Plant Celt Physiol. 40(8): 773-783 (1999)
JSPP © 1999
Sucrose-Controlled Transport and Turnover of er-Amylase in Rice (Oryza
sativa L.) Cells
Toshiaki Mitsui 1 ' 4 , Tadeusz Loboda 1 5 , Ikuko Kamimura1, Hidetaka Hori', Kimiko Itoh 2 and
Shin-ichiro Mitsunaga3
1
2
3
Laboratories of Molecular Life Sciences, Graduate School of Science and Technology, Niigata University, Niigata, 950-2181 Japan
Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Niigata, 950-2181 Japan
Joetsu University of Education, Joetsu, Niigata, 943-8512 Japan
We studied the sucrose-controlled intracellular transport and turnover of a-amylase molecules in suspensioncultured cells of rice {Oryza sativa L.) employing pulse labeling techniques with [3SS] amino acids. The secretion of
two classes of a-amylase isoforms, a-amylase 1-1 (encoded
by RAmylA) and II-4 (encoded by RAmy3D) was differentially controlled by sucrose. In rice cells labeled with
[3SS] amino acids under different sucrose-supplemented conditions, sucrose preferentially prevented the extracellular
liberation of [35S]-labeled a-amylase 11-4 molecules from
rice cells at around 2 mM, whereas the de novo protein
synthesis still occurred at this concentration. Pulse-chase
experiments showed that sucrose regulates the intracellular
transport of [35S]a-amylase II-4 molecules and stimulates
the protein turnover. However, cycloheximide, a protein
synthesis inhibitor, was induced the extracellular liberation
and reduced the turnover of [3SS]a-amyIase II-4 molecules
in the presence of sucrose. These results strongly suggested
that newly synthesized sucrose-induced proteins are involved in the posttranslational regulation on sucrose-controlled a-amylase secretion in rice cells.
Key words: a-Amylase isoform — Intracellular transport
— Protein turnover — Rice cells — Secretory protein —
Sucrose.
Multiple isoforms of a-amylase are present in cereal
seeds. These isoforms have been shown to be encoded by a
multiple gene family (Huang et al. 1992b, Mitsui and Itoh
1997). Many genes of cereal a-amylase have been cloned
and sequenced (Rogers and Milliman 1983, Chandler et al.
1984, Huang et al. 1984, Lazarus et al. 1985, Rogers 1985,
Baulcombe et al. 1987, Knox et al. 1987, Whittier et al.
Abbreviations: ER, endoplasmic reticulum; MS medium,
Murashige-Skoog medium; SD, standard deviation; TCA, trichloroacetic acid.
4
To whom correspondence should be addressed (fax, -4-81-25262-6641; e-mail, [email protected]).
5
Invited research fellow of the Japan Society for the Promotion
of Science. Present address: Plant Physiology Department, Warsaw Agricultural University, Rakowiecka Str. 26/30 02-528 Warsaw, Poland.
773
1987, Huttly et al. 1988, Khusheed and Rogers 1988, OuLee et al. 1988, Rahmatullah et al. 1989, Huang et al.
1990a, b, 1992a, O'Neill et al. 1990, Jacobsen and Close
1991, Sutliff et al. 1991, Kim and Wu 1992, Rushton et al.
1992, Yu et al. 1992). The deduced amino acid sequence
suggested that the enzyme precursors contain the putative
signal sequences in the N-termini for targeting into the lumen of endoplasmic reticulum (ER) without exception.
Cereal a-amylase is known to be a typical secretory protein
in plants. The functional expression of the a-amylase enzyme is quite complicated: after the transcription of genetic
information and the RNA processing and transport, the
enzyme protein is synthesized at the rough ER, the protein
synthesized is transported from the ER to the Golgi complex and modified in the organelle, and then the mature
protein is transported to the plasma membrane and discharged to the extracellular space (cf. Akazawa et al. 1988).
In germinating cereal seeds, a-amylase is secreted from the
scutellar epithelium and the aleurone layer into the endosperm tissue.
A general feature observed with germinating cereal
seeds is that reserve starch in the endosperm tissues is hydrolytically broken down by a-amylase and other amylolytic enzymes and that the glucose produced is mobilized to
the scutellar tissues, where it is reconverted to sucrose and
eventually transported to the embryo axis to sustain seedling growth. The sugar molecules released during the step
of starch breakdown in cereal seeds may regulate the expression of a-amylase in rice cells. Indeed, metabolic regulation of expression of a-amylase genes has been observed
in cereal seedlings (Thomas and Rodriguez 1994, Yu et al.
