Institut für Biochemie (S.W., T.K.) and Institut für
Genetik (T.K.), Universität zu Köln, Köln, Germany;
and Paradigm Genetics, Research Triangle Park, North Carolina
(J.P.W.)
The addition of primary amines to the
growth medium of the unicellular green alga Chlamydomonas
reinhardtii disrupts cell wall assembly in both vegetative and
zygotic cells. Primary amines are competitive inhibitors of the
protein-cross-linking activity of transglutaminases. Two independent
assays for transglutaminase confirmed a burst of extracellular activity
during the early stages of cell wall formation in both vegetative cells
and zygotes. When non-inhibiting levels of a radioactive primary amine
(14C-putrescine) were added to the growth medium, both cell
types were labeled in a reaction catalyzed by extracellular
transglutaminase. The radioactive label was found specifically in the
cell wall proteins of both cell types, and acid hydrolysis of the
labeled material released unmodified 14C-putrescine.
Western blots of the proteins secreted at the times of maximal
transglutaminase activity in both cell types revealed a single highly
cross-reactive 72-kD band when screened with antibodies to guinea pig
tissue transglutaminase. Furthermore, the proteins immunoprecipitated
by this antiserum in vivo exhibited transglutaminase activity. We
propose that this transglutaminase is responsible for an early cell
wall protein cross-linking event that temporally precedes the oxidative
cross-linking mediated by extracellular peroxidases.
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INTRODUCTION |
Cell wall formation in the unicellular green alga
Chlamydomonas reinhardtii has been intensely scrutinized for
over two decades (for review, see Woessner and Goodenough, 1994
).
During its life cycle, C. reinhardtii elaborates two
structurally and biochemically distinct cell walls. The vegetative wall
(V-wall) surrounding both vegetative and gametic cells has salt-soluble
glycoproteins comprising the outer wall layers, and salt- and
detergent-insoluble components found only in the inner wall layers.
During mating, gametic lytic enzyme (GLE) cleaves off the V-wall, and
the early zygotes assemble a desiccation-resistant, insoluble zygote
wall (Z-wall). Both the V- and Z-walls lack the abundant complex
polysaccharides typical of higher plant extracellular matrices
(cellulose, hemicelluloses, and pectins), facilitating analyses of
structural wall protein assembly in C. reinhardtii.
GLE-treated vegetative cells and early zygotes secrete developmentally
specific Hyp-rich glycoproteins (HRGPs), evolutionary homologs of
higher plant HRGPs that self-assemble in a predictable, ordered manner.
This self-assembly has been well documented in vitro for the
salt-extractable outer V-wall HRGPs: when the salt is dialyzed away
from these proteins, they form a crystalline structure similar to that
found in muro (Hills et al., 1975; Goodenough et al., 1986). In
addition, salt-extracted vegetative cells added to the dialysis bag can
nucleate the assembly of the soluble HRGPs to reform a crystalline
layer (Adair et al., 1987).
One of the terminal steps in the formation of both the V-wall and
Z-wall is insolubilization of many of the assembled HRGPs. In previous
work (Waffenschmidt et al., 1993), we determined the time of
insolubilization for each wall and demonstrated that insolubilization is due, at least in part, to the formation of isodityrosine
cross-links, a mechanism also proposed to be responsible for
insolubilization of higher plant HRGPs (Fry, 1982; Cooper and Varner,
1983). Here we present evidence that another cross-linking enzyme,
transglutaminase, is also involved in the assembly and insolubilization
of both walls.
Transglutaminases (TGases; E.C. 2.3.2.13) catalyze the covalent linkage
of protein-bound glutaminyl residues with primary amines (e.g.
polyamines) or with protein-bound lysines to create intermolecular
(
-glutamyl) lysyl cross-links. These cross-links are stable,
resistant to all known proteases and to boiling in 1% (w/v)
SDS. The family of TGases is widespread, with examples found in
bacteria, fungi, plants, and animals. Known biological functions
involving TGase include formation of the fibrin clot (Lorand and
Conrad, 1984), coagulation of seminal plasma to form the copulation
plug in rodents (Williams-Ashman, 1984), keratinization of epidermis
and hair (Rice et al., 1992), assembly of the fertilization envelope in sea urchin (Battaglia and Shapiro, 1988), formation of the
teleost fish eggshell (Oppen-Bernsten et al., 1990), and construction
of a desiccation-resistant fungal spherule (Klein et al., 1992). In
each instance, TGase catalyzes the cross-linking of structural proteins
into a large insoluble network.
Recently, it was proposed that TGase is involved in cytoskeletal
rearrangement during pollen germination and pollen tube formation in
apple trees (Del Duca et al., 1997), and intracellular and chloroplast
membrane-bound TGase activity have been demonstrated in several
instances. Both the large subunit of Rubisco (Margosiak et al., 1990)
and chlorophyll a/b binding proteins (Del Duca et al., 1994)
have been identified as major TGase substrates. A potential role for
TGase in cross-linking plant extracellular proteins has been suggested
in two studies (Falcone et al., 1993; Serafini-Fracassini et al.,
1995). The present study demonstrates that C. reinhardtii secretes an extracellular TGase immunologically related to guinea pig
tissue TGase whose substrates include cell wall HRGPs, and that
inhibition of this activity disrupts normal wall assembly.
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MATERIALS AND METHODS |
Wall Regenerating Vegetative Cells (RVC)
Vegetative cells (CC-621), grown in 600 mL of
Tris-acetate-phosphate (TAP; Harris, 1989) medium to a density of
3 × 106 cells mL
1,
were pelleted at 4,000g for 10 min in a rotor (model HB4,
Sorvall, Newtown, CT). The cells were resuspended in 30 mL of TAP
medium and 30 mL of gametic lytic enzyme (GLE; prepared as described by
Jaenicke et al. [1987]), and incubated for 1 h at room
temperature. GLE was removed by two successive washes in TAP medium
(pelleting in the rotor by bringing it up to 4,000g and
immediately back down), the cells were resuspended in 200 mL of TAP
medium (or TAP medium and inhibitor with the pH adjusted to 7.4) and
allowed to regenerate a new cell wall.
Developing Zygotes (DZ)
Zygotes were formed by mixing 100 mL (5 × 107 cells mL1) of gametes
of each mating type (CC-620 and CC-621). After a 30-min period of
mating, the cells were pelleted, washed, and resuspended in 200 mL of
high-salt medium lacking nitrogen (Harris, 1989) or the same medium
plus inhibitor with the pH adjusted to 7.4.
