins are indeed differentially cleaved in a manner that is important for DCG biosynthesis, the regulation of processing is unlikely to be based on the class of sequence at each site or on the specific proteases that are involved. The latter conclusion assumes that a processing motif, even in a novel context, is still targeted by the same enzyme. Although this was impossible for us to test directly, we could ask whether cleavage occurred at the same peptide bond. The motif identified in Grl4p and Grl5p, in which processing occurs between Gly and Thr, is the most clearly dissimilar from that in Grl1p. We found that processing occurred at the identical peptide bond when the 11-residue peptide from pro-Grl5p was translocated to replace the region surrounding Lys188 in pro-Grl1p (Table II, line 19). In this case, the non-native cleavage motif appears sufficient to direct processing site selection. On the basis of these data, it is unlikely that processing by specific enzymes at functionally distinct sites is essential for DCG biogenesis.


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Fig. 3.   Processing of pro-Grl1p in the presence of protease inhibitors. A, newly synthesized proteins were pulse-labeled with [3H]lysine for 15 min in the presence or absence of 20 µM subtilisin (Subt.) inhibitor III, 10 µM cathepsin (Cath.) inhibitor III, 50 µM benzyloxycarbonyl-Phe-Ala-CH2F. Grl1p was immunoprecipitated from detergent cell extracts, resolved by SDS-PAGE, and visualized by autoradiography. Under these conditions only the pre-processed form is soluble and therefore detectable. Time 0 corresponds to the beginning of the pulse. B, cells were treated as in A, except combinations of protease inhibitors were used. Band intensities are shown below B. These are displayed relative to the earliest "no inhibitor" sample, arbitrarily set at 100. The units are arbitrary.

In P. tetraurelia, it has been suggested that different classes of processing sites might be cleaved with different kinetics (30). To examine this directly in our system, we used biosynthetic pulse labeling and immunoprecipitation to compare the rate of processing of pro-Grl1p with that of the Grl1p-Grl5p chimera. The rates of processing were indistinguishable (Fig. 2, B and C), as judged by the disappearance of the proprotein. We cannot detect the processed products by immunoprecipitation due to their insolubility, making it impossible for us to quantify the processing efficiency. Nonetheless, the similarity in the steady-state accumulation of the mature products in wild type and variants suggests that processing, rather than degradation, is responsible for proprotein disappearance. These results suggested that the differences in primary sequence surrounding the cleavage sites do not play a major regulatory role in kinetics of T. thermophila DCG cargo processing. It should be noted that the core lattice structure in T. thermophila is simpler than that in P. tetraurelia, and it is possible that an additional level of regulation exists for processing of the P. tetraurelia orthologs. In addition, the time resolution of the assay is limited by the 5-min sampling time intervals and the 15-min labeling period.

These results demonstrated that an enzyme or enzymes were capable of processing a very wide range of substrates but did not rule out the possibility that some targets were relatively disfavored. Such differences could underlie the precise cleavage seen in wild type cells. Specifically, the kinetics of processing might be altered if a processing enzyme were being forced to cleave at a site less favorable for binding or cleavage relative to the wild type. We therefore estimated the rate of processing of a set of the pro-Grl1p variants, as above. By this measure, processing proceeded at a rate indistinguishable from the wild type in all cases. The extent of processing also appeared to be normal, since there was no substantial accumulation of precursor in any of the mutant strains (not shown).

Inhibitors Specific to Both Subtilisin and Cathepsin Proteases Delay Grl1p Processing-- Shaw et al. (13) recently reported that regulated secretory vesicle (rhoptry) formation in Toxoplasma gondii, an organism that like Ciliates belongs to the order Alveolata, was disrupted in the presence of inhibitors targeting subtilisin or cathepsin family proteases. To ask if related proteases might be involved in processing of pro-Grl1p during the formation of DCGs in T. thermophila, we biosynthetically pulse-labeled cells undergoing regranulation in the absence or presence of such inhibitors. As shown in Fig. 3A, inhibitors against both subtilisin and cathepsin family proteases caused a delay in the processing of pro-Grl1p. Combination of the inhibitors produced additive effects (Fig. 3B) demonstrating that it is likely that both subtilisin and cathepsin family proteases are involved in DCG maturation.

Sequences Surrounding Processing Sites Are Important for Lattice Assembly-- As discussed earlier, proprotein processing and core assembly are tightly linked in T. thermophila DCGs, and correct assembly is essential to rapid extrusion of the granule cargo upon exocytosis. The similarity in the extent and apparent kinetics of processing between the variant and wild type Grl1 proproteins suggested that core assembly and extrusion in the variants should be identical to wild type. However, it was also possible that core assembly depended on features of the processing site other than its proteolytic cleavage. To address the issue of assembly, we examined core structures by electron microscopy. Function in bulk cultures was implicitly addressed via preparation of DCG cargo protein following dibucaine stimulation, as described above. To extend this to the level of individual cells, we evaluated cargo extrusion based on a visual assay. When wild type cells are exposed to the secretatogue Alcian blue, each cell rapidly discharges the contents of its several thousand docked DCGs (37). As a result, each cell becomes trapped in a robust proteinaceous capsule formed of those contents, easily seen by light microscopy (Fig. 5B). Capsule formation indicates extensive synchronous extrusion of DCG cargo but does not demonstrate that 100% of the DCGs have undergone exocytosis.