1996). Perata et al. (1997) have reported that sugar and
hormonal signaling interact in the regulation of gibberellin-induced gene expression in barley embryos, although
the effects of sugar are independent of their effects on gibberellin synthesis and the sensitivity to endogenous ABA or
induction of ABA synthesis. Kashem et al. (1998) have
reported that ABA stimulates the glucose uptake and the
synthesis of sucrose in rice scutellar tissues as well as in the
suspension-cultured cells derived from the rice embryo,
and that the expression of a-amylase 1-1 in these cells is
coordinated by glucose and ABA. In germinating cereal
seeds, the hormonal and metabolic regulation of a-amylase
expression appear to proceed in an intricate manner.
774
Sucrose controls transport and turnover of a-amylase
Rice suspension-cultured cells actively synthesize and
secrete a-amylase molecules into the culture media (Chiba
et al. 1990, Simmons et al. 1991, Mitsui et al. 1993). We
have purified and characterized ten a-amylase isoforms
derived from rice suspension-cultured cells, A, B, E, F, G,
H, I, J, Y, and Z which have different pi values. Isoform
molecules of a-amylase are classified into two major classes, class-I (A, B, Y, Z) and class-II (E, F, G, H, I, J), and
further into four subgroups, group-1 (A, B), group-2 (Y,
Z), group-3 (E), and group-4 (F, G, H, I, J), and a-amylase
1-1, II-3 and II-4 are shown to be encoded by RAmylA,
RAmy3E and RAmy3D, respectively (Mitsui et al. 1996).
In cultured rice cells, sugars repress the expression of aamylase genes, RAmy3D (aAmy3) and RAmy3E {aAmyS)
(Yu et al. 1991, Huang et al. 1993). Recently, several ciselements for response to sugar have been found in the
promoter of rice a-amylase RAmy3D (Hwang et al. 1998,
Lu et al. 1998), and the regulation occurs at the level of
both transcription and mRNA stability (Huang et al. 1993,
Sheu et al. 1996, Chan and Yu 1998). However, there is
little information concerning the posttranslational regulation of a-amylase expression by metabolic sugars.
Here, we report the mechanism underlying the sugarcontrolled secretion of a-amylase molecules determined
employing suspension culture system of rice embryo. We
found that sucrose entails a regulatory role in the process
of intracellular transport and turnover of a-amylase molecules.
Materials and Methods
Chemicals—Cycloheximide was purchased from Wako (Osaka,
Japan) and monensin from Sigma-Aldrich Japan (Tokyo, Japan).
Cell culture—The procedure of rice cell culture (Oryza sativa
L. cv. Nipponkai) was essentially the same as that described previously (Mitsui et al. 1993). About 2 g of rice cells were grown in
a 500 ml Sakaguchi flask containing 120 ml of Murashige-Skoog
(MS) medium (Murashige and Skoog 1962) containing 3 % (w/v)
sucrose, 2mgliter~' 2,4-dichlorophenoxyacetic acid, and 5 mg
liter" 1 thiamine-HCl, placed on a reciprocal shaker operated at
110 strokes min" 1 with 70 mm amplitude, at 28°C in darkness.
Established suspension-cultured cells were subcultured at 7-day
intervals. All the procedures were performed under aseptic conditions.
Assays—The standard assay of a-amylase was performed as
described previously (Mitsui et al. 1993). One enzyme unit was
denned as described by Okamoto and Akazawa (1978). Protein
contents were determined by the dye binding procedure of Bradford (1976) using bovine gamma globulin as a standard. Sugar
contents were measured by the phenol-H2SO4 method (Dubois et
al. 1956).
Pulse and pulse-chase labeling experiments—In the standard
pulse labeling system, 20 mg of rice cells were incubated in 1 ml
of MS media containing [35S]Met and [35S]Cys (400 kBq, 37 TBq
mmol" 1 ) for 18 h at 30°C on a reciprocal shaker operated at 130
strokes min" 1 with a 30 mm amplitude. In the case of pulse-chase
labeling experiments, rice cells were pulse-labeled with [35S]Met
and [3SS]Cys (400 kBq, 37 TBq mraoP') for 18 h in the absence of
sucrose, and then chased with 1 mM non-radioactive Met and Cys
for 10 to 48 h at 30°C. After pulse or pulse-chase incubation, cells
were removed and homogenized in a small mortar using 1 ml of
MS medium containing 0.1% (v/v) Triton X-100, and centrifuged
at 15,000 x g for 20 min to collect the cell extracts. Further details
and alterations of the incubation conditions are given in each
figure legend. [35S]-labeled proteins in each of the cell extracts
and the culture media were subjected to immunoprecipitation to
quantify the amount of each a-amylase isoform.