Spectrophotometric Assays
Insolubilization of the wall (in both vegetative and zygotic
cells) was monitored by measuring chlorophyll release following Nonidet
P-40 treatment (Waffenschmidt et al., 1993).
H2O2 concentration and
peroxidase activity were determined using the methods described in
Waffenschmidt et al. (1993).
In Vitro TGase Assays
Putrescine Method
At each time point, a 500-µL aliquot of cells was pelleted (2 min at 12,000 rpm in a microcentrifuge) and 40 µL of the supernatant was mixed with 200 µL of the test solution (150 mM
Tris-HCl, pH 7.4, 4 mM CaCl2, 30 mM glutathione, 1 mg mL1
N,N-dimethyl casein, and 0.01 mCi
mL1
[1,4-14C]putrescine). The specific activity of
the labeled putrescine was 0.05 mCi mL1, 117 mCi mmol1. The samples were incubated for 30 min at 25°C and the reaction was stopped by addition of 1 mL of
ice-cold 10% (w/v) TCA. The precipitate was washed three times
with ice-cold acetone, resuspended in 100 µL of 0.1 N NaOH, added to 1 mL of scintillation fluid, and
counted in a BG Betaszint 5000 liquid scintillation counter. Two
control samples were also analyzed in the same manner. One control was
a 40-µL aliquot of supernatant that had been heated for 15 min at
60°C to kill the enzyme. The second control was just test solution
with no supernatant added. Both control samples yielded background
levels of 60 to 80 cpm and all incorporation values were then corrected
for these levels.
The incorporation time courses were done with a 1-mL sample of
supernatant collected from RVC at the time of maximal TGase activity as
determined above. This sample was mixed with 5 mL of the test solution,
incubated at 25°C and at various time points aliquots were removed,
and processed to measure incorporation. Similarly, a second
incorporation time course was generated by taking aliquots from a
mixture of 0.5 mL of supernatant and 5.5 mL of test solution.
Hydroxamate Formation Method
Twenty-five microliters of cell-free supernatant exhibiting
maximal TGase activity as determined by method A were added to a
350-µL reaction cocktail containing 200 mM
Tris-acetate-buffer, pH 6.0, 30 mM
carbobenzoxy-L-glutamylglycine, 5 mM
CaCl2, 10 mM glutathione, and 100 mM hydroxylamine, as described previously (Folk and Cole,
1966). This was mixed well and incubated for 5 min at 37°C, then 375 µL of 1 M FeCl3 in 5% (w/v)
TCA were added. The suspension was mixed, the precipitate was removed
by centrifugation (12,000 rpm, 2 min), and the supernatant was assayed
for iron-hydroxamate-complex formation at 525 nm in a spectrophotometer.
Inhibition of TGase in Vitro
The supernatant isolated from RVC or DZ at the time of maximum
enzyme activity (as determined by the putrescine method detailed above)
was used as a crude preparation of TGase. Enzyme activity was
determined by the hydroxamate formation assay and this value was set to
100%. Then, EGTA, Zn2+(buffered with Tris, pH
7.6), N-ethylmaleimide, or
p-chloromercuribenzoate was added to aliquots of the
supernatant, enzyme activity was remeasured, and the values were
converted to percentages of control activity.
CaCl2 was omitted from the TGase reaction
cocktail for the EGTA experiment. These assays were performed in
triplicate and the average percentages are presented.
In Vivo Incorporation of 14C-Putrescine
One-hundred milliliters of RVC or DZ was prepared as described
above except that the final resuspension was in medium containing 2.5 µCi of 14C-putrescine. One milliliter of each
suspension was removed, immediately pelleted (2 min at 12,000 rpm), and
100 µL of the supernatant added to the scintillation cocktail. The
counts per minute of input label was determined in a scintillation
counter and set to 100%. At 20-min intervals, 250 µL was removed to
test for detergent sensitivity (as above). At the same time, the cells
were pelleted out of a 1-mL aliquot (2 min at 12,000 rpm), washed eight
to 10 times to eliminate non-incorporated radioactivity, and finally resuspended in 1 mL of medium. One-hundred microliters of this cell
suspension was added to the scintillation cocktail and counted. The
resultant counts per minute were converted to percentages of the total input.
After the last time point was collected (200 min for zygotes, 180 min
for vegetative cells) all of the remaining cells were pelleted,
extensively washed, and resuspended in 30 mL of medium. A 1-mL aliquot
was taken and the total incorporation was determined as described
above. The remaining cells were pelleted.
The vegetative cell pellet was first resuspended in 30 mL of 2 M NaClO4 and incubated for 20 min at
room temperature. The sodium perchlorate solubilizes the outer
crystalline wall layers and kills the cells, but does not lyse them
(Goodenough et al., 1986). One-hundred microliters of the supernatant
was taken after the cells were pelleted, and added to scintillation
cocktail for counting. The cell pellet was washed three times to remove
the NaClO4, and then resuspended in 30 mL of GLE
for 1 h. After pelleting the protoplasts, a 100-µL aliquot of
the supernatant was removed and counted.
The pelleted zygotes were extracted with 30 mL of 0.3% (w/v)
NaClO2 in 0.12% (w/v) acetic acid at
70°C for 2 h while bubbling with nitrogen (Jaenicke et al.,
1987). This treatment solubilizes the isodityrosine cross-links within
the wall (Waffenschmidt et al., 1993). The suspension was pelleted and
100 µL of the supernatant was analyzed in the scintillation counter.
Identification of 14C-Putrescine-Labeled Wall Proteins
One-hundred milliliters of GLE-treated and washed vegetative cells
was resuspended in TAP medium and 5 µCi of
14C-putrescine (this concentration, 0.43 µM, is far below the inhibiting levels of putrescine).
After 3 h of regeneration, the cells were pelleted and extracted
with 2 M NaClO4 as described above.
The supernatant was lyophilized and resuspended in 2 mL of water. Both
the perchlorate-solubilized proteins and the lyophilized medium were
dialyzed against water, and two aliquots (400 and 800 µL) were
removed, precipitated with 10% (w/v) TCA in 50% (w/v) acetone, resuspended in loading buffer, and subjected to SDS-PAGE. After fixing for 30 min in 25% (v/v) isopropyl alcohol and 10% (w/v) acetic acid and shaking for 30 min in Enhance (Amersham, Uppsala), the gel was dried and exposed to Kodak XAR-5 film.
