Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637
Phosphoglucomutase (PGM) is a ubiquitous
highly conserved enzyme involved in carbohydrate metabolism. A number of recently discovered PGM-like
proteins in a variety of organisms have been proposed
to function in processes other than metabolism. In addition, sequence analysis suggests that several of these
may lack PGM enzymatic activity. The best studied
PGM-like protein is parafusin, a major phosphoprotein
in the ciliate Paramecium tetraurelia that undergoes
rapid and massive dephosphorylation when cells undergo synchronous exocytosis of their dense-core secretory granules. Indirect genetic and biochemical evidence also supports a role in regulated exocytotic
membrane fusion. To examine this matter directly, we
have identified and cloned the parafusin homologue in
Tetrahymena thermophila, a ciliate in which protein
function can be studied in vivo. The unique T. thermophila gene, called PGM1, encodes a protein that is
closely related to parafusin by sequence and by characteristic post-translational modifications. Comparison of
deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like
proteins have PGM activity. We evaluated the activity
and function of PGM1 through gene disruption. Surprisingly, PGM1 cells displayed no detectable defect
in exocytosis, but showed a dramatic decrease in PGM
activity. Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like
proteins in regulated exocytosis is unlikely to precede
membrane fusion.
THE functions of proteins that are secreted via regulated exocytosis are as wide-ranging as tissue coordination by hormones in metazoans and defense
from predators in ciliated protists (Harumoto and Miyake,
1991 In Paramecium tetraurelia, microinjection of protein phosphatases, including calcineurin, triggers exocytosis of dense-core secretory granules in vivo. This also occurs in a cell-free
system. Furthermore, phosphatase inhibitors and antibodies to calcineurin inhibit exocytosis (Momayezi et al., 1987 Parafusin is phosphorylated by several mechanisms in vitro, but it is not clear which are physiologically relevant. The
most unusual is the attachment of phosphoglucose to the
protein by the enzyme UDP-Glc:glycoprotein Glc-1-phosphotransferase (glucose phosphotransferase)1. Parafusin is
the only detectable protein acceptor for this enzyme in
vitro (Satir et al., 1990 Cloning of parafusin (Subramanian et al., 1994 Another disputed issue has been the localization of
parafusin. Proteins that are involved in rapid exocytotic
responses are expected to be localized near the site of fusion at the plasma membrane. Immunolocalization of parafusin has been complicated by uncertainty over whether
the protein was indeed PGM, and potential interference
by antigenically related proteins (Murtaugh et al., 1987 Although a number of issues remain unresolved, parafusin is the best-studied of a group of PGM-like proteins
that have been proposed, based on their localization or reversible posttranslational modification, to play roles that
are unrelated to the namesake enzymatic activity of PGM.
Adaptation of abundant metabolic enzymes for other specific functions is well known, for example in the case of the
lens crystallins (Wistow et al., 1987 Restriction enzymes were from New England Biolabs (Beverly, MA). All
other reagents were from Sigma Chem. Co. (St. Louis, MO), unless otherwise noted.
Cells and Cell Culture
Cells were grown at 30°C with agitation in 1% proteose peptone, 0.1%
yeast extract (both from Difco, Detroit, MI), 2% glucose, and 0.003% ferric EDTA. Tetrahymena thermophila strains derived from inbred strain B
were used in this work, and are listed in Table I. Strain B2086 is considered to be wild-type, and all initial strains are wild-type with respect to
regulated exocytosis. Strains CU428.1 and B2086 were obtained from Peter Bruns (Cornell University, Ithaca, NY).
Table I.
Strains Used in This Work
). In contrast, the cytosolic machinery involved in vesicle targeting and membrane fusion appears to be highly conserved throughout eukaryotes (Bennett and Scheller,
1993
). The mechanism of exocytosis must involve a set of
proteins that can respond rapidly to changes in cytosolic
signals, frequently involving a rise in the concentration of
cytosolic Ca2+ (Calakos and Scheller, 1996
). The activity
of some of these proteins may be modulated by reversible
modification. In particular, phosphorylation or dephosphorylation-linked regulation of exocytosis has been suggested in disparate systems, though in many cases the
modified proteins have not been identified (Sudhof and
Jahn, 1991
; Calakos and Scheller, 1996
).
).
A potential target for dephosphorylation was identified as
parafusin (a.k.a. PP63, PP65) (Subramanian et al., 1994
).
Parafusin is a major phosphoprotein in resting Paramecium cells and is dramatically dephosphorylated when
cells are stimulated with secretagogues (Gilligan and Satir, 1982
). Because exocytosis is rapid and synchronous in this
organism, stop-flow methods could be used to determine
that dephosphorylation occurs within 80 ms of stimulation,
a similar time-scale to that of membrane fusion (Höhne-Zell et al., 1992
). After exocytosis, rephosphorylation begins within 5 s, and the protein returns to its resting state in
~20 s (Zieseniss and Plattner, 1985
). Dephosphorylation was only observed in Paramecium capable of exocytosis; it
was blocked by exocytosis-inhibiting drugs, and was not
seen in exocytosis-deficient mutants (Gilligan and Satir,
1982
; Zieseniss and Plattner, 1985
). In addition, microinjection of antibodies to parafusin inhibited both exocytosis
and dephosphorylation in vivo (Stecher et al., 1987
).
