From the Department of Biological Sciences, Tokyo Metropolitan University, Minami-osawa 1-1, Hachioji, Tokyo, 192-0397 Japan
Received for publication, April 11, 2000, and in revised form, September 18, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SH-EP is a vacuolar cysteine proteinase from
germinated seeds of Vigna mungo. The enzyme has a
C-terminal propeptide of 1 kDa that contains an endoplasmic reticulum
(ER) retention signal, KDEL. The KDEL-tail has been suggested to
function to store SH-EP as a transient zymogen in the lumen of the ER,
and the C-terminal propeptide was thought to be removed within the ER
or immediately after exit from the ER. In the present study, a protease
that may be involved in the post-translational processing of the
C-terminal propeptide of SH-EP was isolated from the microsomes of
cotyledons of V. muno seedlings. cDNA sequence for the
protease indicated that the enzyme is a member of the papain
superfamily. Immunocytochemistry and subcellular fractionation of
cotyledon cells suggested that the protease was localized in both the
ER and protein storage vacuoles as enzymatically active mature
form. In addition, protein fractionations of the cotyledonary microsome
and Sf9 cells expressing the recombinant protease indicated that
the enzyme associates with the microsomal membrane on the luminal side.
The protease was named membrane-associated
cysteine protease, MCP. The possibility that a
papain-type enzyme, MCP, exists as mature enzyme in both ER and protein
storage vacuoles will be discussed.
The endoplasmic reticulum
(ER)1 is the port of entry of
proteins into the endomembrane system. In this organelle, there are a
number of soluble proteins, membrane proteins, and molecular chaperones
which are involved in folding, glycosylation, assembly, and maturation
of nascent proteins (1-3). The soluble proteins localized in the lumen
of the ER have a retention signal, KDEL or HDEL, at the C terminus
(4-6), and this signal is known to be recognized by the K(H)DEL
receptor on the Golgi complex, which mediates retrieving K(H)DEL-tailed
proteins to the ER. The molecular mechanisms of the ER retention of
soluble proteins are conserved through animal, plant, and yeast
cells (7-9).
In several kinds of plants, unique papain-type proteinases possessing a
C-terminal KDEL sequence have been identified (10-16). One such
KDEL-tailed cysteine protease, designated SH-EP, was first isolated
from cotyledons of germinated Vigna mungo seeds as the
enzyme responsible for degradation of storage proteins accumulated in
protein storage vacuoles (PSV) of cotyledon cells (10, 17). SH-EP is
synthesized on membrane-bound ribosomes as a 43-kDa precursor through
co-translational cleavage of the signal peptide, and the precursor is
processed to the 33-kDa mature enzyme via 39- and 36-kDa intermediates
during or after transport to the vacuoles (18, 19).
The function of the KDEL-tail on a cysteine protease whose final
destination is the PSV has been an interesting question. Based on
analysis of the heterologous expression of SH-EP and a KDEL deletion
mutant of SH-EP in insect Sf9 cells and subcellular fractionation of cotyledon cells, it was proposed that the KDEL-tail of
SH-EP functions to store the enzyme as a transient zymogen in the ER,
and that the conversion of the 43-kDa SH-EP into the 42-kDa form in/at
the ER is accompanied by the removal of the C-terminal propeptide
containing the KDEL-tail (20). From these observations, the protease
responsible for removal of the C-terminal propeptide of SH-EP has been
supposed to exist in the lumen of the ER (20). Recently, Toyooka
et al. (21) showed that in cotyledon cells of V. mungo seedlings, a proform of SH-EP synthesized in the ER
accumulated at the edge or middle regions of the ER where the transport
vesicle was formed. The vesicle, containing a large amount of
pro-SH-EP, termed KV, budded off from the ER, bypassed the Golgi
complex, and fused to PSV (21). It was proposed that the KDEL-tail of
SH-EP functions as the signal for accumulation of pro-SH-EP at the edge
or middle regions of the ER where formation of KV proceeds (21).
In this study, the protease involved in the processing of 43-kDa SH-EP
(KDEL-attached form) into 42-kDa SH-EP (KDEL-removed form) was purified
to homogeneity from microsomes of cotyledons of V. mungo
seedlings, and a cDNA clone for the enzyme was isolated. Immunocytochemical and sucrose gradient analyses of cotyledon cells
were carried out to determine the intracellular localization of the
protease. The protease was also expressed in insect Sf9 cells in
order to observe the characteristics of the membrane association of the
enzyme. The possible dual functions of this enzyme in cells will be discussed.
Plant Materials--
V. mungo seeds were germinated
on layers of wet filter paper at 27 °C in darkness, and cotyledons
were collected on day 3 of post-imbibition.
SDS-PAGE and Immunoblotting--
SDS-PAGE was conducted on
12.5% gels, and immunoblotting was performed as described elsewhere
(18).
MCP Assay--
An active site mutant of 43-kDa SH-EP in which
the active site Cys152 was replaced by Gly was expressed in
Escherichia coli BL21(DE3), recombinant 43-kDa SH-EP (C152G)
was prepared as described (22) and used as substrate to assay the
enzymatic activity of MCP. Ten microliters (2 µg) of the substrate
and 10 µl of enzyme solution were mixed and incubated at 27 °C for
16 h. After incubation, 20 µl of SDS-PAGE sample buffer (20 mM Tris-Cl, pH 6.8, 40% glycerol, 2% SDS, 0.1%
bromphenol blue, 10% 2-mercaptoethanol) was added and the mixture was
boiled for 3 min. The 42-kDa SH-EP (C152G) generated from the substrate
by MCP activity was detected with SDS-PAGE and Coomassie Brilliant Blue
(CBB) R staining.
Purification of MCP and Determination of Its N-terminal Amino
Acid Sequence--
All manipulations were conducted at 0 to 4 °C.
Cotyledons (200 g) of day 3 dark-grown seedlings were homogenized with
600 ml of 0.1 M Tris-Cl (pH 7.4) containing 0.44 M sucrose, 1 mM EDTA, and 0.1 mM
MgCl2. The homogenate was centrifuged at 800 × g for 10 min and then at 4,500 × g for 30 min. The supernatant was again centrifuged at 100,000 × g for 60 min, and the precipitate was washed with
homogenization buffer and resuspended with 120 ml of the buffer. The
resuspended solution was sonicated (3 × 1 min, 30 W, UR-20P, Tomy
Seiko Co., Ltd.), and centrifuged at 100,000 × g for
60 min. The precipitate was washed with the homogenization buffer and
solubilized with 50 mM sodium phosphate buffer (pH 7.2)
containing 1% n-octyl- Construction and Screening of cDNA Libraries--
Total RNA
was prepared from day 3 cotyledons as described previously (24).