Analysis of Self-Assembly Competency
Sixty milliliters of GLE-treated and washed vegetative cells
(1 × 106 cells mL1)
was divided into two equal volumes and pelleted; one half was resuspended in TAP medium and the other half in TAP medium plus 50 mM putrescine. After 3 h, each sample was pelleted and
extracted with 3 mL of 2 M NaClO4.
The supernatant from each sample was saved and lyophilized as well.
These lyophilates were resuspended in 3 mL of 2 M
NaClO4 and, along with the
sodium-perchlorate-solubilized components, dialyzed overnight against
water. This dialysis leads to wall crystal formation when the protein
concentration is greater than or equal to 1 mg
mL1 (Goodenough et al., 1986). Wall crystals
were visible in the dialysis tubing, pelleted out of solution, and
subjected to SDS-PAGE. The gel was stained using the periodic
acid-Schiff reagent as described in Monk et al. (1983).
In Vitro Nucleation Assay
A 100-mL culture of vegetative cells was divided into three
samples (A, B, and C). The cells in samples B and C were GLE treated and washed as usual. Sample B was resuspended in TAP medium and C was
resuspended in TAP medium plus 50 mM putrescine. After
3 h all samples were fixed in paraformaldehyde (1% [w/v]
in 15 mM HEPES, pH 7.0) for 1 h, incubated for 2 h in 25 mM Gly, pH 7.0, washed, and resuspended in 2 M sodium perchlorate. The extracted cells were pelleted,
washed, and mixed with 50,000 cpm of labeled outer wall glycoproteins
solubilized in 2 M sodium perchlorate; the labeled proteins
were obtained by perchlorate-extracting vegetative cells regenerating
in the presence of 14C-putrescine. Additional
unlabeled outer wall glycoproteins (0.2 mg mL1)
were added to increase the protein concentration. The three samples
were dialyzed overnight and then transferred to a test tube for five
successive washes in water. Each sample was then perchlorate extracted
and 100 µL of the soluble fraction was added to 1 mL of scintillation
cocktail and assayed in the scintillation counter.
Electron Microscopy
Quick-freeze, deep-etch electron microscopy of vegetative cells
regenerated for 3 h in the presence or absence of 50 mM putrescine was performed as described in Heuser (1980).
Examination of Cross-Linking on the Plasma Membrane
To label RVC, the same protocol as described in "In Vivo
Incorporation of 14-C-Putrescine" was followed.
One-milliliter aliquots of cells were removed at 5-min intervals. The
cells were pelleted and extracted with sodium perchlorate to remove
soluble wall glycoproteins. The extracted cells were pelleted
again and lysed with 5 mL of 0.006 digitonin in 5 mM KPO4 buffer, pH 7.2, and 1 M KCl.
Cell debris and the chloroplast were removed by centrifugation
for 15 min at 8,000g. Microsomal preparations of the
supernatant were isolated by ultracentrifugation for 1.5 h at
100,000g in a rotor (model 50.3Ti, Beckman Instruments, Fullerton, CA). The pellets were resuspended in 50 µL of loading buffer and subjected to SDS-PAGE on a 7.5% (w/v) running
gel with a 3.5% (w/v) stacker. The gel was fixed, dried, and
exposed to x-ray film as described above.
Recovery of Putrescine from Labeled Protein Conjugates
To 1 mL of a crude cell-free TGase preparation (described above),
20 µCi of 14C-putrescine and 1% (w/v)
dimethylcasein were added. The same amount of label was also added to
100 mL of RVC (prepared as outlined above). Both samples were incubated
for 30 min at room temperature, and the cells were removed from the
second sample by centrifugation for 10 min at 5,000g in a
rotor. The proteins from both samples were precipitated with ice-cold
10% (w/v) TCA, 50% (w/v) acetone, and 10 mM unlabeled putrescine, and then hydrolyzed in 6 N HCl at 110°C under nitrogen for 48 h.
The HCl was removed under vacuum and the remaining sample washed twice
with methanol and finally dissolved in 1 mL of phosphate buffer (30 mM, pH 7.6). The respective solutions were run on
a phosphocellulose column (1 × 6 cm) equilibrated with the
phosphate buffer. The elution was according to Signori et al. (1991) by
first washing with 20 mL of phosphate buffer, then with 20 mL of 100 mM borate buffer (pH 8.1) containing 25 mM NaCl, and finally a 100-mL gradient to a limit
buffer of 200 mM borate and 600 mM NaCl, pH 8.6. Two-milliliter fractions were collected and assayed for radioactivity in a scintillation counter. The
column was calibrated by following the elution of
14C-putrescine.
Immunoprecipitation with Anti-TGase Serum
Forty milliliters of RVC or DZ was harvested at the time of
maximal TGase activity. The cell-free supernatants from these were used
in two experiments. First, a 1-mL aliquot from each supernatant was
mixed with 5 µL of serum against guinea pig tissue TGase (Aeschlimann
et al., 1993), incubated 1 h at 4°C, and the resulting IgG
complex was adsorbed onto 50 µL of protein A-agarose (Sigma
Chemicals, St. Louis) during a 1-h room temperature incubation. After
pelleting the agarose (10 min, 12,000 rpm), 40 µL of supernatant and
40 µL of the pellet (resuspended in 1 mL of TGase assay buffer) were
tested for TGase activity using the putrescine method. Second, the
remainder of each cell-free supernatant was lyophilized and resuspended
in 5 mL of distilled water. Twenty-five-microliter samples of this
resuspension were mixed with an equal volume of Laemmli dissociation
buffer (0.715 M 
-mercaptoethanol, 8 M urea, 0.36 M Suc, 4% [w/v] SDS, 0.04% [w/v]
pyronin y, and 100 mM
Na2CO3) and subjected to
SDS-PAGE. The gel was either silver-stained or electroblotted onto
nitrocellulose. The immunoblot was developed using anti-TGase serum as
the primary antibody and anti-rabbit IgG linked to alkaline phosphatase
(Sigma Chemicals) as the secondary antibody. Color was detected using
nitroblue tetrazolium chloride and 5 bromo-4-chloro-3-indolyl-phosphate
toluidine salt. For the competition experiments 2.5 or 10 µg of
purified TGase from guinea pig (a gift of M. Paulsson, University of
Cologne) was incubated with the primary antibody for 30 min prior to
adding it to the blot.