To determine the total amounts of incorporation of [35S]
amino acids into proteins, the radioactivities of the hot trichloroacetic acid (TCA)-insoluble fraction were measured as follows.
An aliquot of the cell extracts and the culture media were placed
on a glass filter disc (GF-75) (Advantec, Tokyo, Japan). The disc
was soaked in 10% (v/w) TCA solution for 10 min, boiled in 10%
TCA for 3 min, and washed thoroughly in 10% TCA and ethanol.
Finally, radioactivities in the dried disc were measured in a scintillation spectrometer (Aloka liquid scintillation system LSC1000, Tokyo, Japan).
RNA isolation and translation in vitro—The isolation procedure of total RNA from rice cells was identical to the method
described by Shirzadegan et al. (1991). To estimate the amount of
translatable mRNA in the cells, the isolated RNA (4^g) was
translated using the reticulocyte lysate translation system (Amersham Pharmacia Biotech, Tokyo, Japan) with [35S]Met (400 kBq,
29.6 TBq mmol"1) according to the manufacturer's protocol.
Total amounts of the translation products were determined by
measuring the radioactivity incorporation into the hot TCA-insoluble fraction. When RNA isolated from rice cells incubated
without sucrose for 18 h at 30°C was used, radioactivity incorporated into translation products was approximately 50 kBq. The
translation products were finally subjected to the immunoprecipitation to determine the translatable mRNA with respect to
each a-amylase isoform.
Immunoprecipitation of a-amylase isoform—Aliquots (100
to 500 fil) of the above [35S]-labeled proteins synthesized in vivo
arid in vitro were made to 1% (w/v) with SDS, boiled for 1 min,
and then made to 1.5% (w/v) with Triton X-100. After adding 5
volume of buffer A (20 mM Tris-HCl (pH 7.5), 0.14 M NaCl, 1
mM EDTA) containing 0.1% Triton X-100, the mixture was incubated with 25 /ig each of polyclonal anti-AB or anti-H antibodies (IgG), for 1 h at 30°C. As reported previously (Mitsui et al.
1996), anti-AB antibodies bind specifically to a-amylase I and
anti-H antibodies to a-amylase II-4, respectively. Ten iA of Protein A-Sepharose swollen gel was added, and the whole mixture
incubated for 1 h with vigorous shaking. The suspension was then
applied to a small column to retain the Protein A-Sepharoseimmunoprecipitate complex, and the column was washed sequentially with buffer A containing 0.1% Triton X-100, buffer A
containing 1 M NaCl, buffer A, and distilled water. Finally the
immunoprecipitate was eluted from a column with 70 fil of SDS
sample buffer consisting of 10 mM Tris-HCl (pH 6.8), 2% SDS,
5% (v/v) mercaptoethanol, 10% (w/v) glycerol, and 0.005%
(w/v) bromophenol blue. Eluates were boiled for 1 min to dissociate a-amylase molecules from IgG, and subjected to SDSPAGE (Laemmli 1970), followed by fluorography (Laskey and
Mills 1975) or autoradiography using a radioisotope imaging
analyzer (BAS-5000, Fuji Film, Tokyo, Japan).
Results
Effects of sucrose on the incorporation of [35S]-labeled
Met and Cys into the molecules of a-amylase in rice cell
Sucrose controls transport and turnover of a-amylase
culture were determined. Rice cells were incubated with
[35S] amino acids for 18 h under three different conditions
of sucrose supplementing, followed by the quantitative immunoprecipitation of extracellular and intracellular [35S]labeled class-I and -II a-amylases. The extracellular [35S]aamylases were not caused by the broken cells, because (a)
the synthesis rate of [35S] a-amylases was linear during the
incubation period, and (b) the component of [35S]-proteins
in the media was completely different from that of the cell
extracts (data not shown). As shown in Figure 1, 44 mM
sucrose markedly inhibited the incorporation of [35S] amino acids into a-amylase I molecules and the extracellular
liberation of [35S]-labeled enzymes, although total incorporation of [35S] amino acids into proteins was maximum
at 44 mM of sucrose (see Table 1). On the other hand, the
inhibitory effect of sucrose on a-amylase II-4 was greater
than that on a-amylase I. In the presence of 44 mM sucrose, [35S]-labeled a-amylase II-4 was hardly detectable in
the culture media. Since mannitol (up to 165 mM) did not
775
exhibit any inhibitory effect on the magnitude of synthesis
and extracellular/intracellular ratio of [35S] a-amylase II-4
(Fig. 1), it is unlikely that the sucrose effect is due to the
general osmotic stress.