Poly(A)+ RNA was obtained from total RNA using Oligotex(dT)
30 super (Daiichi Pure Chemical). Double-stranded cDNA was
synthesized from poly(A)+ RNA using the Timesarver cDNA
Synthesis Kit (Amersham Pharmacia Biotech). Two primers
(GCIAAT/CGCICAG/CAAGAGCICC) and (T/CTC-G/AAAIGCG/ATTG/ATTCAT) were synthesized on the basis of the N-terminal amino acid sequence of
MCP (ANAQKAP) and conserved sequences of known papain-type proteases
(MNNAFE), respectively. Using these primers together with
double-stranded cDNA as a template, the polymerase chain reaction was carried out in a 50-µl volume for 30 cycles
(94 °C, 1 min; 50 °C, 2 min; and 72 °C, 2 min). The DNA
fragment (0.25 kb) amplified by polymerase chain reaction was
radiolabeled with [ Antibodies--
In order to prepare the recombinant pro-MCP,
full-length MCP cDNA was subcloned into pET17 vector (Novagen)
cleaved by NotI. The vector harboring MCP cDNA was
digested with NheI and BbeI to eliminate the DNA
region encoding the signal sequence. The vector was blunted using a
DNA-blunting kit (Takara), self-ligated, and transformed directly into
E. coli BL21(DE3). Expression of pro-MCP, isolation of
inclusion bodies accumulating pro-MCP, and purification of recombinant
proMCP by ion-exchange column chromatography were carried out as
described (22). The recombinant pro-MCP (1 mg) was immobilized to 3 ml
of ECH-Sepharose 4B (Amersham Pharmacia Biotech) according to the
manufacturer's instructions, and the MCP-immobilized Sepharose was
packed into a column. Affinity purification of the antibody to pro-MCP
from the antiserum against pro-MCP was performed as described
previously (21). Antibody to SH-EP and antibody specific to N-terminal
prosequence of SH-EP were prepared as described (21). Antiserum to
Preparation of Microsomes from Cotyledon Cells--
Day 3 cotyledons of V. mungo seedlings were gently ground in a
mortar and pestle with 3 volumes of 0.1 M Tris-Cl (pH 7.4) containing 0.44 M sucrose, 1 mM EDTA, and 0.1 mM MgCl2. The homogenate was centrifuged at
800 × g for 10 min and then at 4,500 × g for 30 min. The supernatant was again centrifuged at
100,000 × g for 60 min, and the precipitate was washed
twice with the homogenization buffer and used as the microsomal fraction.
Electron Microscopy and Immunocytochemistry--
Day 3 cotyledons of V. mungo seedlings were cut into approximately
1-mm3 cubes and fixed with 4% formaldehyde, 2%
glutaraldehyde in 50 mM potassium phosphate (pH 7.4) at
4 °C for 4 h. The tissue pieces were dehydrated in a graded
methanol series and embedded in a hard formulation of LR White resin.
Ultrathin sections mounted on nickel grids were blocked with 10%
bovine serum albumin in TBST (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.2% Tween 20) for 15 min at room temperature.
The sections were then incubated with affinity-purified antibody
against MCP for 30 min at room temperature. Sections were washed with
TBST and indirectly labeled with 10-nm colloidal gold anti-rabbit IgG
in TBST for 15 min at room temperature. The grids were then washed with
TBST followed by distilled water, and then stained with 5% uranyl
acetate for 20 min. The grids were examined and photographed with a
transmission electron microscope (model 1010EX; JEOL) at 80 kV.
Ultrastructural analysis was carried out according to Hara-Nishimura
et al. (27).
Preparation of Recombinant Baculovirus, Expression of MCP in
Sf9 Cells, and Subsequent Analysis--
The DNA insert of
full-length MCP cDNA was cut out of pBluescript SK+ phagemid vector
using NotI, and subcloned into pVL1392 baculovirus transfer
vector cleaved by the same enzyme. Using the pVL1392 vector harboring
MCP cDNA and a BaculoGoldTM Transfection Kit
(Pharmingen), recombinant baculovirus containing MCP cDNA was
prepared and amplified according to the manufacturer's instructions.
Preparation of recombinant baculovirus of SH-EP and production of MCP
and SH-EP in Sf9 cells were conducted as described (20). After a
3-day infection with MCP or SH-EP baculovirus, Sf9 cells were
peeled off from 25-cm2 culture dishes with serum-free
Grace's medium (Life Technologies, Inc.). The resuspended cells were
centrifuged at 200 × g for 5 min, and the pellet was
resuspended with serum-free Grace's medium and centrifuged again. The
pellet was then resuspended with 0.5 ml of 0.1 M Tris-Cl
(pH 7.4) containing 0.4 M sucrose, 1 mM EDTA, and 0.1 mM MgCl2, and was sonicated for 1 min
at 30 W. A 50-µl aliquot of the sonicated cells was retained as a
whole cell extract, and the remainder of the sonicated cell preparation
was centrifuged at 3,000 × g for 5 min. The
supernatants were again centrifuged at 100,000 × g for
1 h. The supernatant was used as the soluble protein fraction of
the cells, while the precipitate was washed with 0.1 M
Tris-Cl (pH 7.4) containing 0.4 M sucrose, 1 mM
EDTA, and 0.1 mM MgCl2, solubilized with 0.45 ml of 8 M urea in 0.1 M Tris-Cl (pH 8.0) and
used as the membrane protein fraction of the cells. For measurement of
MCP activity of recombinant MCP, the precipitate from the
centrifugation at 100,000 × g was washed with 0.1 M Tris-Cl (pH 7.4) containing 0.4 M sucrose, 1 mM EDTA, and 0.1 mM MgCl2, and
solubilized with 0.5 ml of 50 mM sodium phosphate buffer
(pH 7.2) containing 1% Triton X-100 and 10 mM 2-mercaptoethanol. The solubilized solution was again centrifuged at
100,000 × g for 1 h, and the supernatant was used
for MCP assay.