). More conventional phosphorylation by kinases has also been demonstrated, but no functional role has yet been established for any posttranslational modifications of parafusin (Kissmehl et al., 1996
).
) revealed that it is related to PGM (EC 2.7.5.1). This ubiquitous enzyme catalyzes the interconversion of glucose-
1-phosphate and glucose-6-phosphate, an important step
in glycogen metabolism, trehalose synthesis, galactose metabolism, and glycoprotein synthesis (reviewed in Boles et al.,
1994
). Although the enzyme has no known role in signal
transduction or secretion, a related protein in PC12 cells and rat synaptosomes was shown to undergo posttranslational modification concurrent with exocytosis (Veyna et al.,
1994
). It has been argued that parafusin does not have
PGM activity (Andersen et al., 1994
; Subramanian et al.,
1994
). However, this claim has recently been cast into
doubt by the demonstration of PGM activity in transgenically expressed and purified parafusin (Hauser et al., 1997
).
This activity may explain at least one source of phosphorylation in vivo, since PGM in other species is phosphorylated on its active site serine.
;
Stecher et al., 1987
; Satir et al., 1989
; Höhne-Zell et al., 1992
;
Subramanian and Satir, 1992
; Wyroba et al., 1995
; Hauser et al., 1997
). Indeed, recent cloning results indicate that at least two closely related isoforms are present in Paramecium (Hauser et al., 1997
).
). To address these issues for a PGM-related protein, we have turned to another ciliate, Tetrahymena thermophila. Like Paramecium, Tetrahymena is capable of synchronous fusion of its dense-core secretory granules. A major experimental advantage
in T. thermophila is that techniques for gene disruption are
well-developed, so that the in vivo role of identified proteins can be evaluated. Importantly, regulated exocytosis
is nonessential for laboratory growth, so any mutants disrupted in this pathway, as might be expected in the case of parafusin mutations, are expected to remain viable. Disruption analysis is facilitated by the fact that Tetrahymena
appears frequently to have maintained genes as single micronuclear copies, where Paramecium has duplicated copies or gene families. We here describe the identification
and functional analysis of a parafusin homologue in Tetrahymena thermophila.
Materials and Methods
Cloning of PGM1 cDNA and Genomic DNA
Based on known codon usage in Tetrahymena thermophila (Martindale,
1989), degenerate primers (Integrated DNA Technologies, Coralville, IA)
were designed to amplify parafusin homologues via the polymerase chain
reaction. Primers were designed to correspond to regions conserved between GAL5 (Saccharomyces cerevisiae), PGM1 (Homo sapiens), and parafusin (P. tetraurelia). A total of four forward and four reverse primers
were designed and used in all 16 possible combinations. The primers correspond to residues 126-133, 142-149, 321-327, 339-344, 388-384, 369-362, 410-403, and 427-421 of the deduced Pgm1p protein sequence (see Fig. 4). PCR was performed on a Tetrahymena thermophila
gt10 cDNA library generously provided by Tohru Takemasa (University of Tsukuba, Japan),
or on Tetrahymena thermophila strain CU428.1 genomic DNA. The 50-µl
reactions contained 108 plaque-forming units or 10 ng genomic DNA, 2.5 U Taq polymerase (Promega Corp., Madison, WI), 10 mM Tris-HCl pH
8.3, 50 mM KCl, 2.5 mM MgCl2, 250 µM each dNTP, and 1 µM each
primer. 35 cycles of 92°C, 0.5 min; 40, 45, 50, or 55°C, 1 min; 72°C, 0.5 min,
were performed. A total of 12 bands of the expected sizes were cloned into pCRII (Invitrogen, San Diego, CA) and sequenced; 5 products were
unrelated to PGM, the other 6 (3 amplified from genomic DNA, 3 from
cDNA, using 6 of the 8 primers in all) were related to various PGMs. These
were determined to be identical in overlapping regions and derived from a
single gene.
The largest PCR product, 1,265 bp (corresponding to bases 559-1824 of
Fig. 2) was used to screen a cDNA phage library using the Genius system
(Boehringer Mannheim, Indianapolis, IN), which resulted in the isolation
of a single clone containing a 1.7-kB insert. After subcloning into pBluescript SK (+) (Stratagene, La Jolla, CA), sequencing revealed that the 5
end was truncated. On the assumption that some full-length clones were
present in the library, we conducted PCR on the cDNA library using a
gt10 primer and a primer complementary to the portion of the gene we had already sequenced. Cloning and sequencing of the resultant 900-bp
product revealed that it contained a putative start codon.
Genomic sequences 5 of the cDNA were obtained by inverse PCR
(Ochman et al., 1988
) on genomic DNA digested with EcoRI. The forward
primer corresponded to bases 306-275, and the reverse primer to bases
1250-1273 of Fig. 2. PCR was performed on 200 ng of circularized genomic
DNA using the same reagents and cycling times as above, but with a 1.5-min extension step. These reactions produced a single product which was
the expected size (3 kB) based on Southern blotting. This was cloned into
pCRII and partially sequenced. The PGM1 sequence reported represents
the consensus obtained from at least three independent templates, each
sequenced on both strands.