Effects of sucrose on the level of translatable mRNA
of two a-amylase isoforms was examined using the reticulocyte lysate translation system. As shown in Figure 2 (a
and b), the molecular mass of in vitro translation product
of a-amylase II-4 mRNA was 3 kDa larger than that of aamylase-I mRNA (43 kDa; Miyata and Akazawa 1982),
although the molecular size of the mature form of both
enzyme isoforms were indistinguishable (44 kDa; Mitsui et
al. 1996). The translatable mRNA level for a-amylase II-4
encoded by RAmy3D was greatly decreased in the culture
cells supplemented with 44 mM and 88 mM sucrose (Fig. 2b,
c), although the total amount of translatable mRNA increased in the presence of sucrose (Fig. 2c). Interestingly,
the level of translatable mRNA for a-amylase I encoded by
RAmylA was not affected regardless of the addition of
Extracellular ;
Intracellular .0
44
88
Sucrose (mM)
u-Amylase-l
fj
•
Intracellular
0
44
88
Sucrose (mM)
0
82
165
Mannitol (mM)
a-Amylase-IM
Extracellular
0.8-
I
0.6-
|0.4H
i
CO
8 0.2 H
ll
' i " ' i ™ ^ i — ' i — — r ^ — i ™ ' i — 'i
0
44
88
0
44
88
0
82 165
Sucrosa (mM) Sucros* (mM) | Minnltol (mM)
a-Amylaa«-4
<x-Amylu*-IM
Fig. 1 Effects of sucrose on incorporation of [33S]-labeled amino acids into a-amylase I and a-amylase II-4 molecules in rice cells. Rice
cells were incubated with [35S]-labeled Met and Cys under different concentrations of sucrose (0, 44, and 88 mM) or mannitol (0, 82, and
165 mM) in the MS media for 18 h at 30°C. Amounts of extracellular and intracellular [35S]a-amylase molecules synthesized were
quantified by immunoprecipitation using anti-AB and anti-H antibodies which specifically recognize a-amylase I and a-amylase II-4,
respectively. Immunoprecipitates were subjected to SDS-PAGE, followed by fluorography. (a) Fluorograms of the immunoprecipitated
[35S]a-amylase I and II-4. (b) The amounts of [35S]a-amylases were quantified by densitometric analyses of fluorograms. The total
radioactivity of [35S]a-amylase without sugars in each experimental series was normalized to 1 unit. The values are the mean±SD
(standard deviation) of three experiments, (c) Ratio of extracellular as intracellular [35S]a-amylase was calculated from the results
presented in panel b. The extracellular/intracellular ratio without sugars in each experimental series was arbitrarily denned as 1.
Sucrose controls transport and turnover of a-amylase
kD
..
pre-u-A-l - •
kD
45 pre-a-A-ll •
••W. '
~-45
-4
—36
—36
I
Total Proteins
FJ <*-Amylase I
gJa-Amylase 11-4
0 mM
44 mM
88 mM
Sucrose Concentration
Fig. 2 Effects of sucrose on accumulation of translatable mRNA for encoding a-amylase I and II-4 in rice cells. RNA was isolated
from rice cells incubated under different concentrations of sucrose (0, 44, and 88 mM) in the MS media for 18 h at 30°C, and subjected
to the in vitro translation analysis using reticulocyte lysate with [35S]Met. Levels of translatable mRNA were estimated by measuring
incorporation of [3SS]Met into the translated proteins. To quantify the amount of translatable mRNAs for a-amylase I and II-4, total
[35S]-labeled proteins obtained were subjected to the quantitative immunoprecipitation, followed by SDS-PAGE and fluorography. (a
and b) Fluorograms; the molecular mass of a-amylase I (a) and II-4 (b) precursors produced by translation in vitro was estimated to be
43 kDa and 46 kDa, respectively, (c) Quantitative results; each amount of translatable mRNA for total proteins, a-amylase I and II-4
in rice cells incubated without sucrose (0 mM) was normalized to 100%. The data represent the average of duplicate experiments.
sucrose (Fig. 2a, c).