In Vitro Processing of Recombinant 43-kDa SH-EP (C152G) by
Papain--
Papain (10 µg/µl, 30 milliunits/µg) was purchased
from Roche Molecular Biochemicals, and the enzyme was diluted to the
concentration of 10 or 1 ng/µl with 50 mM sodium
phosphate buffer (pH 7.2) containing 1% Triton X-100 and 10 mM 2-mercaptoethanol. Recombinant 43-kDa SH-EP (C152G) was
prepared as above, and 10 µl (2 µg) of the substrate was mixed with
10 µl of diluted papain solution. After 12 h incubation of the
mixture at 27 °C, 20 µl of SDS-PAGE sample buffer was added and
the mixture was boiled for 3 min. Processed product of the substrate by
papain was detected with SDS-PAGE followed by Coomassie Brilliant Blue
staining or by immunoblotting with antibody against mature SH-EP or
N-terminal prosequence of SH-EP.
Identification of Enzymatic Activity of MCP--
Recombinant
43-kDa SH-EP can be converted into the mature enzyme by autocatalysis
(22). For the assay of the enzymatic activity of MCP responsible for
the processing of the C-terminal proregion of SH-EP, an active site
mutant, 43-kDa SH-EP (C152G), was used as a substrate to eliminate the
possibility of autocatalytic processing of the substrate during
incubation with the enzyme solution. The 43-kDa SH-EP (C152G) has also
been shown to be correctly folded (22). When the substrate was
incubated with the buffer, no degradation products of the substrate was
detected (Fig. 1A, buffer
lane), indicating that the substrate was stable and there was no
contaminating protease in the preparation of the substrate.
Coincubation of the substrate with the soluble protein fraction and the
luminal protein fraction from microsomes showed no effect on the
substrate (Fig. 1A, 100,000 × g
sup and lumen lanes). The substrate was cleaved
into the 42-kDa form when it was incubated with the membrane protein
fraction from microsomes (Fig. 1A, membrane lane). The results of the MCP assay indicated that the enzymatic activity catalyzing the substrate into the 42-kDa form is localized in the
membrane protein fraction of microsomes from cotyledons. This protease
activity was inhibited by E-64 but not by diisopropyl fluorophosphate,
pepstatin A, or EDTA (Fig. 1, B and C),
suggesting that the enzyme involved in this proteolysis is a cysteine
protease whose active site cysteine can be irreversibly bound to the
epoxy group of E-64. In addition to the conversion of the substrate to
42-kDa form by MCP activity, the total amount of the substrate was
decreased during incubation (Fig. 1, B and C).
This loss of protein will be due to degradation of the substrate by
proteinase(s) which coexist in the membrane protein fraction from
microsome.
To investigate whether there was C-terminal cleavage of the substrate
by the MCP activity, and to determine the intracellular localization of
the enzyme, the microsomes were further separated by isopycnic sucrose
density gradient and subsequent analysis of each fraction from the
gradient was conducted. The major enzymatic activity of MCP was
detected in fractions 5 and 6 (Fig.
2A). When the reaction mixture
consisting of the substrate and each fraction from the gradient was
analyzed by SDS-PAGE/immunoblotting with anti-KDEL antibody, the
antibody labeled only the substrate (Fig. 2B), and the
density of the substrate bands decreased at fractions 5 and 6. In
addition, the 42-kDa SH-EP (C152G) generated by the enzymatic activity
of MCP was not recognized by the antibody. These suggest that the
conversion of 43-kDa SH-EP (C152G) to the 42-kDa form by MCP activity
was accompanied by the loss of the C-terminal prosequence containing
the KDEL-tail. When the fractions from the sucrose gradient were
analyzed by SDS-PAGE/immunoblotting with anti-BiP antiserum, an intense
band of BiP was detected in fractions 5 to 7 where the MCP activity was
detected (Fig. 2C), suggesting that MCP is mainly localized
in the ER.
Purification of MCP--
Using the assay method described above,
MCP was purified from microsomes of day-3 cotyledons of V. mungo seedlings. n-Octyl- Cloning of MCP cDNA--
Homologous proteases with an
N-terminal amino acid sequence identical to that of MCP were searched
for with FASTA, and the search results indicated that MCP belongs to
the papain protease family. These search results were supported by the
inhibitory effect of E-64, a potent inhibitor of papain-type protease,
on MCP activity. Primers were set to the N-terminal amino acid
sequences of MCP and to a conserved motif of the papain-type proteases. A DNA fragment amplified by polymerase chain reaction was used for
screening of a cDNA library from day 3 cotyledons of V. mungo seedlings. The nucleotide sequence of the full-length
cDNA for MCP consisted of 1414 bp (accession number AB038598), with an open reading frame of 1045 bp encoding 364 amino acid residues with
a calculated molecular mass of 39491.32 Da. The deduced amino acid
sequence of MCP had a putative signal sequence of 20 amino acid
residues (29) and an N-terminal prosegment of 98 amino acid residues.
One putative Asn-linked glycosylation site was found in the amino acid
sequence of MCP. The phylogenetic tree and amino acid sequence
alignment of 25 papain-type proteases are presented in Fig. 4,
A and B, respectively. Proteases with high
homology to MCP belonged to the same subfamily (Fig. 4A) and
a unique conserved region (LPAN/HAQKAPILPT) was found at the N-terminal
region of mature MCP (Fig. 4B). Interestingly, the N-terminal amino acid residue of MCP was located 14 or 15 amino acids
upstream from those of proteases belonging to other papain subfamily enzymes.
Intracellular Localization of MCP--
In general, the papain-type
proteases are known to be destructive enzymes and to be localized in
vacuoles/lysosomes or to be secreted. However, MCP was purified from
the microsomal fraction and is thought to be localized in the ER.
Therefore, we carried out sucrose gradient and immunocytochemical
analyses of cotyledon cells to determine the intracellular localization
of MCP. Each fraction of the sucrose gradients of microsomes from the
cotyledons was analyzed by SDS-PAGE/immunoblotting using anti-MCP,
anti-BiP, and anti- Association of MCP with Membranes--
Experiments to examine the
possible membrane association of MCP by ultrasonic wave treatment of
microsomes from the cotyledons and subsequent ultracentrifugation were
carried out since MCP activity was detected in the membrane protein
fraction from microsomes (Fig. 1A). MCP was mainly detected
in the membrane protein fraction of microsomes (Fig.
8A), whereas most of the SH-EP
was presented in the luminal soluble protein fraction (Fig.