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RESULTS |
Inhibition of Cell Wall Insolubilization with Primary Amines
The effect of exogenously added compounds on cell wall
insolubilization can be measured by a simple spectrophotometric assay for detergent sensitivity (Waffenschmidt et al., 1993). Mature V-walls
and Z-walls rendered cells detergent insensitive, and a plot of percent
cell lysis versus time reflects the progress of wall insolubilization.
During the course of our work on peroxidase-mediated cross-linking, we
discovered that the addition of Gly ethyl ester delayed
insolubilization of both V-walls and Z-walls. Since transglutaminases, which have been shown to generate covalent intermolecular cross-links in animal systems, are competitively inhibited by primary amines such
as Gly ethyl ester (Lorand et al., 1979), we tested several other
primary amines (cadaverine, putrescine, spermine, and spermidine) in
cultures of RVC or DZ. Cadaverine, spermine, and spermidine all
inhibited or delayed insolubilization of both wall types, but all three
ultimately led to cell death and thus were not utilized further (data
not shown). Putrescine, like Gly ethyl ester, did not cause any obvious
cell lethality as determined by visual inspection under the light microscope.
Figure 1 shows detergent-sensitivity
plots for RVC and DZ in medium containing various concentrations of
putrescine. Prior to time 0 in Figure 1A, the vegetative cells were
incubated for 1 h in GLE to remove the V-wall. At the end of this
incubation, the cells were washed free of GLE (time 0) and began to
synchronously assemble a new V-wall. As indicated by the control curve
in this panel, a detergent-resistant V-wall is normally completed
within 2 h (Robinson and Schlösser, 1978; Waffenschmidt et
al., 1993). When vegetative protoplasts were resuspended in media with
increasing concentrations of putrescine, a proportional decrease in the
number of cells completing the insolubilization program was observed. Washing the putrescine away from the treated cells did not reinitiate the wall-insolubilization program (data not shown).
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Figure 1.
Effect of putrescine on cell wall
insolubilization. A, Vegetative cells were treated with GLE for 1 h and the resultant protoplasts were pelleted, washed, and resuspended
in fresh medium (time 0) without (control) or with various
concentrations of putrescine. Aliquots were taken at 30-min intervals
and analyzed for detergent sensitivity by mixing with an equal volume
of 0.2% (w/v) Nonidet P-40 and pelleting the cells and cellular
debris in a microfuge. The A440 of the
supernatant was read to quantitate chlorophyll release from lysed
cells, and the highest absorbance reading was set to 100% cell lysis.
The other values were converted to percentages accordingly. B, Equal
numbers of gametes of both mating types were mixed (time 0), allowed to
mate for 30 min, then pelleted, washed, and resuspended in fresh medium
lacking (control) or containing various concentrations of putrescine.
At 30-min intervals, aliquots were removed and analyzed for detergent
sensitivity as above. Error bars at each time point indicate the
results from three independent assays.  , Control;  , 5 mM putrescine ;  , 10 mM putrescine; +, 50 mM putrescine.
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In Figure 1B, gametes of the opposite mating type were mixed together
at time 0, and within 30 min the culture contained synchronous DZ. The
early zygotes were wall-less and assembled a new detergent-resistant Z-wall over the next 3 h (control curve). The DZ were pelleted at
30 min and resuspended in increasing concentrations of putrescine. As
seen in the RVC, greater concentrations of putrescine resulted in
proportionally fewer cells becoming insolubilized. The concentrations of putrescine found to be most effective at blocking the
insolubilization of both walls in C. reinhardtii were
directly comparable to the levels of putrescine required to inhibit the
TGase activity in assembly of the sea urchin fertilization envelope
(Battaglia and Shapiro, 1988).
In Vitro TGase Assays
With suggestive evidence that TGase activity might be involved in
the wall insolubilization program, our next step was to perform in
vitro TGase assays. To determine whether the culture medium of RVC or
DZ contains TGase activity, small aliquots were taken over time from a
culture of RVC or DZ, the cells were pelleted, and the supernatant
assayed for TGase activity in vitro by measuring 14C-putrescine incorporation into dimethyl casein
(Battaglia and Shapiro, 1988). At each time point, aliquots were also
removed to test for detergent sensitivity,
H2O2 concentration, and
peroxidase activity. The results for both RVC and DZ are shown in
Figure 2. Figure 2A documents a sharp
peak of TGase activity around 60 min after GLE removal from
vegetative cells. The peak of TGase activity, unlike the peak of
H2O2 secretion and
peroxidase activity (indicated in the figure by the arrow), preceded
the time of V-wall insolubilization. Likewise, Figure 2B shows a peak
of TGase activity in DZ that also precedes the time of Z-wall
insolubilization and the peak of
H2O2 and peroxidase
activity (arrow). Figure 2C shows the incorporation time courses for a
crude cell-free preparation of TGase isolated from RVC at the time of
maximum enzyme activity, as determined from the data presented in
Figure 2A. Curve A represents incorporation values from a crude
preparation that was twice as concentrated as the sample used to
generate curve B. The two curves clearly demonstrate enzymic behavior.
The activity for the V-wall TGase was 78.4 pmol incorporation
h1 mg1 and for the
Z-wall enzyme, 90 pmol h1
mg1. The Km
values were 1.8 mM (RVC) and 2.0 mM (DZ). Vmax
for the RVC enzyme was 180 pmol h1
mg1 casein1 and for the
DZ enzyme 185 pmol h1
mg1 casein1. These
values for Km and
Vmax are somewhat misleading because the crude enzyme preparation we assayed contains natural substrates for
the TGase (wall proteins secreted into the media) that compete with the
substrate (casein) we were using to measure activity. Thus, truly
meaningful characterization of the C. reinhardtii TGase
enzyme activities will have to be done with more purified enzyme
preparations.
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Figure 2.