All but two of the mutants accumulated DCGs that were indistinguishable from the wild type in both shape and organization of contents (Fig. 4). The exceptions were Delta 161-187 and +6:Leu185, Leu186, Glu187, Lys188, Glu189. The former contained cores with a variety of shapes and electron densities, in which no organized protein lattices were detectable. The defect in cells expressing +6:Leu185, Leu186, Glu187, Lys188, Glu189 was more subtle. The mutants contained DCGs with an elliptical or egg shape rather than the extended wild type rods. Nonetheless, the contents were organized in protein crystals as in the wild type.


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Fig. 4.   Morphology of DCGs in wild type (wt) and mutant cells. Thin sections were imaged by transmission electron microscopy. With the exception of strains expressing +6:Leu185, Leu186, Glu187, Lys188, Glu189, and Delta 161-187, mutant strains make DCGs that are indistinguishable from the wild type. Bar, 200 nm.

In strong confirmation of predictions based on previous correlative studies, we found that defects in DCG morphology were associated with functional abnormalities. As judged by the Alcian blue assay, all variants with wild type DCGs were reproducibly equivalent to wild type in capsule formation (wild type = 91%; mutants = 88-98%, not shown). The two mutants with aberrant DCG morphologies had exocytic defects. Cells expressing Delta 161-187 showed no encapsulation following stimulation with Alcian blue. In contrast, cells expressing +6:Leu185, Leu186, Glu187, Lys188, Glu189 were initially encapsulated to the same level as wild type. However, within minutes ~75% of the mutant cells had escaped from their capsules, which appeared as empty shells, while virtually all wild type cells remained imprisoned. We hypothesize that rapid escape from capsules is due to a structural weakness in the capsules themselves and is related to the aberrant core structure in these mutants.

However, the quick-escape phenomenon might also be expected if the mutants contained fewer secretory granules or if exocytic release following stimulation was incomplete or delayed. To rule out these possibilities, we visualized DCGs in cells expressing +6:Leu185, Leu186, Glu187, Lys188, Glu189 by indirect immunofluorescence, both before and after stimulation, using a monoclonal antibody directed against a granule cargo protein unrelated to Grl1p. The cells showed an extensive array of docked DCGs at the cell surface (Fig. 5A), comparable to wild type. Furthermore, after stimulation the cells showed virtually complete degranulation, indicating that the extent of exocytosis was not decreased. We note that a subtle delay in exocytic release would not be detected by this assay, since the wild type response may occur in milliseconds while the assay measures exocytosis over a period of ~30 s.


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Fig. 5.   Degranulation and regranulation of wild type and mutant cells. A and C, DCGs were visualized by indirect immunofluorescence using a monoclonal antibody directed against a DCG cargo protein p80. Cells were visualized before stimulation (t = 0 minutes) and at various intervals following the triggering of massive exocytosis by Alcian blue. A, wild type (wt) and 17 (17) (+6:Leu185, Leu186, Glu187, Lys188, Glu189). The extent of degranulation of cells expressing the mutant protein is similar to the wild type, but the cells show a reproducible delay in the rate of regranulation. B, capsule formation resulting from Alcian blue stimulation, visualized by phase contrast microscopy. The upper image shows a wild type cell entrapped in a capsule formed from extruded DCG cargo. More than 90% of cells remain trapped at 5 min post-stimulation. After the same interval, the large majority of mutants expressing 17(+6:Leu185, Leu186, Glu187, Lys188, Glu189) have escaped from their now-empty capsules, an example of which is shown in the lower image. C, wild type and 1(Delta 161-187). Upper panel, wild type cells, as in A, show extensive degranulation followed by DCG replacement. Lower panel, in the mutant expressing Delta 161-187, DCG cargo localizes chiefly to cytoplasmic puncta that are not docked at the plasma membrane and that do not respond to an exocytic stimulus. Bar, 10 µm.

Finally, we asked whether the assembly of abnormal lattices in +6:Leu185, Leu186, Glu187, Lys188, Glu189 proceeded at a reduced rate. Wild type cells, following degranulation, can synthesize a replacement set of DCGs within several hours (Fig. 5) (37). We made side-by-side comparisons of wild type and a variety of mutant strains by fixing cells at intervals following degranulation and detecting new DCGs by indirect immunofluorescence. Among the mutants, only the strain expressing +6:Leu185, Leu186, Glu187, Lys188, Glu189 was distinct from wild type, demonstrating a reproducible delay in regranulation (Fig. 5A). Since we detected no delay in proprotein processing in this mutant, the result suggests that assembly itself is delayed in cells expressing a Grl1p variant with a subtle shift in the cleavage site.