8B). To see whether MCP is associated with the membrane on
the luminal side or the cytoplasmic side from microsomes, the
microsomes of cotyledons were treated with proteinase K with or without
detergent and analyzed by SDS-PAGE immunoblotting with anti-MCP
antibody (Fig. 8C). MCP was intact in the absence of Triton
X-100, but the addition of the detergent to the reaction mixture
resulted in degradation of MCP by proteinase K, indicating that MCP
associates with the microsomal membrane on the luminal side. It should
be noted that SH-EP was presented as a proenzyme in the microsomes, but
MCP as the mature form (Fig. 8, A and B). These
suggest that MCP associates with the microsomal membrane as the mature
enzyme.
Next, full-length MCP cDNA was expressed in insect Sf9 cells
to see whether the association of MCP with the membrane is due to the
nature of the MCP protein itself or is a localization specific for
cotyledon cells. When the total proteins of Sf9 cell expressing MCP were analyzed by SDS-PAGE/immunoblotting using anti-MCP antibody, intense bands of 32.5, 35, 40, 42, and 44 kDa, and weak bands of 37 and
38 kDa were detected (Fig.
9A). The 32.5-kDa polypeptide is the putative mature MCP in Sf9 cells, and the 44-kDa
polypeptide corresponds to the proform of MCP. It could not be
determined whether the other MCP-related polypeptides were
intermediates of MCP or degraded products of pro-MCP. When the extracts
of the Sf9 cells were separated into luminal protein and
membrane protein fractions, the 32.5-kDa MCP was detected only in the
membrane protein fraction, whereas the 35-, 37-, and 38-kDa
polypeptides were observed only in the luminal protein fraction. When
SH-EP, a soluble enzyme, was expressed in Sf9 cells and the
cells were treated as above, SH-EP-related polypeptides were detected
only in the luminal protein fraction (Fig. 9B), suggesting
that the separation of luminal and membrane protein fractions was
successful. These expression of MCP and its subsequent specific
localization in this heterologous cell system strongly suggest that the
membrane association of MCP is a result of the nature of the MCP
polypeptide itself.
To observe whether MCP heterologously expressed in Sf9 cells is
enzymatically active, MCP activities in membrane protein fractions from
Sf9 cells expressing MCP and uninfected (wild type) Sf9
cells were measured. When the recombinant 43-kDa SH-EP (C152G) was
incubated with membrane proteins from uninfected Sf9 cells, the
substrate was stable (Fig. 9C). In contrast, after
incubation of the substrate with membrane proteins from Sf9
cells expressing MCP, the recombinant 43-kDa SH-EP (C152G) was
converted to 42-kDa form (Fig. 9C), suggesting that MCP
expressed in Sf9 cells is correctly folded and activated. No MCP
activity was detected in membrane proteins from uninfected Sf9
cells, however, it has been revealed that 43-kDa SH-EP expressed in
Sf9 cells is cleaved to 42-kDa form by endogenous protease(s) with removal of C-terminal region (20). The possibility may be
suggested that the endogenous protease in Sf9 cells which is responsible for such conversion was lost or inactivated during the
preparation of membrane protein fraction from Sf9 cells, or that
the enzymatic activity cannot be detected by the MCP assay system used
in this study.
Processing of Recombinant 43-kDa SH-EP (C152G) by
Papain--
Processing pattern of the recombinant 43-kDa SH-EP
(C152G) by papain was observed to see whether general cysteine protease has proteolytic activity that converts the substrate to 42-kDa form,
since MCP was revealed to be a member of papain family. When the
recombinant 43-kDa SH-EP (C152G) was incubated with papain, the
substrate was processed to 33-kDa form. Immunoblot analysis of the
processed SH-EP with antibody to mature SH-EP or to N-terminal propeptide of SH-EP indicated that the 33-kDa SH-EP cleaved by papain
was not immunoreactive to the antibody specific to N-terminal prosequence of SH-EP (Fig.
10C) but to antibody against
mature SH-EP (Fig. 10B). These suggest that the recombinant
43-kDa SH-EP (C152G) was processed to the 33-kDa form, which is
corresponding to the mature enzyme, through intermolecular proteolysis
by papain. This result is consistent with the report that the
recombinant 43-kDa SH-EP (C152G) was intermolecularlly processed to the
mature form by active mature SH-EP (22).
Although MCP was initially isolated as a protease which is
involved in post-translational cleavage of the C-terminal propeptide of
SH-EP containing the KDEL-tail within the ER, immunocytochemical analysis of cotyledon cells revealed that MCP is localized in PSV as
well as the ER, and the amino acid sequence of MCP deduced from its
cDNA indicated that the enzyme is a member of papain family which
are potentially degradative enzymes. The involvement of MCP in
degradation of storage proteins is consistent with the report that an
MCP homologue from Vicia faba was isolated as an enzyme
responsible for globulin hydrolysis in cotyledons of germinated V. faba seeds (30, 31). It has been shown that there are
some kinds of proteinases in the cotyledons of germinated V. mungo seeds (17, 28) and other papain-type proteinases are thought to exist in the cotyledons, however, only MCP showed the proteolytic activity that converts the recombinant 43-kDa SH-EP (C152G) to the
42-kDa form. In addition, general cysteine proteinase, papain, cleaved
the recombinant 43-kDa SH-EP (C152G) to the 33-kDa form through removal
of the N-terminal prosequence of the proenzyme. This processing profile
is apparently different from that of the recombinant 43-kDa SH-EP
(C152G) by MCP. This suggest the possibility that the processing
activity for the conversion of the recombinant 43-kDa SH-EP (C152G) to
the 42-kDa form is unique to MCP.