Southern and Northern Blotting
Probe templates were prepared by PCR amplification of cloned sequences followed by purification on QIAprep columns (Qiagen, Chatsworth, CA). Probes labeled with either digoxigenin (Boehringer Mannheim; for Southern analysis) or 32P (DuPont NEN, Wilmington, DE; for Northern analysis) were then prepared by randomly primed DNA synthesis.
Genomic DNA was prepared as described (Gaertig et al., 1993) except
that after isopropanol precipitation, DNA was further purified by dissolving in 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 µg/ml RNaseA for 10 min at 65°C, and then separated from contaminants on a Q-100 column
(Qiagen, Chatsworth, CA) according to the manufacturer's directions.
DNA was quantified by spectroscopic analysis (A260/A280 ratios were typically 1.75-1.85). Southern blotting was according to manufacturer's instructions (Genius, Boehringer Mannheim) loading 20 µg of DNA per
lane. For low-stringency blots, hybridization was conducted in 50% formamide, 5× SSC, 2.0% DIG blocking reagent (Boehringer Mannheim),
0.1% N-lauroylsarcosine, 0.2% sodium dodecyl sulfate, at 20°C for 16 h.
This was followed by four 15-min washes in 2× SSC containing 0.1% SDS,
also at 20°C. RNA purification and Northern blotting were performed as
previously described (Chilcoat et al., 1996
).
Disruption of PGM1
PGM1 was disrupted by replacement of the genomic sequences 76 to
+2182 relative to the start codon (resulting in removal of the first 486 residues) with a construct containing the neo2 resistance cassette (Chilcoat et al.,
1996
; Gaertig et al., 1994
), which confers paromomycin resistance in Tetrahymena. Electroporation (ECM 600; BTX Corporation, San Diego, CA)
was used to transform mating pairs of B2086 and CU428.1, as previously
described (Gaertig et al., 1995
). Initial drug selection was with 120 µg/ml
paromomycin, added 6 h after transformation. 3-6 transformants were obtained per transformation (n = 3). Because only some of the macronuclear PGM1 copies were replaced initially, cells were grown in increasing concentrations of paromomycin for stringent selection of the pgm1-1:: neo2 allele. Several transformants were cloned and grown in paromomycin (up to 4 mg/ml) for 400 generations.
Enzyme Assay
To measure PGM activity, 500-ml cultures were grown to 1-2 × 105 cells/
ml, then rapidly chilled and pelleted at 200 g for 5 min in conical bottles.
All subsequent steps were performed cold. Cells were washed once in
10 mM Hepes-KOH, pH 6.9, and the pellet volume measured. The pellet
was then washed and suspended in 2 vol of Buffer A (0.3 M sucrose, 10 mM
Hepes-KOH, pH 6.9, 36 mM KCl, 2 mM MgCl2, 2 mM EGTA; Fluka,
Ronkonkoma, NY) with the protease inhibitors leupeptin (0.5 µg/ml;
Boehringer Mannheim), antipain (12.5 µg/ml), E-64 (10 µg/ml), and chymostatin (10 µg/ml). The resuspended pellet was homogenized by passage
through a ball-bearing cell cracker (Hans Issel, Palo Alto, CA) with a
nominal clearance of 0.0005 in. In some cases this crude lysate was fractionated by centrifuging in a Beckman Optima TLX ultracentrifuge with a TLA120.2 rotor at 890 g for 10 min (to generate S1 and P1 fractions), 16,000 g for 15 min (S2 and P2) and 300,000 g for 15 min (S3 and P3). Pellets were washed and suspended in the starting volume of Buffer A. Paramecium tetraurelia was grown as a monoxenic culture, and the bacteria
were depleted by a brief period of cell starvation before cell homogenization (Sonneborn, 1970). Paramecium homogenates were prepared identically to those of Tetrahymena, and S. cerevisiae (strain GPY60) lysates
were prepared as described (Baker et al., 1990
).
PGM activity was determined using an assay based on (Joshi, 1982).
200-µl reaction volumes contained a final concentration of 2 mM Glc-1-P,
7.9 nM Glc-1-6-biP, 0.5 mM NADP, 5 mM MgSO4, 40 mM imidazole-HCl,
pH 7.4, and 0.2 U Glc-6-P dehydrogenase. Ribose-5-phosphate was added
to some reactions to a final concentration of 2 mM. After all reaction components were assembled they were incubated at 4°C for 5 min to ensure
complete activation of PGM. Samples were then warmed to 30°C; after a
2-min equilibration the absorbance at 340 nm was recorded every 10 s for
12 min on a microplate reader (Bio-Tek 340, Highland Park, VT). This resulted in linear plots of absorbance vs. time for all measurements used. This assay was found to be linear for solutions of 4-400 mU/ml PGM.
Each assay was calibrated with purified rabbit PGM equilibrated at 4°C
for 30-120 min in 40 mM imidazole, pH 7.4, 2 mg/ml BSA (Calbiochem-Novabiochem, La Jolla, CA). Protein concentrations were determined using bicinchoninic acid (Pierce, Rockford, IL).