Table 1 shows the effects of various sugars on the
synthesis of [35S]a-amylase II-4 in rice cells. Total protein
synthesis was significantly stimulated by supplementation
of 1.5% (w/v) sugar, i.e., sucrose (44 mM), maltose (42
mM), glucose (83 mM), fructose (83 mM), and galactose
(83 mM), but not by mannitol (82 mM). The a-amylase II4 synthesis was almost equally prevented by sucrose, maltose, glucose and fructose. On the other hand, the effect of
galactose was relatively weak compared with the above
sugars (Table 1). Figure 3 shows the effects of different
concentrations of sucrose on the synthesis of [35S]a-amylase II-4 in rice cells. Sucrose markedly inhibited the a-amylase II-4 synthesis at around 5 mM. Surprisingly, sucrose
effectively prevented the extracellular liberation of a-amylase II-4 molecules (Fig. 3a, b) and reduced the extracellular/intracellular ratio (Fig. 3c) at around 2 mM.
In agreement with classical studies performed by Jami-
eson and Palade (1968) who demonstrated that the synthesis and the subsequent intracellular transport of secretory proteins in pancreatic exocrine cells are governed by
separable mechanisms, we found previously that in rice
scutellar tissues a-amylase molecules produced are discharged extracellularly without ongoing protein synthesis
(Mitsui and Akazawa 1986). The sharp decrease of the
extracellular/intracellular ratio of [35S]a-amylase in the
presence of sucrose (Fig. 1, 3) may indicate that sucrose
preferentially arrests the intracellular transport processes
including exocytosis of the protein molecules.
In order to test this intriguing possibility, we performed pulse-chase experiments. At the first chase incubation of 10 h, the extracellular liberation of [35S]a-amylase II-4 (Fig. 4a, Ml) was reduced and the intracellular
accumulation of the enzyme molecules (Fig. 4a, Tl) was
increased slightly by supplementation of sucrose, although the total amount of [35S]a-amylase II-4 was scarcely
Sucrose controls transport and turnover of a-amylase
777
Extracellular:
0
1.1
2.2
5.5
11
22
44
88
0
1.1
2.2
5.5
11
22
44
88
Intracellular:
Sucrose (mM)
0.8--
|
Intracellular
^
0.7-
fj
Extracellular
3
0.6~
ID
0 5-
h
(+
r
J
1* 0.4-
n*
1
1
0.20.1 -
0
1L • • • •
1.1
_
2.2
5.5
11
•
22
44
88
22
44
88
Sucrose (mM)
c
1 n
•2
w 1.6OC
(5 1.4trace
3
•g
a
5
0.8-
8
0.4-
2
x
0.6-
0.2-
UJ
1=77?)
0
1.1
2.2
5.5
11
Sucrose (mM)
Fig. 3 Effects of different concentrations of sucrose on the synthesis of [35S]a-amylase II-4 molecules. Rice cells were incubated with
[35S] amino acids under different concentrations of sucrose for 18 h at 30°C. (a) Fluorograms of the extracellular and intracellular
[35S]a-amylase II-4 molecules synthesized, (b) The amounts of [35S]a-amylases were determined by the quantitative immunoprecipitation
as described in the legend of Figure 1. Each value is the mean±SD of three experiments, (c) Ratio of extracellular as intracellular
[35S]a-amylase was calculated from the results presented in panel b.
changed (Fig. 4b, Exp. 1). For the second incubation of 14
h, the extracellular liberation of [35S]a-amylase II-4 was not
detectable in the presence of sucrose (Fig. 4a, M2), and for
the third incubation of 24 h, the extracellular liberation of
[35S]a-amylase II-4 continued to be prevented perfectly by
sucrose (Fig. 4a, M3). In addition, the degradation of
intracellular [35S]a-amylase II-4 molecules was found to
be occurred after 10 h of sucrose treatment, as shown in
Figure 4b. These results clearly indicate that sucrose arrests
the intracellular transport of a-amylase molecules and
stimulates their turnover in the cells.
We next examined the effect of the carboxylic ionophore monensin, a potent perturbant of the structure
and function of the Golgi complex (Kimura et al. 1993).