The present results of sucrose gradient centrifugation and
immunocytochemistry of cotyledon cells suggest the possibility that MCP
exists as an active mature enzyme in the ER in association with the
membrane, although papain-type proteases have not been expected to be
localized in the ER lumen since the ER is a site for folding and
oligomerization of nascent proteins (1-3). However, MCP was detected
as the mature enzyme in a microsome preparation in which SH-EP was
presented as the proenzyme (Fig. 8, A and B), suggesting the enrichment of the ER in the microsomal preparation, and
supporting the possibility that mature MCP is localized in the ER. Most
papain-type proteases are synthesized as zymogens with the large
N-terminal proregion which is essential for correct folding of the
enzyme (32), contains intracellular transport signal (33), and plays a
role in inactivation of the protein (34-36). The activation mechanisms
of the proform of papain-type proteases have been elucidated mainly for
propapain (32, 35), procathepsin L (37, 38), and procathepsin B (39),
and the mechanisms are largely conserved among these proenzymes. The
N-terminal proregion occluding the active site with antiparallel
peptide chains is removed by autocatalytic proteolysis triggered by
acidification (40, 41). We have also succeeded in in vitro
activation of recombinant pro-SH-EP (22), but recombinant pro-MCP was
not converted to the mature enzyme under the conditions of acidic and
nutral pH (data not shown). An activation mechanism different from that
of pro-SH-EP, procathepsin L, and propapain may account for the
activation of pro-MCP. In fact, the in vitro activation mechanism of proforms of proteases belonging to the same subfamily as
MCP has not yet been resolved. Therefore, the processing profiles of
MCP was analyzed by heterologous expression system in insect Sf9
cells. It is notable that mature MCP was detected only in the membrane
protein fraction prepared from Sf9 cells expressing MCP, and
that the other MCP-related polypeptides of 35, 37, and 38 kDa were
detected only in the soluble protein fraction (Fig. 9A). In
addition, MCP activity derived from expressed MCP was detected in the
membrane protein fraction (Fig. 9C). This suggests that the
membrane association of pro-MCP in Sf9 cells is needed for
correct processing to the mature form, and pro-MCP in the soluble
protein fraction is processed abnormally or degraded to the 35-, 37-, and 38-kDa polypeptides. Membrane association may have a crucial role
in post-translational processing of pro-MCP.
A hydropathy plot for the MCP protein did not show the existence of a
hydrophobic transmembrane domain on the MCP polypeptide (data not
shown). Membrane association of papain-type proteases has been reported
in procathepsin L (42, 43) and the soybean cysteine protease P34
(44-46). Procathepsin L associates with microsomal membranes at pH 5 via a 6-amino acid residue region in the N-terminal prosequence, which
is similar to the vacuolar targeting sequence of yeast proenzymes, and
the binding is essential for transport of the proenzyme from the Golgi
complex to lysosomes through a mannose 6-phosphate-independent pathway
(43). However, MCP must associate with the membrane through part of the
mature enzyme, rather than the proregion, since mature MCP was detected
in the membrane protein fraction (Figs. 8A and
9A), suggesting that the membrane association process for
MCP differs from that for procathepsin L. A candidate amino acid
sequence of MCP for association with the microsomal membrane is the
N-terminal region of mature MCP, LPANAQKAPILP (Fig. 4B). The
region is positioned upsteam from the N-terminal amino acid residues of
the papain-type proteases belonging to the other subfamily, is strongly
conserved only among the proteases of the MCP subfamily, and shows high
hydrophobicity. This may suggest that this amino acid sequence has some
functional role, and investigations to see the biological function of
the sequence are now underway in our laboratory. P34, a papain-type protease from soybeans, was first identified as a membrane-associated protease of oil bodies of cotyledon cells of soybean seeds (44, 45),
but later reports indicated that p34 is localized in vacuoles, and that
disruption of the cells resulted in the release of P34 from vacuoles
and subsequently P34 associates with the membranes of oil bodies (46).
It was suggested that the association of P34 might be only a fortitous
event or that the association reflects some aspect of the functions of
P34 (46). The possibility of a merely fortitous association of MCP with
the membrane will be ruled out by experiments involving proteinase K
treatment of microsomes and subsequent immunoblotting (Fig.
8C). The results strongly supported the conclusion that MCP
detected as a membrane-associated protein is localized in the luminal
side of the microsomes, and that MCP does not simply bind
nonspecifically to the microsomal membrane on the cytoplasmic side
during preparation of the microsomes. The specific association of MCP
with the luminal membrane is also indirectly supported by the results
of immunocytochemistry showing that MCP is localized along the membrane
of the ER (Fig. 6B) and on peripheral regions and membranous
structures of specific areas of PSV where degradation of storage
proteins is progressing (Fig. 7B). In addition, the
heterologous expression of MCP in Sf9 cells and subsequent
sublocalization within the cells revealed that the MCP protein itself
possesses the property of associating with the membrane (Fig. 9). From
these observations, the features of membrane association of MCP are
considered to reflect some biological role. In the ER lumen, soluble
proteins are able to move freely, but the movement of
membrane-associated proteins is thought to be restricted and it is
considered likely that membrane association will result in
compartmentalization of the proteins in some region of the lumen.
Molecular chaperones in the ER bind to nascent proteins until the
nascent proteins are correctly folded or oligomerized (1-3). Membrane
association of MCP on the luminal side of the ER may reduce the
opportunity for assembly with nascent proteins in the lumen of the ER
prior to binding of chaperones to the proteins, resulting in the
protection of MCP against nonspecific proteolysis of the protein.
Although the in vivo substrates of MCP in the ER lumen
remain to be identified, at least MCP was revealed to process the
C-terminal prosegment of SH-EP in vitro. One hypothesis is
that MCP in the ER lumen is involved in cleavage of the
C-terminal region of reticuloplasmins possessing K(H)DEL-tails,
resulting in escape from the ERD2 retention system and degradation of
the proteins for turnover processes in the vacuoles. We herein
postulate that MCP is localized in both the ER and PSV as a
membrane-associated protease, and plays dual roles in cells, in the
post-translational cleavage during the C-terminal processing of
pro-SH-EP, and in the degradation of storage proteins in PSV.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thioglucoside and 10 mM 2-mercaptoethanol. The resuspended solution was again
centrifuged at 100,000 × g for 60 min, and the
supernatant was used as the starting material for column chromatography
procedures. The solution was applied to a column (1.6 × 20 cm) of
DEAE-cellulose (Whatman DE-52) that had been pre-equilibrated with 50 mM sodium phosphate buffer (pH 7.2) containing 0.1%
n-octyl-
-D-thioglucoside and 10 mM
2-mercaptoethanol. The loaded column was first washed with the same
buffer and subsequently eluted with a linear gradient (100 ml/100 ml)
of 0 to 0.5 M KCl in the buffer. The eluate was collected
in 10-ml fractions at a flow rate of 1.5 ml/min. The active fractions
(60 ml) were placed in a dialysis tube and the volume was reduced to 3 ml by use of polyethylene glycol 20,000. The concentrated solution was
loaded onto a column (1.0 × 75 cm) of Sephacryl S-200 (Amersham
Pharmacia Biotech) which was pre-equilibrated with 50 mM
sodium phosphate buffer (pH 7.2) containing 0.1%
n-octyl-
-thioglucoside, 10 mM
2-mercaptoethanol, and 0.2 M KCl. The column was eluted with the buffer at a flow rate of 10 ml/h, and 5-ml fractions were
collected. The eluate (2 ml) containing the active fraction was
dialyzed against distilled water, and the dialyzed solution was
lyophilized to a powder. The powder was dissolved in 40 µl of
SDS-PAGE sample buffer. After boiling for 3 min, the sample solution
was subjected to 12.5% SDS-PAGE and electroblotted onto a
polyvinylidene difluoride membrane (Millipore) (23). The membrane was
stained with 0.1% Coomassie Brilliant Blue in 50% methanol and
7% acetic acid. The band corresponding to MCP was cut out from
the membrane, and the N-terminal amino acid sequence was determined
using an automated sequence analyzer (Model 477A, Applied Biosystem).