In Vivo Labeling of PGM1
An overnight culture of cells was pelleted for 5 min at 200 g, then washed and suspended in Buffer H (5 mM Hepes-KOH, pH 7.5, 0.65 mM CaCl2, 0.1 mM MgCl2) for overnight starvation at 30°C without shaking. Starved cells are optimal for labeling and secretion. Cells were concentrated by centrifugation to a final concentration of 2-9 × 106 cells/ml and labeled by addition of 1/9 vol [32P]orthophosphoric acid (DuPont-NEN, 8500-9120 Ci/mmol), to a final concentration of 1 mCi/ml, and incubated at room temperature for 45 min. To induce secretion, 1/5 vol of saturated picric acid was added, followed within ~2 s by the addition of 2 vol of 95°C SDS-PAGE sample buffer (190 mM Tris-HCl, pH 6.95, 15% glycerol, 6% SDS, 15 mM EDTA, 0.002% bromophenol blue). Samples were heated to 95°C for 10 min, separated by SDS-PAGE, and electrophoretically transferred to nitrocellulose. Membranes were then washed (3 × 1 min) with 1× TBS (20 mM Tris-HCl, pH 7.6, 137 mM NaCl), dried, and exposed to film (Kodak XOMAT AR) for 12-60 h or to PhosphorImager screens (Molecular Dynamics, La Jolla, CA) overnight. After exposure, membranes were stained for total protein using Ponceau S. All experiments were performed at least three times, with similar results.
In Vitro Glucose Phosphotransferase Assay
Cells were grown to 3-6 × 105/ml, pelleted for 5 min at 200 g, washed and
suspended in Buffer B (50 mM Bis-Tris-HCl, pH 6.8, 105 mM NaCl, 5 mM
CaCl2, and 1 mM NaH2PO4), and homogenized as for PGM assays. 25 µg
of this lysate was incubated in Buffer B with 2.8 µCi [-35S]UDP-Glc (2.8 mCi/ml, 345 Ci/mmol; DuPont NEN, Wilmington, DE) and 0 or 200 µM
glucose-1-phosphate, plus 0 or 200 µM UDP-glucose in a total volume of
50 µl for 30 min at room temperature (Marchase et al., 1993
). 2 vol of 95°C
SDS-PAGE sample buffer were added, and samples were visualized as
described above.
Exocytosis Assays
Cells were grown to 3-6 × 105/ml, washed and suspended in DMC (0.2 mM
sodium phosphate, pH 6.9, 0.17 mM sodium citrate, 0.1 mM MgCl2, 0.65 mM
CaCl2) at a concentration of 106/ml, and incubated for 6-9 h at room temperature. Aliquots of cells were stimulated by the addition of 1/4 vol Alcian Blue 8 GX in water (concentrations given in text) for 15 s. Cells were then fixed and processed for immunofluorescence using mAb 4D11, which
recognizes the granule core protein p80, as previously described (Turkewitz and Kelly, 1992). Cells in randomly chosen fields were scored as either unstimulated or stimulated based on the presence or absence of
docked granules.
In both Paramecium tetraurelia and Tetrahymena thermophila, synchronous exocytosis of dense-core secretory granules can be triggered by a transient increase in intracellular
calcium. In Paramecium, exocytosis has been correlated
with dephosphorylation of parafusin, an abundant phosphoprotein. To see whether a similar protein exists in Tetrahymena, we labeled cells with [32P]orthophosphate. One
major phosphoprotein of the same apparent molecular
mass as parafusin (~60 kD) was observed in unstimulated cells, and was massively dephosphorylated in stimulated
cells (Fig. 1). This suggested that a parafusin homologue
with a conserved function could be studied in Tetrahymena.
To clone the gene encoding the putative homologue as
well as any related proteins, we identified protein sequences
conserved between Paramecium parafusin, human PGM,
and yeast PGM, and designed eight degenerate PCR primers corresponding to those regions. PCR amplification using combinations of these primers, in conjunction with
Tetrahymena cDNA or genomic DNA, generated six products encoding a PGM-like protein. Each overlapped with
all of the others, and overlapping sequences were identical, indicating that they were derived from a single gene.
The largest product hybridized to a 1.7-kB clone from a
cDNA library, whose sequence was identical to that of the
gene identified by PCR. We extended the search for potential homologues with low stringency Southern blots of genomic DNA, using probes made both from S. cerevisiae
PGM (GAL5) as well as the isolated Tetrahymena cDNA
clone. Both probes detected a single macronuclear gene
(see Fig. 3). Based on these lines of evidence, it appears
that Tetrahymena has only a single gene homologous to
PGM and parafusin, which we named PGM1 for reasons described below. Northern blots (see Fig. 6 C) were also
consistent with a single gene.
Sequence Analysis
Both cDNA and genomic DNA sequences were determined. The PGM1 sequence displayed characteristics of a
Tetrahymena thermophila gene: several short introns and
the 5 and 3
UTRs were rich in AT-rich sequence, and
codon usage showed the expected bias (Fig. 2) (Horowitz
et al., 1987
; Martindale, 1989
). Southern blotting of total
Tetrahymena DNA indicated two bands hybridizing with PGM1, which differed in intensity 20-fold (Fig 3). This is
the expected difference between macronuclear and micronuclear alleles, since the macronucleus contains 45 copies
of each gene, while the micronucleus is diploid. The micronuclear allele appeared to contain a ~2.0 kB insertion,
known as an IES. This was confirmed by direct cloning of
the IES, which was determined to lie within intron 4 (Fig.
2). As expected, this micronuclear copy persisted in clones
whose macronuclear alleles were disrupted (not shown).