TGase, peroxidase, and
H2O2 levels during cell wall formation. A,
Vegetative cells were treated with GLE for 1 h, washed, and
resuspended in fresh medium (time 0). Aliquots were removed at 20-min
intervals and analyzed for detergent sensitivity (%), TGase (cpm),
peroxidase activity, and H2O2 production. TGase
activity was assayed by measuring 14C-putrescine
incorporation into dimethyl casein. Peroxidase activity and
H2O2 accumulation were determined
spectrophotometrically using vanillin and KI, respectively, as
substrates. Only the time point (arrow) of maximum peroxidase activity
and highest H2O2 level is indicated in this
figure. , % Cell wall lysis; , counts per minute. B, Equal
numbers of gametes of each mating type were mixed (time 0) and aliquots
were taken at 20-min intervals to assay for detergent sensitivity (%),
TGase (cpm), peroxidase activity, and H2O2
accumulation. Again, only the time point of peak peroxidase activity
and H2O2 accumulation is indicated (arrow).
, % Cell wall lysis; , counts per minute. C, Incorporation time
courses were derived from aliquots of supernatants collected from RVC
at the time of maximum TGase activity. Curve A ( ) presents the
incorporation for an undiluted aliquot of supernatant, while curve B
( ) shows the incorporation for a sample that is diluted 1:1 with
buffer. Error bars represent the results from three independent assays.
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Response of C. reinhardtii TGase to Various
Inhibitors
Using the supernatants from aliquots taken at the times of
maximum TGase activity in RVC and DZ (as determined by
14C-putrescine incorporation), we tested this
enyzme activity for Ca2+ dependence and
sensitivity to thiol inhibitors and Zn2+ with the
hydroxamate-formation assay of Folk and Cole (1966). The hydroxamate
assay offers direct evidence, independent of the 14C-putrescine/dimethyl casein assay, of a
C. reinhardtii TGase activity capable of cross-linking
hydroxylamine (amine donor) to a
carbobenzoxy-L-glutaminylglycine (substrate),
forming an iron-hydroxamate complex. The rate of hydroxamate formation
in RVC was 30.8 pmol h1
mg1 and in DZ was 45.1 pmol
h1 mg1.
While the vast majority of animal TGases show a strong
Ca2+ dependence, the plant TGases examined to
date do not (Falcone et al., 1993; Serafini-Fracassini et al., 1995).
Animal TGases are also inhibited by low levels (<50 µM)
of Zn2+; the effect of Zn2+
on plant TGases has not been studied. As shown in Table
I, C. reinhardtii TGases, like
those of higher plants, do not appear to have a strict requirement for
Ca2+ and are only slightly inhibited by 1 mM Zn2+. The lack of
inhibition by Zn2+ is not surprising since the
growth medium for both vegetative cells and zygotes normally
contains 76 µM Zn2+
(Harris, 1989). The thiol inhibitors N-ethylmaleimide and
p-chloromercuribenzoate completely blocked both C. reinhardtii enzymes, which is typical for TGases.
In Vivo TGase Assays
Given the presence of TGase activity in culture
supernatants, we next looked for TGase activity associated with
wall-regenerating cells. For these experiments, RVC or DZ were
resuspended in medium containing 14C-putrescine.
The concentration of putrescine in these cultures (0.22 µM) was far below the inhibiting levels shown in Figure 1
because in this case we were testing whether the labeled putrescine could be cross-linked by TGase onto cell surface substrates (e.g. wall
proteins). At each time point, an aliquot was removed and the cells
were pelleted, washed, and assayed for
14C-putrescine incorporation. Another aliquot was
taken to determine detergent sensitivity. Figure
3 shows the two curves (incorporation and
detergent sensitivity) for RVC (Fig. 3A) and DZ (Fig. 3B). The
incorporation values are given as percentages of the counts per minute
originally added to the medium. Both cell types were labeled with
14C-putrescine, and the maximum rates of
incorporation occurred within the time of highest TGase activity, as
determined in vitro with the culture supernatants (see Fig. 2).
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Figure 3.
14C-Putrescine incorporation during
cell wall formation. A, Vegetative cells were treated with GLE for
1 h, washed, and resuspended in fresh medium containing a
non-TGase-inhibiting concentration of 14C-putrescine (time
0). Every 20 min aliquots were taken and the cells were either tested
for detergent sensitivity, as described in Figure 1, or pelleted,
washed, and assayed for radioactivity. Incorporation is presented as a
percentage of the original quantity of 14C-putrescine added
to the medium. B, Gametes of both mating types were mixed in equal
numbers. After 30 min of mating, a non-inhibiting quantity of
14C-putrescine was added to the medium. Samples were taken
every 20 min and the cells were tested for detergent sensitivity and
incorporation of radioactivity as above. Error bars represent the
results from three independent assays. , % Cell wall lysis; , % incorporation over 20 min.
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Identification of Potential TGase Substrates
The in vitro assays indicated that the TGases are extracellular,
suggesting that the in vivo labeling shown in Figure 3 may have
occurred on cell wall substrates. We took the remaining vegetative cells from the in vivo labeling experiment described above and treated
them with sodium perchlorate. This salt extraction solubilizes glycoproteins of the outer layers of the V-wall, leaving behind the
glycoproteins of the insoluble inner layer (Hills et al., 1975). As
seen in Table II (which presents data
averaged over six separate extractions), when this salt-soluble
fraction was assayed for radioactivity, 53% of the total counts per
minute incorporated into the RVC were released. Next, the
salt-extracted cells were incubated with GLE, which cleaves off the
insoluble inner V-wall layer (Goodenough and Heuser, 1985; Imam and
Snell, 1988; Waffenschmidt et al., 1988). This treatment released
another 39% of the total counts per minute. This leaves 8% of the
counts still on the cell surface, possibly attached to the radiating wall fibers left behind after GLE cleavage (Goodenough and Heuser, 1985). Since the Z-wall is not salt-soluble or susceptible to GLE, we
used acidified chlorite to cleave the phenolic cross-links within the
Z-wall (Jaenicke et al., 1987; Biggs and Fry, 1990; Waffenschmidt et
al., 1993). This released 86% of the counts incorporated into DZ
(Table II).