The rate of de novo DCG biosynthesis could not be determined for cells expressing Delta 161-187. These cells demonstrated a pattern of immunofluorescence strikingly different from all other strains. The cell cytoplasm showed abundant punctate labeling unlike the wild type pattern (Fig. 5B). These puncta appear to be vesicles distinct from DCGs, since there was no hint of non-docked dense core bodies when cell sections were analyzed by electron microscopy (not shown). These cells therefore appear to accumulate DCG protein in at least two compartments, since they also contain the docked dense-core structures shown in Fig. 4. The pattern of cytoplasmic puncta was not altered when cells were stimulated by the secretagogue (Fig. 5). The high level of background due to these vesicles made it impossible to visualize replacement of docked DCGs in these cells.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There are two possible interpretations of the results presented here. The first is that a single enzyme is responsible for processing both wild type and variant Grl1 proteins. This would imply that the enzyme is highly promiscuous and also that the mechanism of site selection is relatively independent of the local sequence. Although protease inhibition suggests that more than one family of processing enzymes may be active in pro-Grl1p cleavage, one of these inhibitors could be acting on a regulatory protease involved in zymogen activation and therefore indirectly on processing. The second explanation is that the number of processing enzymes in this system is greater than previously suspected. While each protease may have a limited range of substrates, the collection is capable of cleaving a wide range of exposed peptide bonds. Cleavage following Lys188 might be catalyzed by a single enzyme, but other enzymes can act at nearby accessible residues. In this scenario, accessibility could sharply delimit the stretches of amino acids that are targets for a variety of enzymes. This is consistent with previous results from in vitro digestion of pro-Grl1p with chymotrypsin or elastase, each of which generated a species very similar in size to the in vivo product (17).3 This scenario is also consistent with a proposed model of the proprotein 3° structure based on sequence comparisons and 2° structure predictions (41), in which at least one of the processing sites lies within a linker between two tightly folded domains. The relative precision of processing site selection within a limited accessible zone, both in wild type and variants, may arise from competition between DCG endoproteases, each of which recognizes a narrow range of substrates. A simple hypothesis to explain these findings is that paralogous proteases, similar in their activity and regulation, have diverged at the active site pocket to collectively accommodate many substrates. Alternatively, more than one protease family may be directly involved.

In either case, the presence of multiple processing enzymes in T. thermophila DCGs could serve to generate small peptides that would not have been identified by the approaches used to date. Similarly, new DCG peptides continue to be detected even in well studied mammalian neuroendocrine cells (39). There is indirect evidence in ciliates that small peptide products may be generated, since we have never detected all the products of the GRL genes that are predicted based on known processing sites. Of the nine predicted products of the known GRL genes, we have detected only six (15). This is consistent with a significant fraction of pro-Grl proteins, including residues 19-188 of pro-Grl1p (1-18 composes the signal sequence; (29)), being degraded to small polypeptides. Such degradation may occur in part via amino- and carboxypeptidases, and indeed we cannot eliminate the possibility that the N termini of mature wild type Grl1p and/or variants thereof are generated by endoproteolysis and subsequent N-terminal trimming. This uncertainty does not compromise our conclusions that processing does not depend on a conserved motif and can occur following a wide range of residues.

We previously identified a gene encoding a cysteine protease, targeted to the secretory pathway, whose transcript abundance increases dramatically during massive regranulation.4 This was a promising candidate for a DCG-processing enzyme, but disruption of the gene had no obvious consequences for lattice assembly.5 However, the results reported here hint that the absence of any single processing enzyme would be unlikely to show a defect in DCG assembly, although it might lead to the generation of alternate polypeptides during granule maturation.

Our results demonstrate that cleavage at precise sites is not required for DCG biogenesis. We are left with the question of why the motifs at the cleavage sites in Grls and tmps have apparently been conserved. We found that gross changes in processing site selection can impair granule lattice formation and exocytic capacity, but cannot rule out the possibility that less dramatic alteration produces defects not detected by our assays. Alternatively, conserved sequences may reflect biological activities following exocytosis, rather than requirements during assembly or expansion. It is tempting to speculate that a collection of small peptides may play important roles in this regard. Detection and analysis of peptide contents should allow us to clarify this issue, en route to characterizing the processing enzymes in this organism.

    ACKNOWLEDGEMENTS

We thank the members of this laboratory including G. Bowman, M. Cacciaoca, N. Elde, A. Haddad, and particularly A. Cowan for help and insights. We also thank T. Steck for careful reading of this manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM-50946.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to the memory of Andre Adoutte, a pioneer in the appreciation of ciliate biology whose work was a direct inspiration for our fascination with DCG biogenesis.

Dagger To whom correspondence should be addressed: Dept. of Molecular Genetics and Cell Biology, 920 E. 58th St., the University of Chicago, Chicago, IL 60637. Tel.: 773-702-4374; Fax: 773-702-3172; E-mail: apturkew@midway.uchicago.edu.

Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M207236200

2 A. Turkewitz, unpublished data.

3 N. Bradshaw, unpublished data.

4 A. Haddad and A. Turkewitz, unpublished data.

5 A. Haddad, unpublished data.

    ABBREVIATIONS

The abbreviations used are: DCGs, dense core granules; PCs, prohormone convertases; tmps, trichocyst matrix proteins.

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