The longest ORF in PGM1 is predicted to encode a 587-residue protein with a calculated mass of 65.5 kD. This
product, Pgm1p, is closely related to P. tetraurelia parafusin (60% identity) and known eukaryotic PGMs (40-
50% identity). We note that T. thermophila and P. tetraurelia, while both ciliates, diverged at least several hundred
million years ago (Wright and Lynn, 1997). To evaluate
the significance of the observed differences between these
proteins, we used the known crystal structure of rabbit muscle PGM (Dai et al., 1992
) as a framework upon which to
compare a group of PGM-related proteins. These included
Paramecium parafusin and H. sapiens PGM5, both of
which have been speculated to lack enzymatic activity in
part on the basis of sequence analysis (Andersen et al.,
1994
; Subramanian et al., 1994
; Moiseeva et al., 1996
) and
O. cuniculus PGM2, a sarcoplasmic reticulum-associated
phosphoprotein (Lee et al., 1992
). Strikingly, all six residues predicted to be involved in substrate binding and enzymatic catalysis in conventional PGM (Dai et al., 1992
)
are completely conserved in these genes (Figs. 4 and 5), arguing that all are enzymatically active. As has been previously noted, parafusin contains several insertions and deletions relative to mammalian PGMs (overlined in Fig. 4); it has been hypothesized that these differences account for
a non-PGM activity in parafusin (Subramanian et al.,
1994
). Tetrahymena Pgm1p contains insertions and deletions at the same positions. However, neither the size nor
the sequence of the insertions is conserved between Tetrahymena and Paramecium. Moreover, molecular modeling predicts that all insertions and deletions in the ciliate proteins relative to the mammalian enzyme occur at the
end of helices or in regions of random coil, and are located
at the periphery of the molecule (Fig. 5). Thus, residues
predicted to be involved in enzyme catalysis are maintained
in both parafusin and Pgm1p, and variable regions lie in
parts of the molecule that are not likely to be important
for either folding or enzymatic activity.
Disruption of PGM1
To analyze the in vivo role of PGM1, a disruption vector
(Gaertig et al., 1994) was used to target the PGM1 gene.
The vector was designed so that its recombination with the
PGM1 locus eliminated the first 80% of PGM1, including
the start codon (Fig. 6 A). Disruption occurs by replacement of macronuclear PGM1 sequences with neo2, which
confers resistance to paromomycin. Tetrahymena have both a micro- and a macronucleus, but only the latter is transcribed during vegetative growth. The macronucleus contains ~45 identical copies of most genes (Bruns, 1986
). Immediately after transformation, vector integration results
in replacement of a small number of the macronuclear
copies. Transformed clones are then grown in increasing
concentrations of paromomycin, to select for cells containing a progressively higher fraction of disrupted alleles, through a process called phenotypic assortment (Bruns,
1986
). After phenotypic assortment, we verified the complete replacement of macronuclear PGM1 through Southern and Northern blotting (Fig. 6, B and C). A second disruption strategy was also used, in which neo2 was inserted
in the 5
NspV site of PGM1 (not shown). Transformants created using either construct showed identical phenotypes. Furthermore, Southern blotting using a probe derived from neo2 demonstrated that the targeting construct
had not integrated elsewhere in the genome (not shown).
After complete gene disruption, cells were transferred to
drug-free media for subsequent analysis.
PGM1 cells displayed no noticeable defects in growth or morphology.
Pgm1p Phosphorylation In Vivo and In Vitro
A well-characterized biochemical property of parafusin in
Paramecium lysates is that it serves as the acceptor of a
glucose phosphotransferase (Satir et al., 1990). To determine whether this property was shared by Tetrahymena
Pgm1p, we prepared cell lysates and incubated them with
the donor molecule for this reaction, [
-35S]UDP-glucose.
A single band with the mobility predicted for Pgm1p was
labeled in lysates prepared from wild-type cells (Fig. 7 A). In contrast, no band was labeled in parallel experiments
done with
PGM1 lysates.
Labeling of PGM in this reaction mix could also be due
to the presence of a breakdown product, [-35S]Glc-1-P,
which can label the active site of PGM (Marchase et al.,
1987
). To eliminate this possibility, cell lysates were incubated with [
-35S]UDP-glucose in the presence of a 200-fold excess of either unlabeled UDP-Glc or Glc-1-P (Fig. 7
B). UDP-Glc, but not Glc-1-P, nearly abolished the labeling, indicating that glucose phosphotransferase activity and
not active site labeling was primarily being observed.
Our initial observation was that a ~60-kD phosphoprotein underwent dephosphorylation in vivo upon exocytosis
induction in Tetrahymena. To establish this parafusin homologue as Pgm1p, the experiment was repeated with
PGM1 cells. As anticipated, the parafusin homologue at
that position was no longer present (Fig. 7 C). In summary,
similarity between parafusin and Pgm1p is seen both in sequence and in two kinds of characteristic phosphorylation. No other protein in T. thermophila had this combination
of qualities.