32P]dCTP using a Random Primer
DNA Labeling kit (Takara), and then used as a probe for screening the
cDNA library from the day 3 cotyledons. DNA sequencing was
conducted using a BigDye Dye Terminator Sequencing kit (PerkinElmer
Life Sciences) and an ABI 310 DNA sequencer (Applied Biosystem).
-amylase was prepared as described previously (25). Antiserum raised
against maize BiP (26) was provided from Dr. Eliot Herman (USDA/ARS,
Beltsville). Monoclonal antibody, 1D3, recognizing the C-terminal KDEL
sequence was purchased from StressGene (Victoria, Canada).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (37K):
[in a new window]
Fig. 1.
The enzymatic activity of MCP acting to
process the 43-kDa SH-EP (C152G) to the 42-kDa form
(A) and effects of inhibitors on the MCP activity
(B). A, cotyledons (30 g) of day 3 dark-grown seedlings were homogenized with 62.5 ml of 0.1 M
Tris-Cl (pH 7.4) containing 0.44 M sucrose, 1 mM EDTA, and 0.1 mM MgCl2. The
homogenate was centrifuged at 800 × g for 10 min and
then at 4,500 × g for 30 min. The supernatant was
again centrifuged at 100,000 × g for 60 min. The
supernatant was used as the 100,000 g sup.
fraction, and the precipitate was washed with homogenization
buffer and resuspended with 10 ml of the buffer. The resuspended
solution was sonicated (3 × 1 min, 30 W) and centrifuged at
100,000 × g for 60 min. The supernatant was used as
the luminal proteins of the microsomes, and the precipitate was washed
with the buffer and solubilized with 50 mM sodium phosphate
buffer (pH 7.2) containing 1% Triton X-100 and 10 mM
2-mercaptoethanol. The resuspended solution was again centrifuged at
100,000 × g for 60 min, and the supernatants were used
as the membrane protein fraction of the microsomes. The enzymatic
activity of MCP was assayed as described under "Experimental
Procedures." B, several kinds of inhibitors were added to
the reaction mixture which contained the substrate and membrane protein
fraction described in A. C, same reaction mixtures with
B were analyzed by SDS-PAGE/immunoblotting with anti-SH-EP
antibody. The concentrations of the inhibitors were:
L-trans-epoxysuccinyl-leucylamido-(4-guanidinobutane)
(E-64), 10 µM; diisopropyl fluorophosphate
(DFP), 1 mM; pepstatin A, 10 µM;
EDTA, 1 mM.
View larger version (71K):
[in a new window]
Fig. 2.
Sucrose gradient centrifugation of microsomes
from cotyledons of day-3 V. mungo seedlings and
subsequent analysis. Microsomes prepared from 25 g of day 3 cotyledons were resuspended in 1.5 ml of buffer A (0.1 M
Tris-Cl, pH 7.4, 0.44 M sucrose, 1 mM EDTA, and
0.1 mM MgCl2). The suspension was centrifuged
at 200 × g for 5 min, and the supernatant was layered
on a continuous 0.6-1.6 M sucrose gradient and centrifuged
at 100,000 × g for 17 h. After the centrifugation
was completed, the gradient was fractionated into 0.7-ml fractions.
Fifty microliters of each fractionated sample was pooled for the
analysis described in C. The remaining sample (0.65 ml) was
mixed with 0.7 ml of buffer A, and the mixture was sonicated (1 min, 30 W). After centrifugation at 100,000 × g for 60 min,
the precipitate was washed with buffer A and solubilized with 0.5 ml of
50 mM sodium phosphate buffer (pH 7.2) containing 1%
Triton X-100 and 10 mM 2-mercaptoethanol. The solution was
again centrifuged at 100,000 × g for 60 min, and the
supernatants were used for the procedures described in A and
B. After incubation of each sample with the substrate, the
mixture was separated by SDS-PAGE in duplicate. One gel was stained
with Coomassie Brilliant Blue for measurement of MCP activity
(A), and the other was immunoblotted with anti-KDEL antibody
to check for the removal of the C-terminal KDEL from the substrate
(B). Each fraction from the sucrose gradient was analyzed by
SDS-PAGE immunoblotting with antiserum against maize BiP
(C).
-D-thioglucoside was used as a detergent during the purification procedures instead of
the Triton X-100 used in the MCP assay (Fig. 1A), since
Triton X-100 often interfered with the column chromatography
procedures. The enzyme was purified to homogeneity by DEAE-cellulose
column chromatography and gel filtration. All active fractions from the DEAE-cellulose column chromatography were applied to gel filtration (Fig. 3A, upper panel). The
proteolytic activity to the recombinant 43-kDa SH-EP (C152G) was
detected in fractions 18 and 19 (Fig. 3A). The molecular
mass of MCP was 32 kDa as judged by its mobility in SDS-PAGE (Fig.
3B). The N-terminal amino acid sequence of the enzyme was
determined to be Leu-Pro-Ala-Asn-Ala-Glu-Lys-Ala-Pro-Ile (Fig.
4B).
View larger version (36K):
[in a new window]
Fig. 3.
Separation of MCP by column chromatography
(A) and SDS-PAGE of purified MCP
(B). A, the active fraction from
DEAE-cellulose column chromatography was subjected to gel filtration on
Sephacryl S-200 (upper panel). Experimental details are
described under "Experimental Procedures." The results of
measurements of MCP activity in separated fractions were presented in
the lower panel. Inset in upper panel, MCP
activity. B, fractions 16-21 in A was separated
by SDS-PAGE and the gel was silver stained. Arrow indicates
MCP polypeptide.