Since 53% of the counts incorporated into RVC are found within the
well-studied outer layers of the V-wall (see Woessner and Goodenough,
1994), we wished to determine which salt-soluble components are
substrates for TGase. Vegetative cells that had regenerated for 3 h in the presence of non-inhibiting concentrations of
14C-putrescine were pelleted and extracted with
sodium perchlorate, and the supernatant was lyophilized. Samples of
both the salt extract and the supernatant were analyzed by SDS-PAGE and
autoradiography. As shown in Figure 4,
most of the previously identified salt-soluble glycoproteins
(Goodenough et al., 1986) were labeled and present in both the extract
(lane A) and the supernatant (lane B). There were, however, other
polypeptides in the supernatant that were not labeled (lane C), which
is consistent with the finding that TGases recognize only certain
protein-bound Gln as acceptor substrates (Folk and Cole, 1966; Dutton
and Singer, 1975).
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Figure 4.
Identification of
14C-putrescine-labeled wall proteins. Vegetative cells were
treated with GLE for 1 h, washed, and resuspended in fresh medium
with a non-inhibiting quantity of 14C-putrescine. After
3 h, the cells were pelleted and extracted with
NaClO4. Both the salt-extracted proteins and the medium
from which the cells were pelleted were dialyzed against distilled
water, TCA precipitated, and analyzed by SDS-PAGE and autoradiography.
Lane A shows the autoradiograph of the NaClO4-extracted
proteins, and lane B shows the labeled proteins in the supernatant.
Lane C shows an equal loading of the supernatant sample that has been
silver-stained to reveal all of the proteins actually present. The
outer wall salt-soluble glycoproteins (GP) that have been previously
identified and characterized (Goodenough et al., 1986) are indicated
adjacent to lane A.
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Involvement of TGase in V-Wall Assembly
Having identified outer wall glycoproteins as potential TGase
substrates, our next goal was to define the role of TGase in the
assembly of these components. One well-documented trait of the
salt-soluble glycoproteins is that, upon dialysis of the salt, they can
self-assemble into a crystalline array similar to that found in muro
(Hills et al., 1975; Goodenough et al., 1986). We first examined
whether inhibition of TGase had any effect on self-assembly. RVC were
grown in the presence or absence of 50 mM putrescine and
after 3 h the cells were pelleted, resuspended, and sodium perchlorate extracted; the supernatants were lyophilized and
resuspended in sodium perchlorate. All four samples were then dialyzed
to remove the salt and allow self-assembly into wall crystals.
Crystals, observed in all four samples, were pelleted and analyzed by
SDS-PAGE as shown in Figure 5.
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Figure 5.
Putrescine's effects on self-assembly of outer
wall proteins. GLE-treated vegetative cells were washed and resuspended
in medium with or without inhibiting levels of putrescine. After 3 h, the cells in both cultures were pelleted and the two supernatants
were lyophilized and resuspended in sodium perchlorate, while the cells
were sodium perchlorate extracted. All four samples, the supernatants
and the two salt extracts, were dialyzed against distilled water to
remove the chaotrope and initiate self-assembly of outer wall proteins
into crystals. The crystals of all four samples were analyzed by
SDS-PAGE and the gel was stained with periodic acid-Schiff reagent.
Lane A is a standard of purified outer wall crystals. The next two
lanes are the crystals from the salt extracts of control (lane B) and
putrescine-inhibited (lane C) cells. The last two lanes show the
crystals from the control (lane D) and putrescine-inhibited (lane E)
supernatants. The previously identified outer wall glycoproteins are
indicated along the outside of lanes A and E. Periodic acid
Schiff-staining of GP1.5 is concentration dependent and there is too
little of this glycoprotein (GP) in the standards (lane A) for it to be
visibly stained.
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Only wall crystals were loaded on the gel, so everything on the gel was
assembly competent and the material derived from equal numbers of
cells. The salt-extracted components of RVC in the absence of
putrescine displayed all of the glycoproteins in abundance (lane B),
whereas in the presence of inhibiting levels of putrescine, relatively
few glycoproteins were extracted (lane C). Reciprocally, few outer wall
glycoproteins were present in the supernatant of control RVC without
putrescine (i.e. the majority had assembled onto the cell surface, lane
D), while the supernatant from RVC in putrescine contained abundant
outer-wall glycoproteins (lane E). Inhibition of TGase therefore does
not block the potential for self-assembly, but it appears to block the
ability of outer wall glycoproteins to assemble onto the cell.
The assembly of outer wall glycoproteins onto the cells can be studied
in vitro as well (Hills et al., 1975; Adair et al., 1987). When
"shells" (vegetative cells that have been perchlorate extracted and
concomitantly killed) are dialyzed together with the solubilized
glycoproteins, the glycoproteins assemble onto the shells, reforming a
normal V-wall (Adair et al., 1987). The experiment documented in Figure
6 compared shells from normal vegetative
cells and RVC in the absence or presence of inhibiting concentrations
of putrescine. Each type of shell was dialyzed with
perchlorate-solubilized glycoproteins labeled with
14C-putrescine. After assembly, the shells were
washed, re-extracted with sodium perchlorate, and the solubilized
fractions counted for radioactivity. Shells from untreated vegetative
cells and from RVC unexposed to putrescine nucleated assembly of five
to six times the amount of labeled glycoproteins, as did shells from RVC in the presence of putrescine (Fig. 6). This provides quantitative support for the hypothesis that TGase is involved in rendering the
cells competent to nucleate the assembly of outer wall glycoproteins.
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Figure 6.
Quantitative determination of putrescine's
effects on cell wall assembly. A culture of vegetative cells was
divided into three equal volumes (A, B, and C). Volume A was the
non-GLE-treated control, while volumes B and C were GLE-treated,
washed, and allowed to regenerate for 3 h in medium lacking
putrescine (B) or containing inhibiting levels of putrescine (C). After
3 h all samples were sodium-perchlorate-extracted, removing the
soluble outer wall proteins and leaving behind extracted cells. These
cells were pelleted, washed, and mixed with radioactively labeled
assembly-competent outer wall proteins in sodium perchlorate. The three
samples were dialyzed overnight against water to initiate assembly onto
the cell surface. After extensive washing to remove unassembled
proteins, the three samples were re-extracted with sodium perchlorate
and the radioactivity of the solubilized components was determined.
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Direct visual proof that TGase activity is essential to wall assembly
in RVC was provided by the electron micrographs shown in Figure
7. These are images of the wall on RVC
that have been incubated in medium with or without inhibiting
concentrations of putrescine. After regeneration in the absence of
putrescine (Fig. 7A), the insoluble V-wall had all of the layers
described in detail by Goodenough and Heuser (1985): a recognizable
crystalline layer (W6), globular components (W4), and a highly
cross-linked inner layer (W2) all interwoven on long W1/W7 fibers
radiating out from the plasma membrane. In contrast, the wall found on
RVC incubated in putrescine displayed only radiating short fibers (Fig.