PGM Activity and PGM1
To determine whether Pgm1p was enzymatically active, we
measured PGM activity in crude cell lysates prepared from
wild-type or PGM1 cells (Fig. 8). PGM activity in
PGM1
cells was reduced 92% (±1.8%) relative to wild-type cells,
indicating that Pgm1p accounts for most of the PGM activity in T. thermophila. Since data presented above argue that
T. thermophila does not contain other PGM-like genes,
the residual PGM activity observed in
PGM1 cells might
be accounted for by unrelated phosphomutase(s) with broad specificity. Similarly, S. cerevisiae contains proteins other than PGMs that can catalyze interconversion of glucose-1-P and glucose-6-P (Boles et al., 1994
; Hofmann et al.,
1994
). We asked whether various monosaccharide phosphates, known to act as competitive inhibitors for unrelated phosphomutases, would affect the apparent PGM activity in wild-type or
PGM1 lysates. Supporting our
hypothesis, ribose-5-phosphate was found to be a more
potent inhibitor of the residual activity present in
PGM1 cell lysates (82% inhibition) than of the activity in wild-type lysates (30% inhibition; Fig. 8) or purified rabbit PGM
(35% inhibition; not shown). Similar differential inhibition
was seen with mannose-1-P (not shown). This argues that
the residual PGM activity in
PGM1 cells comes from enzymes with substantially different properties from classically
defined PGMs. Candidates include phosphopentomutase, phosphomannomutase, and N-acetylglucosamine phosphomutase.
Localization
To examine the localization of Pgm1p in Tetrahymena, cell
homogenates were fractionated by centrifugation, and
PGM activity was determined. Less than 0.1% of the total
PGM activity could be pelleted (Table II). It remains possible that posttranslational modification of Pgm1p induces
a weak or transient membrane attachment. A second potential complication is that a membrane-localized population of PGM might have a latent activity, as has been reported for a sarcoplasmic reticulum-associated PGM (Lee
et al., 1992). However, a variety of treatments which had
been shown to activate latent PGM activity in that system,
including nonionic detergents, high salt, and denaturants,
failed to uncover latent activity in Tetrahymena membranes. Therefore, Pgm1p appears to be a soluble protein
with no detectable membrane association.
Table II. Phosphoglucomutase Activity in Subcellular Fractions |
Effect of PGM1 Disruption on Exocytosis
Tetrahymena contain a large number of dense-core granules, called mucocysts, whose exocytosis can be easily triggered and monitored. Virtually all granules in resting cells
are docked in positions from which they can fuse and release their contents within seconds of stimulation. This
dramatic release can be detected by immunofluorescence
visualization of docked granules using a mAb against a secreted granule protein. Before stimulation, both wild-type
and PGM1 cells displayed an identical pattern of docked granules, indicating that granule synthesis, targeting, and
docking had taken place (Fig. 9). Cells were then visualized after 15 s of stimulation, and individual cells in randomly chosen fields were scored as either unstimulated or
having undergone extensive exocytosis. Under optimal stimulation conditions,
PGM1 cells displayed the same response as wild-type cells, both showing virtually complete
exocytosis in >97% of the cells (Fig. 10). A number of suboptimal stimulation conditions (lower concentrations
of secretagogue) were tested to see whether the sensitivity
of the
PGM1 cells might be altered. Again, the responses of
PGM1 and wild-type cells were indistinguishable. In summary,
PGM1 cells appeared identical to wild-type in both
the extent of exocytosis and the stimulation threshold.
Several other tests were performed to assess the PGM1
exocytotic response. At higher concentrations of secretagogue, >90% of both wild-type and
PGM1 cells were
seen to become encapsulated, an assay that has previously
been used to indicate extensive and synchronous secretion
(Tiedtke, 1976
). We tested the response of
PGM1 cells to
other secretagogues including dibucaine, the calcium ionophore A23187, electrical shock, and picric acid. Secretion was evaluated by several methods: immunofluorescence as
previously described; observing cell behavior (secreted protein causes cells to clump in a distinctive fashion); isolating
secreted proteins (Turkewitz et al., 1991
); and electron microscopy (Chilcoat et al., 1996
). In all cases, the responses
of
PGM1 cells were indistinguishable from those of wild-type (data not shown).
Parafusin has been the subject of a large number of papers, and its potential role in regulated exocytosis has been
widely cited. The most significant evidence supporting such
a role includes the demonstration of dramatic dephosphorylation on a <80-ms time scale, indicating that this modification occurs rapidly enough to be playing a regulatory
role in membrane fusion, and that the dephosphorylation
does not occur in exocytosis-deficient mutant cells (Zieseniss and Plattner, 1985). The potential significance of these
findings was underscored by the finding that a related protein undergoes posttranslational modification upon depolarization of rat brain synaptosomes, suggesting that this
step represented an evolutionarily maintained aspect of
regulated exocytosis for both dense-core granules and synaptic vesicles (Veyna et al., 1994
). This model also fits with
data obtained in numerous systems that indicate a role for
protein phosphatases during exocytotic triggering (Verhage et al., 1995
; Raufman et al., 1997
). Nonetheless, these
data can be broadly described as circumstantial, and no direct evidence has established a functional role for parafusin or its dephosphorylation.
We approached these questions by taking advantage of an organism whose physiological and experimental features make it highly advantageous for studies of regulated exocytosis. Like Paramecium, Tetrahymena can undergo rapid synchronous exocytosis of dense-core secretory granules under controlled conditions. In addition, recent methodological advances facilitate targeted gene disruption, thereby allowing in vivo analysis of protein function. We have identified and cloned the parafusin homologue in T. thermophila. Results of gene disruption demonstrate that the PGM1 gene encodes enzymatically active PGM. The data do not support a role for Pgm1p in regulated exocytosis.