View larger version (65K):
[in a new window]
Fig. 4.
Phylogenetic tree (A) and
alignment of amino acid sequences (B) of papain-type
proteases. A, the phylogenetic tree was constructed by
the neighbor-joining method (47) without gap regions which were
generated for maximum matching. Numbers along each branch
are bootstrap values (48). Proteases of the MCP subfamily are
bracketed. B, after alignment of the amino acid
sequences of papain-type proteases, a portion around the N-terminal
amino acid residues of the mature forms of the proteases were analyzed.
The line above the sequence indicates the determined
N-terminal amino acid sequence of MCP. The open arrowhead
indicates the N-terminal amino acid residue of MCP. The closed
arrowheads indicate N-terminal amino acid residues of papain,
SH-EP, aleurain, cathepsin L, cathepsin B, REP-1, EPB p34, proteinase
A, and oryzain . The open circle indicates the active
site cysteine. Accession numbers of sources are: MCP, AB038598; french
bean CP, Z99953; soybean isoform B, U71380; soybean isoform A, U71379;
pea CP, X54358; fava bean CP, U59465; vetch CPR2, Z30338; tobacco CP,
Z14028; arabidopsis RD19, D13042; soybean CP, Z32795; mouse cathepsin
L, P06797; Maize CP, 99936; barley aleurain, X58859; arabidopsis RD21,
D13043; rice oryzain
, D90406; barley EPB, U19359; rice REP-1,
D76415; barley EPA, Z97023; rice REP-A, D76416; vetch proteinase A,
Z34895; french bean EP-C1, X56753; mung bean SH-EP, X15723; soybean
p34, J05560; papaya papain, M15203; mouse cathepsin B, M14222.
-amylase antibodies.
-Amylase has been
resolved to be localized in vacuoles of cotyledon cells of V. mungo seeds (25).2
The major band of MCP was detected in fractions 5-7, BiP in fractions 5-8, and
-amylase in fractions 6-9 (Fig.
5, A-C), suggesting that MCP
in the microsomal fraction was localized in the ER and vacuoles. In
addition, MCP was detected as an enzymatically active mature form of 32 kDa even in ER fractions (Fig. 5, A and B, fractions 6 and 7). These suggest the possibility that MCP exists
as an active enzyme in the ER. When immunogold labeling of cotyledon cells with affinity purified antibody to MCP was conducted, gold particles were localized in the ER although the staining was not very
dense (Fig. 6, A and
B). Gold particles were also localized in PSV (Fig.
7, A and B) in
addition to the ER. Localization of gold particles from anti-MCP
antibody on Golgi complex suggested that vacuolar sorting of MCP is
mediated through Golgi-dependent pathway (Fig.
6C). Interestingly, MCP was abundant in specific regions of
PSV where degradation of storage proteins is occurring. Ultrastructural
analysis of cotyledon cells indicated that degradation of storage
proteins starts at the inside of PSV, and the size of the white-colored
regions gradually enlarges as degradation progresses (Fig.
7C, PSV1 to PSV3). These findings strongly suggested that,
in PSV, MCP plays a role in the early stage of degradation of storage
proteins.
View larger version (58K):
[in a new window]
Fig. 5.
Sucrose gradient centrifugation of microsomes
from cotyledons of day-3 V. mungo seedlings.
Microsomes prepared from 25 g of day 3 cotyledons were resuspended
in 1.5 ml of buffer A (0.1 M Tris-Cl, pH 7.4, 0.44 M sucrose, 1 mM EDTA, and 0.1 mM
MgCl2). The susupension was centrifuged at 200 × g for 5 min, and the supernatant was layered on a continuous
0.6-1.6 M sucrose gradient and centrifuged at 100,000 × g for 17 h. After the run was completed, the
gradient was fractionated into 0.7-ml fractions. Each fraction from the
gradient was analyzed by SDS-PAGE/immunoblotting using anti-MCP
antiserum (A), anti-BiP antibody (B), and
anti- -amylase antiserum (C).
View larger version (90K):
[in a new window]
Fig. 6.
Electron micrographs showing the localization
of MCP in the endoplasmic reticulum (A and
B) and Golgi complex (C). Gold
particles from anti-MCP antibody were localized in the ER and Golgi
complex of cotyledon cells of germinated V. mungo seeds.
Asterisks, unidentified cell compartments.
View larger version (76K):
[in a new window]
Fig. 7.
Electron micrographs showing the localization
of MCP in protein storage vacuoles (A and
B), and the ultrastructure of cotyledon cells
(C). A, gold particles from anti-MCP
antibody were localized in specific regions of PSV where storage
proteins were degraded. B, a magnified image showing
localization of the gold particles in white-colored regions
of PSV. C, white-colored regions in PSV were
enlarged with progression of storage protein degradation (PSV1 to
PSV3).
View larger version (31K):
[in a new window]
Fig. 8.
Membrane association of MCP in the microsomes
of cotyledon cells. Microsomes prepared from 30 g of day 3 cotyledons were resuspended in 10 ml of buffer A (0.1 M
Tris-Cl, pH 7.4, 0.44 M sucrose, 1 mM EDTA, and
0.1 mM MgCl2). The suspension solution was
sonicated (3 × 1 min, 30 W) and centrifuged at 100,000 × g for 60 min. The supernatant was used as the luminal
protein fraction, and the precipitate was washed with homogenization
buffer, solubilized with 8 M urea, and used as the membrane
protein fraction. Both the luminal and membrane fractions were analyzed
by SDS-PAGE and immunoblotting with anti-MCP antibody (A) or
anti-SH-EP antibody (B). Microsomes prepared from 15 g
of day 3 cotyledons were resuspended in 3 ml of buffer A. The
suspension solution was incubated with proteinase K at a final
concentration of 40 µg/ml with or without 1% Triton X-100. After
incubation at 37 °C for 1 h, each sample was analyzed by
SDS-PAGE/immunoblotting with anti-MCP antibody (C).
Molecular masses from ovalbumin (43-kDa) and carbonic anhydrolase (30 kDa) (Amersham Pharmacia Biotech) were presented at the right
side of panels in A and B.
View larger version (38K):
[in a new window]
Fig. 9.