7B).
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Figure 7.
Electron micrographs of cell walls from control
(A) and putrescine-treated (B) RVC quick-frozen 3 h after GLE
treatment. P, Plasma membrane; W1, W2, W6, and W7, layers of the
wall.
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Examination of Membrane-Bound Protein Cross-Linking
The electron micrograph images, in accordance with our other
studies, suggested that TGase is involved in an early stage of wall
assembly. Goodenough and Heuser (1985), after examining many electron
micrographs of vegetative wall growth, proposed that the earliest
visible event of V-wall formation is the appearance of long fibers
(W1/W7) radiating from the plasma membrane. To look for TGase-mediated
cross-linking of membrane-bound proteins, we set up an experiment
identical to in vivo labeling, resuspending 100 mL of RVC in
14C-putrescine. At 5-min intervals we collected
aliquots of cells. Each sample was sodium perchlorate extracted to
remove all soluble wall proteins. Then the cells were lysed with
digitonin (solubilizing the plasma membrane), subjected to osmotic
shock, and a microsomal fraction was prepared by ultracentrifugation.
Each microsomal pellet was resuspended in loading buffer and subjected
to SDS-PAGE. Figure 8 presents the
autoradiograph of three pellets from successive time points. No labeled
band is visible by SDS-PAGE until 35 min into the wall formation (lane
1). This molecule is over 200 kD; the largest molecular mass
standard on the gel was 193 kD. Within the next 5 min this band
disappeared and was replaced by a diffuse band that migrated in the
middle of the stacking gel (lane 2). By 45 min, the labeled band had
become so large it could not even run through the stacking gel (lane
3). A signal was found at this position in subsequent time points as
well (data not shown). Our interpretation of these results is that we
were seeing a plasma membrane-bound protein being rapidly cross-linked
by TGase.
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Figure 8.
Cross-linking of membrane-bound proteins.
GLE-treated vegetative cells were washed and resuspended in media with
14C-putrescine. Samples were collected every 5 min and
extracted with sodium perchlorate to remove soluble wall proteins. The
plasma membrane of the cells was solubilized in digitonin, subjected to
osmotic shock, and a microsomal fraction was isolated by
ultracentrifugation. Each microsomal pellet was resuspended in loading
buffer and analyzed by SDS-PAGE and autoradiography. Lane 1 shows the
sample collected at 35 min, lane 2 is the 40-min sample, and lane 3 is
from 45 min. Only the position of the largest molecular mass standard,
193 kD, is indicated to the left of lane 1. The brackets on the right
side of lane 3 delineate the boundaries of the stacking gel. Bands
located in the stacker are typically diffuse.
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Characterization of Incorporated 14C-Putrescine
The results of the hydroxamate formation assay can only be
ascribed to the presence of an extracellular TGase activity in C. reinhardtii. However, it was important to document that the putrescine inhibition and labeling results were not due to amine oxidase activity. Diamine oxidases, enzymes commonly found in plant
cells, can oxidize putrescine to mono- and di-aldehydes that
indiscriminately inhibit many biological processes, and such aldehydes
could also potentially cross-link proteins through non-enyzmatic reactions involving Schiff's base intermediates. If amine oxidases were involved, then the incorporated
14C-putrescine in the in vitro and in vivo
labeling experiments would be oxidized to an aldehyde. Therefore, we
set up two experiments to characterize the incorporated putrescine
molecules. First, 20 µCi of 14C-putrescine was
added to our standard in vitro assay using dimethylcasein and cell-free
supernatant isolated from RVC at the time of maximal TGase activity.
Following a 30-min incubation, the proteins were precipitated out of
solution using TCA in the presence of excess cold putrescine. Second,
the same amount of label was added to 100 mL of RVC in our standard in
vivo assay. After 30 min, the cells were pelleted out of the medium and
perchlorate extracted. These salt-soluble glycoproteins and the
TCA-precipitated proteins from the in vitro assay were hydrolyzed in 6 N HCl and assayed for radioactivity; 86% of the
incorporated radioactivity was recovered for the in vitro experiment
and 87% for the in vivo experiment. Both hydrolysates were dried under
nitrogen, resuspended in phosphate buffer, loaded onto a
phosphocellulose column, and eluted fractions assayed for
radioactivity. The column was calibrated using authentic 14C-putrescine. As shown in Figure
9, the hydrolyzed label from both the in
vitro and in vivo experiments eluted in the same fractions as the
control, proving that the labeled putrescine was incorporated without
metabolic conversion.
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Figure 9.
Characterization of incorporated
14C-putrescine. The 14C-putrescine-labeled
dimethyl casein substrate from a standard in vitro TGase assay with RVC
( ) and the NaClO4-solubilized outer wall proteins
labeled with 14C-putrescine in an in vivo TGase assay ()
were hydrolyzed in 6 N HCl. The hydrolysates were loaded
onto a phosphocellulose column and the eluted fractions were tested for
radioactivity. The control curve is the elution of authentic
14C-putrescine. Error bars indicate the results from three
independent assays.  , Control.
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Immunoprecipitation with Antibodies to Mammalian TGase
Confident that we were analyzing a bona fide TGase, we tested for
antigenic conservation between this algal enzyme and a mammalian counterpart using polyclonal antibodies to guinea pig tissue TGase (Aeschlimann et al., 1993). The proteins secreted into the medium of
RVC and DZ at the times of maximal TGase activity were separated by
SDS-PAGE, blotted to nitrocellulose, and probed with the anti-TGase serum. Figure 10, A and B, indicate
that the serum cross-reacts with a 72-kD band found in the supernatant
of both RVC and DZ. To rule out that this cross-reactivity is due to a
nonspecific interaction, we reprobed the western blot using antiserum
that had been preincubated with increasing amounts of purified guinea pig tissue TGase. Figure 10C shows that the 72-kD signal was competed away in a concentration-dependent manner. Lastly, we used the anti-TGase serum in immunoprecipitations of the cell-free supernatants of both RVC and DZ isolated at the time points of maximum TGase activity. The resulting antigen/antibody complexes were adsorbed onto
protein A-agarose and pelleted out of solution. Following this
immunoprecipitation, TGase activity was detected only in the pelleted
complexes and not in the treated supernatants (data not shown).