Sucrose controls transport and turnover of a-amylase
778
Table 1 Effects of different sugars on the synthesis of [35S] a-amylase molecules in rice
cells
Sugar treatments
No sugar supplementation
1.5% (44 mM) Sucrose
3.0% (88 mM) Sucrose
1.5% (42 mM) Maltose
3.0% (83 mM) Maltose
1.5% (83 mM) Glucose
3.0% (167 mM) Glucose
1.5% (83 mM) Fructose
3.0% (167 mM) Fructose
1.5% (83 mM) Galactose
3.0% (167 mM) Galactose
1.5%(82mM)Mannitol
3.0% (165 mM) Mannitol
Total [35S]-proteins
(Bq)
407 ±49*
712±57
549 ±41
757 ±73
671 ±57
667 ±69
537 ±49
684 ±45
553±57
769 ±94
696 ±53
488 ±53
387 ±49
[35S]a-Amylase II-4(unit) D
Intracellular
Extracellular
0.41 ±0.03*
0.04±0.005
0,01 ±0.001
N.D.
N.D.
0.05 ±0.006
0.05 ±0.004
N.D.
N.D.
0.30±0.02
0.11±0.01
0.42 ±0.04
0.43 ±0.03
0.59±0.07 6
N.D.C
N.D.
N.D.
N.D.
0.12±0.01
0.12±0.01
N.D.
N.D.
0.50±0.04
0.31 ±0.03
0.58±0.06
0.67 ±0.08
Rice cells were incubated with [35S] amino acids under different concentrations (0, 1.5 and 3% (w/v))
of various sugars for 18 h at 30°C.
" Quantitative immunoprecipitation was performed as described in the legend of Figure 1.
* Each value is the mean±SD of three experiments.
c
N.D., Not detectable.
In the pulse-labeling experiments, monensin prevented
the extracellular liberation of [35S] a-amylase II-4. On the
other hand, in the pulse-chase experiments monensin caused marked accumulation of the enzyme molecules intracellularly (Table 2). In addition, the extracellular/intra-
cellular ratio was significantly decreased by treatment with
monensin in both experiments. Furthermore, the degradation of [35S]a-amylase II-4 molecules was shown not to be
induced by monensin (Table 2). The results indicate that
the sucrose-controlled modulation of intracellular trans-
Table 2 Effects of monensin on the intracellular transport and degradation of [35S]aamylase molecules in the sugar-depleted cells
l reatments
[35S]a-Amylase II-4(unit)c
Intracellular
Extracellular
Pulse labeling"
0/iM Monensin
0.1 fiM Monensin
1.0 /iM Monensin
10 fiM Monensin
0.54±0.05rf
0.53±0.03
0.73±0.07
0.54±0.03
0.46 ± 0.04 d
0.44 ±0.04
0.26±0.01
0.12±0.01
0.22
Pulse-chase *
0^M Monensin
0.1 yuM Monensin
1.0/UM Monensin
10/iM Monensin
0.24±0.01
0.36±0.03
0.59±0.06
0.81 ±0.08
0.76 ±0.04
0.64±0.05
1.12±0.10
0.87±0.09
3.17
1.78
1.89
1.07
Extracellular/
intracellular ratio
0.85
0.89
0.36
Rice cells were incubated with [35S] amino acids in the presence of monensin (0 to 10 fiM) under a
sugar-depleted condition for 18 h at 30°C.
* Rice cells were pulse-labeled in the absence of sugar for 18 h at 30°C, and then chased in the
presence of monensin (0 to 10 fiM) under a sugar-depleted condition for 24 h at 30°C.
'Amounts of [35S]a-amylase II-4 molecules were determined by the quantitative immunoprecipitation
as described in the legend of Figure 1.
d
The data represent the average of duplicate experiments.
Sucrose controls transport and turnover of a-amylase
10h
14 h
779
24 h
•M* "
B mM Sue:
11 mM Sue:
44 mM Sue:
—«~—
—
—
Ml Tl Ml M2 T2 M l M2 M3 T3
Exp. 1 Exp. 2
Exp. 3
onUI SUCTOM
Fig. 4 Effects of sucrose on the extracellular liberation and degradation of [35S]a-amylase II-4 molecules. Rice cells were pulse-labeled
in MS media with [33S]Met and Cys in the absence of sugar for 18 h at 30°C, and then chased with 1 mM non-radioactive Met and Cys
under different concentrations of sucrose (0, 11 and 44 mM) for 10 h at 30°C (first incubation). Rice cells were further incubated with
the new chase media for 14 h (second incubation) and 24 h (third incubation), sequentially. Total chase periods in Exp. 1, 2 and 3 were
10, 24 and 48 h, respectively. In the top panel, the closed arrow represents the start of chase and open arrows represent the replacement
with new chase media. Tl, T2 and T3 represent the cell extracts at 10, 24 and 48 h of incubation, respectively. Ml, M2 and M3 represent
the chase media of the first, second and third incubations. Amounts of extracellular and intracellular [3!S]a-amylase II-4 molecules were
quantified by BAS 5000. (a) Autoradiograms of extracellular (Ml-3) and intracellular (Tl-3) [35S]a-amylase II-4 molecules, (b) Quantitative results. The amount of [35S]a-amylase II-4 without chase incubation was normalized to 1 unit. Each value is the mean±SD of
three experiments.
port of a-amylase molecules and their turnover proceed
independently.