A parafusin homologue was identified using a variety of approaches, including degenerate PCR, library screening, low stringency Southern blotting, Northern blotting, PGM activity assays, in vivo stimulus-dependent dephosphorylation, and in vitro glucosylphosphotransferase assays. All of these approaches identified PGM1, but no other related genes. This experimental redundancy implies that we have identified the parafusin homologue, demonstrates that it encodes active PGM, and indicates that there are no other PGM-like proteins in Tetrahymena. The gene product displays several highly characteristic features of parafusin.
While Tetrahymena contains a single PGM, multiple isoforms of the enzyme are found in other eukaryotes. In
mammals, isoforms of metabolic enzymes with apparently
identical functions are commonly observed, sometimes with
differences in tissue distribution or gene regulation. Such
is the case with human PGM (reviewed in Whitehouse et al.,
1992), and a similar situation may account for the number
of isoforms in rabbit. The two isoforms of PGM in the generally compact genome of S. cerevisiae result from an ancient large-scale chromosomal duplication (Boles et al.,
1994
). In P. tetraurelia, where at least two isoforms of parafusin exist, multiple isoforms are the norm rather than an
exception (Madeddu et al., 1995
). Our results strengthen
the impression that single-copy genes are common in T. thermophila.
The identification of a novel PGM from the highly divergent ciliate kingdom led to some interesting sequence-based observations. PGM is an ancient enzyme, highly conserved even between prokaryotes and eukaryotes (Lu and
Kleckner, 1994), and we asked if intron/exon boundary
sites are conserved between T. thermophila PGM1, H. sapiens PGM1, and P. tetraurelia parafusin. Except for the
first intron of Paramecium and the second of Tetrahymena, no boundary sites were conserved (Putt et al., 1993
;
Hauser et al., 1997
). In none of these genes do intron/exon
junctions coincide with polypeptide domain boundaries
(as determined by Dai et al., 1992
).
A number of recently sequenced PGM-like genes have
been predicted to lack enzymatic activity because of the
perceived divergence of important amino acid residues.
The suspected absence of PGM activity was indeed one of
the strongest arguments that these proteins have novel
roles. As a divergent PGM, the Tetrahymena protein is especially useful for evaluating these predictions. When the
deduced product of PGM1 was aligned with these proteins, it was apparent that active site residues were universally conserved. This is in contrast to what was originally
stated for parafusin (Subramanian et al., 1994), but is in
agreement with the recently observed activity of parafusin
expressed in vitro (Hauser et al., 1997
). The short insertions and deletions present in both parafusin and Pgm1p,
relative to mammalian PGMs, are predicted by molecular modeling to lie in regions where small changes will have
little or no effect on enzyme activity. The lack of sequence
conservation between the Tetrahymena and Paramecium
proteins in these regions does not support the proposal
that they endow parafusin with a specialized role in exocytosis, as previously proposed (Subramanian et al., 1994
).
H. sapiens PGM5 (also known as aciculin), is a dystrophin-
and utrophin-associated protein that was suggested, based
in part on sequence analysis, to lack enzymatic activity (Belkin et al., 1994
; Moiseeva et al., 1996
). Nonetheless, it shows complete conservation of active site residues. Similarly, while a Gly-to-Ala substitution in the Mg2+-binding
hairpin of aciculin was perceived as incompatible with enzymatic activity, both the ciliate sequences predict an Ala-to-Gly substitution in a symmetric position of this hairpin.
Given these observations, the reported existence of PGM-like proteins lacking PGM activity may deserve reconsideration.
If phosphorylation plays a regulatory function, the sites
of phosphorylation might be conserved among divergent
family members. In vivo data suggest that (Ser/Thr) kinases/phosphatases may be involved (Momayezi et al., 1987;
Verhage et al., 1995
; Raufman et al., 1997
), and parafusin
can be phosphorylated in vitro by casein kinase and dephosphorylated by calcineurin (Kissmehl et al., 1996
, 1997
).
Disallowing the active site, only 4 of 11 Ser/Thr residues
conserved throughout eukaryotes are expected from the crystal structure to be on or near the surface of the protein: residues 30, 286, 584, and 587 (Tetrahymena Pgm1p
numbering). Only Thr30 and Thr286 are on the same face
of the enzyme as the active site, and molecular modeling
suggests that phosphorylation of Thr30 would block entry
of substrate.
Effect of PGM1 Disruption
We evaluated the function of PGM1 by generating three
independent cell lines in which all macronuclear copies of
PGM1 were disrupted. Lysates prepared from PGM1
cells contain a dramatically reduced level of PGM activity,
and the residual activity appears to be the result of an unrelated enzyme(s). The specific activity of PGM in wild-type Tetrahymena was two- to fivefold higher than that
usually reported in S. cerevisiae, plants, or liver (Hanson and McHale, 1988
; Oh and Hopper, 1990
; Mithieux et al.,
1995
), but was identical to that we observed in Paramecium.