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Figure 10.
Antigenic conservation between the cell wall
TGase of C. reinhardtii and the tissue TGase of guinea
pig. A, The growth media was collected from RVC (left lane) and DZ
(right lane) at the times of maximal TGase activity as determined in
Figure 2. The secreted proteins were TCA-precipitated out of the
medium, separated by SDS-PAGE, and the silver-stained gel is presented.
The positions of the molecular mass markers (193, 112, 86, 70, 57, and 39.5 kD) are indicated by arrowheads. B, Two lanes
identical to those in A were blotted onto nitrocellulose and probed
with antiserum against guinea pig TGase. C, Three samples of the RVC
secreted proteins were isolated, electrophoresed, and blotted as
described above. The left blot was probed with only anti-TGase serum as
in B. The next two blots were probed with the same antiserum after
preincubation with either 2.5 (middle) or 10 µg (right) of purified
guinea pig TGase.
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DISCUSSION |
Delineating the assembly program for a plant extracellular matrix
is a challenging task, yet we have made considerable progress toward
this goal in our studies of cell wall protein interactions in C. reinhardtii. As noted in the introduction, these algal cell walls
lack the complex diversity of matrix molecules found in higher plants.
Nevertheless, matrix assembly in C. reinhardtii is not an
elementary process. To date, three distinct assembly events have been
identified: the early TGase-catalyzed cross-linking described in this
paper, the self-assembly of glycoproteins into a crystalline array
(Hills et al., 1975; Goodenough et al., 1986), and an oxidative
cross-linking reaction (Waffenschmidt et al., 1993). We propose, as
detailed below, that all three events must transpire sequentially to
achieve insolubilization of the V-wall.
Although we have demonstrated the existence of an extracellular TGase
in C. reinhardtii whose activity is essential to cell wall
formation, the role of this enzyme in early wall formation remains
unclear in both RVC and DZ. Nevertheless, the large body of literature
on the V-wall has facilitated a more detailed examination of matrix
assembly in RVC than is currently possible in DZ. Following a crude but
accepted method, we identified most of the soluble outer wall
glycoproteins in RVC as substrates for the TGase when putrescine is
added. Since these would no longer be soluble if indeed they were
covalently cross-linked to each other, our interpretation is that these
glycoproteins carry potential sites for covalent attachment of
putrescine by TGase, but these sites are not the preferred substrate
and are normally inaccessible, unavailable, and/or unused in muro.
In contrast, the labeling of inner-wall components in vivo is likely to
be significant because other data indicate that the TGase activity
appears to be responsible, directly or indirectly, for nucleating the
assembly of the wall. EM images confirm the lack of both the soluble W6
layer and the insoluble W2 layer on the cell surface of RVC incubated
with inhibiting levels of putrescine, and a previous EM study of
wild-type RVC indicated that the assembly of W6 precedes that of the W2
layer (Goodenough and Heuser, 1985). The EM image of
putrescine-inhibited RVC also shows only small fibers radiating out
from the plasma membrane. These short fibers might be precursors of the
much longer W1/W7 molecules. These data, together with the results of
the SDS-PAGE indicating cross-linking, suggest that the sequence of
matrix assembly includes an early TGase cross-linking of
yet-to-be-identified membrane-bound wall molecules (possibly comprising
the W1/W7 radial fibers). This cross-linking somehow facilitates and/or
initiates the self-assembly of the W6 crystalline layer. Assembly of
the inner W2 layer components rapidly follows and, finally, a
peroxidase-mediated oxidative cross-linking of these proteins leads to
full insolubilization of the wall.
When we presented our previous work on peroxidase-mediated
cross-linking in C. reinhardtii walls (Waffenschmidt et al.,
1993), we proposed, based on the model for the hardening of the sea
urchin vitelline layer (Shapiro, 1991), that the early assembly of the crystalline layer of salt-extractable W6 and W4 components might serve
to organize and position the underlying W2 components prior to the
ensuing oxidative cross-linking. Now we can carry the analogy one step
further. In the sea urchin, an early TGase-catalyzed event leads to the
formation of a non-cross-linked "soft envelope" that, in turn,
organizes underlying proteins prior to the oxidative cross-linking
responsible for the hardened fertilization membrane (Battaglia and
Shapiro, 1988). If this sea urchin TGase is inhibited, then a
fertilization envelope is never formed. Likewise, there is an
extracellular TGase in C. reinhardtii RVC whose inhibition prevents assembly of the "soft envelope" of W4 and W6 proteins and,
consequently, insolubilization of the V-wall.
This commonality in matrix insolubilization programs between C. reinhardtii and sea urchin could be entirely fortuitous, but TGases participating in matrix hardening have been found in organisms as distant as bacteria and humans (Rice et al., 1993). Indeed, previous
studies have even used comparisons of TGase amino acid sequences to
develop phylogenetic trees (Tokunaga et al., 1993). One of the most
conserved domains of the TGase enzyme at the amino acid level
corresponds to the active site, and DNA probes from this region have
been used to clone TGases from different organisms (Floyd and Jetten,
1989; Kim et al., 1991). Perhaps the antigenicity of this domain is the
reason why antibodies to animal TGases cross-react with homologs in
filarial parasites (Mehta et al., 1992), higher plants (Del Duca et
al., 1994, 1997), Dunaliella salina
(Serafini-Fracassini et al., 1995), and C. reinhardtii. To
date, only intracellular TGases have been reported in higher plants
(Serafini-Fracassini et al., 1995; Del Duca et al., 1997; Hou and Lin,
1997). However, high-molecular-mass substrates for TGase in non-green
tissue of Helianthus tuberosus were identified by Falcone et
al. (1993), and these could correspond to cell wall structural
proteins. The implication is that there could be extracellular TGases
integral to cell wall formation in higher plants as well, as has been
speculated (Serafini-Fracassini et al., 1995).
We thank Dr. J.E. Heuser for the EM images, Dr. M. Paulsson for
the anti-TGase serum and samples of guinea pig TGase, and Dr. U. Goodenough for guidance and scientific insight during preparation of
this manuscript. The excellent technical assistance of Eva Glees is
gratefully acknowledged.
Received February 24, 1999; accepted July 6, 1999.
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ABSTRACT
INTRODUCTION