The effects of cycloheximide on the sucrose-controlled synthesis and secretion of a-amylase were studied,
employing basically the same experimental protocols as
above. As shown in Table 3, cycloheximide effectively inhibited total protein synthesis de novo at more than 10 fiM
regardless of the presence or absence of sucrose. Cycloheximide also inhibited the synthesis of a-amylase II-4 in
the sugar-depleted rice cells, but not so markedly in the
sucrose-supplemented cells. The extracellular liberation of
a-amylase molecules and the extracellular/intracellular ratio was also markedly increased (Table 3). The pulse-chase
experiments showed that cycloheximide significantly induces the extracellular liberation (Fig. 5a, M2) and the intracellular accumulation (Fig. 5a, T2) of [35S]a-amylase II4 in the sucrose-supplemented cells. Importantly, it was
shown that cycloheximide clearly inhibited the sucroseinduced degradation of intracellular a-amylase molecules
(Fig. 5b). Judging from these experimental results, we infer
Sucrose controls transport and turnover of a-amylase
780
Table 3 Effects of cycloheximde on the synthesis of [35S]a-amylase molecules in rice cells
Total [3SS] -proteins
(Bq)
i reatments
Sugar-depleted cells
O^iM Cycloheximide
1 //M Cycloheximide
10/uM Cycloheximide
100 /xM Cycloheximide
441+79*
379 + 79
48± 4
9± 1
[35S]a-Amylase II-4(unit)a
Intracellular
0.51
0.51
0.06
0.01
44 mM Sucrose-supplemented cells
OfiM Cycloheximide
732±75
1 fiM Cycloheximide
869 + 62
lOyuM Cycloheximide
101± 4
100 juM Cycloheximide
13± 0.4
±0.03b
±0.05
±0.01
±0.00
0.066 ±0.005
0.075 ±0.007
0.030 ±0.002
0.011 ±0.001
Extracellular
0.49
0.49
0.05
0.01
±0.05"
±0.05
±0.004
±0.00
0.003 ±0.000
0.002 ±0.000
0.012±0.001
0.009 ±0.001
Rice cells were incubated with [35S] amino acids in the presence of cycloheximide (0 to
under
a sugar-depleted or 44 mM sucrose-supplemented condition for 18 h at 30°C.
" Quantitative immunoprecipitation was performed as described in the legend of Figure 1.
* Each value is the mean±SD of three experiments.
ing exocytosis and the stimulation of turnover of a-amylase
molecules in the sucrose-supplemented rice cells.
that sucrose-induced and newly synthesized proteins are
involved in the arrest of the intracellular transport includ-
CHX:
IBuMCHX:
108
•«----<«*-« r _4*S-..-^-r~
CHX:
Ml M2 T2 M l M2 T2 M l M2 T2
e mM Sue
11 mM Sue 44 mM Sue
b
11 mMSucroM
0 mM Sucrose
0 6-
0.4-
0 1-
{ |
44 mM Sucros*
0.8-
0.3-
ill
Cydohaxltnlda (|>M)
Fig. 5 Effects of cycloheximide on the extracellular liberation and degradation of [35S]a-amylase II-4 molecules in the sugar-supplemented cells. The pulse-chase experimental system used was the same as that in Fig. 4 (Exp. 2). After pulse-labeling, rice cells were
chased in the presence or absence of cycloheximide (0, 10, and 100 fiM) under different sucrose supplemented conditions (0, 11 and 44
mM) at 30°C. (a) Autoradiograms. (b) Quantitative results. The amount of [35S]a-amylase II-4 without chase incubation was normalized
to 1 unit. Each value is the mean±SD of five experiments.