To test if PGM1 was required for regulated secretion,
we induced exocytosis using a variety of stimulatory conditions. Wild-type and PGM1 cells displayed indistinguishable sensitivity to stimulation, and extent of release. Since
Pgm1p became dephosphorylated under stimulation conditions, our results appear to uncouple these two previously correlated phenomena and indicate that this protein is not essential for exocytosis. Two alternative interpretations deserve mention, although we consider both to be
unlikely. First, the Tetrahymena protein may have lost a
function conserved in other eukaryotes; however, it is then
necessary to explain why rapid dephosphorylation has
been maintained. Second, although we stimulated cells using a variety of different conditions, our quantitative measure was based on cells fixed 15 s after stimulation. Because this period is long relative to the minimum time
required for membrane fusion, it is possible that
PGM1
and wild-type cells have different kinetics of release, which
cannot be detected by our assay. In this case, one would
have to postulate that Pgm1p acts as a kinetic effector of
exocytosis that has no effect on either the threshold or extent of release.
The lack of an observable phenotype in a gene disruption experiment may be due to suppression by an offsetting second site mutation to maintain an advantageous
trait. For the results reported here, this could theoretically
involve selection for PGM activity or regulated exocytosis.
Both seem highly unlikely for the following reasons. In a
variety of organisms, PGM mutants with only mild phenotypes have been obtained (Hanson and McHale, 1988; Boles et al., 1994
; Lu and Kleckner, 1994
; Zhou et al., 1994
), indicating that PGM is not an essential enzyme under many
growth conditions. Similarly, regulated exocytosis in Tetrahymena appears completely dispensable for growth in
the laboratory: a variety of exocytosis-deficient mutants
have been obtained that have no growth defects and are nonreverting (Maihle and Satir, 1985
; Turkewitz et al.,
1991
; Gutierrez and Orias, 1992
). Finally, indistinguishable results were obtained with three independent transformants.
In summary, our results indicate, in contradiction to the
central hypothesis of previous work on parafusin, that
Pgm1p is not required upstream of membrane fusion during regulated exocytosis. Some of the earlier evidence can
easily be reinterpreted in light of these findings. The absence of parafusin dephosphorylation in exocytosis mutants or in the presence of exocytosis inhibitors (Gilligan
and Satir, 1982; Zieseniss and Plattner, 1985
) was originally interpreted as evidence that the genetic or pharmaceutical blocks are epistatic to dephosphorylation of parafusin, which was postulated to be epistatic to exocytosis.
However, the data are equally consistent with a model in
which exocytosis is epistatic to dephosphorylation, leaving
open the possibility that parafusin may function in recovery from exocytosis. The time course of dephosphorylation established in Paramecium may suggest that it is not
membrane fusion per se that triggers dephosphorylation,
but some earlier event.
Possible Functions of Pgm1p Dephosphorylation
The family of PGM-like proteins, some of which have
been shown to undergo changes in phosphorylation, includes a calmodulin-dependent kinase substrate in sarcoplasmic reticulum (Lee et al., 1992), a yeast PGM which
undergoes phosphoglucosylation upon heat shock (Dey
et al., 1994
), and a dystrophin/utrophin-associated PGM
found in adherens junctions (Moiseeva et al., 1996
). A possible link between these proteins may be their sensitivity
to fluctuations in intracellular calcium. Modification appears
to occur under conditions of increased free cytosolic calcium associated with secretion in regulated secretory cells
and contraction in muscle (Clapham, 1995
; Erxleben et al.,
1997
). In addition, localization of calcium-sensitive proteins to the adherens junction suggests that it is also a site
of calcium flux (Burridge et al., 1988
). Therefore, PGM-like proteins may respond (via kinases/phosphatases or
phosphoglucose-transferases/esterases) to calcium levels.
What is the nature and function of this response? Given
the rapidity and extent of the modifications, we considered
that the dephosphorylated enzyme may play a role in demobilization of the high calcium required to trigger exocytosis, for example by increasing the rate of calcium uptake
into the vesicular stores that underlie the plasma membrane of ciliates, or into the ER or SR in mammalian cells.
Recent experiments in mammalian cells suggest that levels
of glucose can influence calcium transport, which could
mean that modulation of PGM activity affects free calcium
levels (Darbha and Marchase, 1996
). Therefore, one might
expect to see effects from perturbation of calcium homeostasis in
PGM cells, but these have not been reported in
either S. cerevisiae (Boles et al., 1994
) or in Tetrahymena
(Chilcoat, N.D., unpublished observation). Further development of these or other in vivo systems may help to address this issue.
Address correspondence to Aaron P. Turkewitz, Department of Molecular Genetics and Cell Biology, The University of Chicago, 920 E. 58th St., Chicago, IL 60637. Tel.: (773) 702-4374. FAX: (773) 702-3172. e-mail: apturkew{at}midway.uchicago.edu
Received for publication 20 August 1997 and in revised form 22 September 1997.
1. Abbreviations used in this paper: PGM, phosphoglucomutase; Glc, glucose; glucose phosphotransferase, UDP-Glc:glycoprotein Glc-1-phosphotransferase.We thank Pam Bounelis and Richard Marchase (University of Alabama
Birmingham) for advice and for the generous gift of [-35S]UDP-Glc, Paul
Gardner for help with DNA sequencing, Denis Nyberg (University of Illinois Chicago) for Paramecium cultures, and Celeste Richardson for S. cerevisiae cultures and lysates. Our thanks also go to Alex Haddad, Phoebe
Rice, Nava Segev, Ted Steck, and Ben Glick for discussion and comments.
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