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
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ABSTRACT |
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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.
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 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.
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. 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.
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Conserved motifs in proGrlps and tmps
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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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, 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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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 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
(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 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 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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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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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 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.
|
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 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.
|
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 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.
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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The abbreviations used are: DCGs, dense core granules; PCs, prohormone convertases; tmps, trichocyst matrix proteins.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Urbé, S., Tooze, S. A., and Barr, F. A. (1997) Biochim. Biophys. Acta 1358, 6-22[Medline] [Order article via Infotrieve] |
2. | Steiner, D. F. (1973) Nature 243, 528-530[Medline] [Order article via Infotrieve] |
3. | Rouille, Y., Duguay, G. J., Lund, K., Furuta, M., Gong, Q., Lipkind, G., Oliva, A. A., Jr., Chan, G. J., and Steiner, D. F. (1995) Front. Neuroendocrinol. 16, 322-361[CrossRef][Medline] [Order article via Infotrieve] |
4. | Brange, J., Ribel, U., Hansen, J. F., Dodson, G., Hansen, M. T., Havelund, S., Melberg, S. G., Norris, F., Norris, K., Snel, L., Sørensen, A. R., and Voigt, H. O. (1988) Nature 333, 679-682[CrossRef][Medline] [Order article via Infotrieve] |
5. | Orci, L., Ravazzola, M., Storch, M.-J., Anderson, R. G. W., Vassalli, J.-D., and Perrelet, A. (1987) Cell 49, 865-868[Medline] [Order article via Infotrieve] |
6. | Bailyes, E. M., Shennan, K. I., Seal, A. J., Smeekens, S. P., Steiner, D. F., Hutton, J. C., and Docherty, K. (1992) Biochem. J. 285, 391-394[Medline] [Order article via Infotrieve] |
7. | Brakch, N., Rholam, M., Boussetta, H., and Cohen, P. (1993) Biochemistry 32, 4925-4930[Medline] [Order article via Infotrieve] |
8. | Steiner, D. F. (1998) Curr. Opin. Chem. Biol. 2, 31-39[CrossRef][Medline] [Order article via Infotrieve] |
9. | Davidson, H. W., Rhodes, C. J., and Hutton, J. C. (1988) Nature 333, 93-96[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Che, F. Y.,
Yan, L., Li, H.,
Mzhavia, N.,
Devi, L. A.,
and Fricker, L. D.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
9971-9976 |
11. | Hook, V. Y., Toneff, T., Aaron, W., Yasothornsrikul, S., Bundey, R., and Reisine, T. (2002) J. Neurochem. 81, 237-256[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Miller, S. A.,
Binder, E. M.,
Blackman, M. J.,
Carruthers, V. B.,
and Kim, K.
(2001)
J. Biol. Chem.
276,
45341-45348 |
13. | Shaw, M. K., Roos, D. S., and Tilney, L. G. (2002) Microbes Infect. 4, 119-132[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Blackman, M. J.,
Fujioka, H.,
Stafford, W. H.,
Sajid, M.,
Clough, B.,
Fleck, S. L.,
Aikawa, M.,
Grainger, M.,
and Hackett, F.
(1998)
J. Biol. Chem.
273,
23398-23409 |
15. |
Collins, T.,
and Wilhelm, J. M.
(1981)
J. Biol. Chem.
256,
10475-10484 |
16. |
Maihle, N. J.,
and Satir, B. H.
(1986)
J. Biol. Chem.
261,
7566-7570 |
17. |
Verbsky, J. W.,
and Turkewitz, A. P.
(1998)
Mol. Biol. Cell
9,
497-511 |
18. | Steers, E., Jr., Beisson, J., and Marchesi, V. T. (1969) Exp. Cell Res. 57, 392-396[Medline] [Order article via Infotrieve] |
19. | Tindall, S. H., DeVito, L. D., and Nelson, D. L. (1989) J. Cell Sci. 92, 441-447[Abstract] |
20. | Shih, S. J., and Nelson, D. L. (1991) J. Cell Sci. 100, 85-97[Abstract] |
21. | Madeddu, L., Gautier, M.-C., Vayssié, L., Houari, A., and Sperling, L. (1995) Mol. Biol. Cell 6, 649-659[Abstract] |
22. | Satir, B. (1977) Cell Biol. Int. Rep. 1, 69-73[Medline] [Order article via Infotrieve] |
23. | Hausmann, K., Fok, A. K., and Allen, R. D. (1988) J. Ultrastruct. Mol. Struct. Res. 99, 213-225 |
24. |
Shih, S. J.,
and Nelson, D. L.
(1992)
J. Cell Sci.
103,
349-361 |
25. | Adoutte, A., Garreau de Loubresse, N., and Beisson, J. (1984) J. Mol. Biol. 180, 1065-1081[Medline] [Order article via Infotrieve] |
26. | Turkewitz, A. P., Madeddu, L., and Kelly, R. B. (1991) EMBO J. 10, 1979-1987[Abstract] |
27. | Gautier, M.-C., Garreau de Loubresse, N., Madeddu, L., and Sperling, L. (1994) J. Cell Biol. 124, 893-902[Abstract] |
28. | Madeddu, L., Gautier, M. C., Le, Caer, J. P., Garreau de Loubresse, N., and Sperling, L. (1994) Biochimie (Paris) 76, 329-335 |
29. | Chilcoat, N. D., Melia, S. M., Haddad, A., and Turkewitz, A. P. (1996) J. Cell Biol. 135, 1775-1787[Abstract] |
30. |
Vayssie, L.,
Garreau De Loubresse, N.,
and Sperling, L.
(2001)
J. Cell Sci.
114,
875-886 |
31. |
Allen, S. L.,
Altschuler, M. I.,
Bruns, P. J.,
Cohen, J.,
Doerder, F. P.,
Gaertig, J.,
Gorovsky, M.,
Orias, E.,
and Turkewitz, A.
(1998)
Genetics
149,
459-462 |
32. | Wuitschick, J. D., and Karrer, K. M. (1999) J. Eukaryotic Microbiol. 46, 239-247[Medline] [Order article via Infotrieve] |
33. |
Cassidy-Hanley, D.,
Bowen, J.,
Lee, J. H.,
Cole, E.,
VerPlank, L. A.,
Gaertig, J.,
Gorovsky, M. A.,
and Bruns, P. J.
(1997)
Genetics
146,
135-147 |
34. | Laemmli, U. K. (1970) Nature 227, 680-682[Medline] [Order article via Infotrieve] |
35. | Towbin, H., Staehlin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract] |
36. | Turkewitz, A. P., Chilcoat, N. D., Haddad, A., and Verbsky, J. W. (2000) Methods Cell Biol. 62, 347-362[Medline] [Order article via Infotrieve] |
37. | Tiedtke, A. (1976) Naturwissenschaften 63, 93[Medline] [Order article via Infotrieve] |
38. | Orias, E., Flacks, M., and Satir, B. H. (1983) J. Cell Sci. 64, 49-67[Abstract] |
39. | Turkewitz, A. P., and Kelly, R. B. (1992) Dev. Genet. 13, 151-159[Medline] [Order article via Infotrieve] |
40. | Gaertig, J., and Gorovsky, M. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9196-9200[Abstract] |
41. |
Gautier, M.-C.,
Sperling, L.,
and Madeddu, L.
(1996)
J. Biol. Chem.
271,
10247-10255 |