Sucrose controls transport and turnover of a-amylase
Discussion
The gene expression of RAmy3D and RAmy3E encoding class-II isoforms has been reported to be suppressed
by metabolic sugars including sucrose (Yu et al. 1991,
Huang et al. 1993), although the expression of RAmylA
(a-amylase 1-1) is less affected (Karrer and Rodriguez 1992,
Yu et al. 1996). We found that the secretion of class-I aamylase isoforms is preferentially regulated by sucrose in
the step of mRNA translation as well as subsequent intracellular transport of a-amylase molecules including exocytosis employing the suspension-cultured cells of rice (Fig. 1,
2). On the other hand, the secretion of class-II a-amylase
isoforms encoded by RAmy3D was shown to be regulated
by sucrose at both the transcriptional and posttranscriptional steps (Fig. 1,2). Based on these experimental results,
thus it now appears clear that the control of the posttranscriptional secretory process by sucrose occurs commonly
in two classes of a-amylase in the rice cells, although the
expression of corresponding genes is differentially regulated.
A question arising is whether or not the inhibition of
synthesis and extracellular release of a-amylase molecules
in cultured rice cells is triggered by other sugars. The results presented in Table 1 show that the synthesis and extracellular liberation of [35S]-labeled a-amylase II-4 molecules were prevented effectively by maltose, glucose and
fructose, whereas the effect of galactose was relatively weak
and mannitol totally unaffected. These four metabolic sugars have been reported to markedly reduce a-amylase
production in rice and barley embryos (Perata et al. 1997,
Kashem et al. 1998, Umemura et al. 1998).
Recently, it has been suggested that sugar sensing occurs in association with the secretory (ER-Golgi) system
(Herbers et al. 1996, Smeekens and Rook 1997). The results
presented in Figure 3 reveal that 2 mM sucrose preferentially prevents the extracellular liberation of [35S]a-amylase
II-4 molecules, although the de novo synthesis of the isoform molecules still occurred at this concentration. The
results obtained by a series of pulse-chase experiments
(Fig. 4) showed that sucrose regulates the posttranslational
secretory processes of the enzyme molecules in rice cells.
Yu et al. (1996) have reported that 25 mM glucose inhibited
the expression of a-amylase gene, aAmy3 (RAmy3D) in the
rice embryo. The modulation of the posttranslational secretory system is thought to be an important event for the
rapid and fine response to sugars in higher plant cells.
As shown in Figure 4, sucrose was found to stimulate
the degradation of [35S]a-amylase II-4 molecules. The results obtained by using monensin suggested that the modulating effects of sucrose on the intracellular transport of
a-amylase molecules and their turnover are independent
(Table 2). It has been reported that sucrose decreased the
half-life of a-amylase mRNA in rice cell culture (Sheu et al.
781
1996). In microorganisms, sugar sensing is tightly connected not only to transcription but also mRNA translation, protein stability and mRNA stability (Ronne 1995).
Altogether, the posttranscriptional regulation of a-amylase
secretion governed by sugar must be complicated.
It is crucially important to clarify how sugar controls
the intracellular transport and turnover of a-amylase molecules in rice cells. As presented in Figure 5 the protein
synthesis inhibitor cycloheximide induced the extracellular
liberation of [35S]a-amylase II-4 and delayed the degradation of the isoform molecules in the sucrose-supplemented
cell culture. It was also observed that the addition of sucrose to the sugar-depleted rice cells induced the de novo
protein synthesis within 1 h (Mitsui et al. unpublished
data). These results strongly suggested that newly synthesized sucrose-induced proteins are involved in the posttranslational regulation on sucrose-controlled a-amylase
secretion in rice cells. Sheu et al. (1994) have reported that
at a high concentration cycloheximide (>0.1 mM) is capable of enhancing a-amylase mRNA accumulation in rice
cells either in the presence or absence of sucrose. The
enhancing effect appeared to be more prominent in the
sucrose-provided rice cells. Recent investigations have
suggested that there exist several sugar-responsible cis-elements in the promoter region of the rice a-amylase gene,
RAmy3D (aAmy3) (Hwang et al. 1998, Lu et al. 1998). The
transcription of the aAmy3 promoter was also suggested to
be enhanced by cycloheximide, probably through an inhibition of repressor synthesis (Lu et al. 1998). Based on
these experimental results, it was concluded that sucroseinduced and newly synthesized proteins may play a key role
in the overall metabolic regulation of a-amylase expression.
This research was supported by Grant-in-Aid no. 10640628
(to T.M.) from the Ministry of Education, Science, Sports and
Culture, Japan.
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(Received November 24, 1998; Accepted May 12, 1999)