Distributions of MCP- and SH-EP-related
polypeptides in insect Sf9 cells (A and
B) and production of active MCP in Sf9 cells
(C). A and B, Sf9
cells were infected with MCP or SH-EP baculovirus, and soluble proteins
and membrane proteins of the cells were prepared as described under
"Experimental Procedures." Whole cell extracts, soluble proteins
and membrane proteins of the cells (10 µl each) were analyzed by
SDS-PAGE/immunoblotting with anti-MCP antibody (A) or
anti-SH-EP antibody (B). C, membrane proteins
were prepared from Sf9 cells infected with MCP baculovirus or
uninfected Sf9 cells for assay of MCP activity as described
under "Experimental Procedures." After samples were separated with
SDS-PAGE, the gel was stained with Coomassie Brilliant Blue. Lane
1, membrane proteins from uninfected Sf9 cells; lane
2, membrane proteins from Sf9 cells infected with MCP
baculovirus; lane 3, MCP assay of membrane proteins from
uninfected Sf9 cells; lane 4, MCP assay of membrane
proteins from Sf9 cells infected with MCP baculovirus;
lane 5, substrate and buffer (50 mM sodium
phosphate buffer (pH 7.2) containing 1% Triton X-100 and 10 mM 2-mercaptoethanol) were mixed and incubated.
View larger version (56K):
[in a new window]
Fig. 10.
In vitro processing of
recombinant 43-kDa SH-EP (C152G) by papain. Two microgram of the
recombinant 43-kDa SH-EP (C152G) was incubated with 10 or 100 ng (3 or
3 milliunits) of papain as described under "Experimental
Procedures." After incubation, the samples were separated by
SDS-PAGE. Proteins in the gel was stained with Coomassie Brilliant Blue
(A), or immunoblotted with antibodies to mature SH-EP
(B), or to N-terminal propeptide of SH-EP (C).
The lanes of 0 ng of papain indicates the incubation of the substrate
with buffer (50 mM sodium phosphate buffer (pH 7.2)
containing 1% Triton X-100 and 10 mM 2-mercaptoethanol).
Solid and open arrowheads indicate the processed
SH-EP and papain, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Eliot Herman, USDA/ARS at Beltsville, for the generous gift of antiserum against maize BiP. We are grateful to Dr. Kenji Yamada (Kyoto University, Japan) for useful discussion and to Dr. Koichiro Tamura (Tokyo Metropolitan University, Japan) for reconstructing the phylogenic tree.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Scientific Research Grants-in-Aid 12740441 from the Ministry of Education, Science and Culture of Japan and by Sasakawa Scientific Research Grant from The Japan Science Society (12-246).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biological
Sciences, Tokyo Metropolitan University, Minami-osawa, Hachioji, Tokyo, 192-0397 Japan. Fax: 81-426-77-2559; E-mail:
okamoto-takashi@c.metro-u.ac.jp.
§ Present address: Department of Infectious Diseases and Tropical Medicine Research Institute International Medical Center of Japan Toyama 1-21-1, Shinjuku, tokyo 162-8655, Japan.
Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M003078200
2 K. Toyooka et al., unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ER, endoplasmic reticulum; PSV, protein storage vacuoles; PAGE, polyacrylamide gel electrophoresis; MCP, membrane-associated cysteine protease.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Galili, G., Sengupta-Gopalan, C., and Ceriotti, A (1998) Plant Mol. Biol. 38, 1-29[CrossRef][Medline] [Order article via Infotrieve] |
2. | Okita, T. W., and Rogers, J. C. (1996) Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 327-350[CrossRef] |
3. |
Vital, A.,
and Denecke, J.
(1999)
Plant Cell
11,
615-628 |
4. | Munro, S., and Pelham, H. B. R. (1987) Cell 48, 899-907[Medline] [Order article via Infotrieve] |
5. | Pelham, H. B. R. (1989) Annu. Rev. Cell Biol. 5, 1-23[CrossRef] |
6. | Napier, R. M., Fowke, L. C., Hawes, C. R., Lewis, M., and Pelham, H. B. R. (1992) J. Cell Sci. 102, 261-271[Abstract] |
7. | Lee, H. I., Gal, S., Newman, T. C., and Raikhel, N. V. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11433-11437[Abstract] |
8. | Denecke, J., De Rycke, R., and Botterman, J. (1992) EMBO J. 11, 2345-2355[Abstract] |
9. | Herman, E. M., Tague, B. W., Hoffman, L. M., Kjemtrup, S. E., and Chrispeels, M. J. (1990) Planta 182, 305-312 |
10. | Akasofu, H., Yamauchi, D., Mitsuhashi, W., and Minamikawa, T. (1989) Nucleic Acids Res. 17, 6733[Medline] [Order article via Infotrieve] |
11. | Becker, C., Shutov, A. D., Nong, V. H., Senyuk, V. I., Jung, R., Horstmann, C., Fischer, J., Nielsen, N. C., and Müntz, K. (1995) Eur. J. Biochem. 228, 456-462[Abstract] |
12. | Guerrero, C., de la Calle, M., Reid, M. S., and Valpuesta, V. (1998) Plant Mol. Biol. 36, 565-571[CrossRef][Medline] [Order article via Infotrieve] |
13. | Tanaka, T., Yamauchi, D., and Minamikawa, T. (1991) Plant Mol. Biol. 16, 1083-1084[Medline] [Order article via Infotrieve] |
14. | Valpuesta, V., Lange, N. E., Guerrero, C., and Reid, M. S. (1995) Plant Mol. Biol. 28, 575-582[Medline] [Order article via Infotrieve] |
15. | Schmid, M., Simpson, D., Kalousek, F., and Gietl, C. (1998) Planta 206, 466-475[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Cercos, M.,
Santamaria, S.,
and Carbonell, J.
(1999)
Plant Physiol.
119,
1341-1348 |
17. | Mitsuhashi, W., Koshiba, T., and Minamikawa, T. (1986) Plant Physiol. 80, 628-634 |
18. | Mitsuhashi, W., and Minamikawa, T. (1989) Plant Physiol. 89, 274-279 |
19. | Okamoto, T., Nakayama, H., Seta, K., Isobe, T., and Minamikawa, T. (1994) FEBS Lett. 351, 31-34[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Okamoto, T.,
Minamikawa, T.,
Edward, C.,
Vakharia, V.,
and Herman, E.
(1999)
J. Biol. Chem.
274,
11390-11398 |
21. | Toyooka, K., Okamoto, T., and Minamikawa, T. (2000) J. Cell Biol. 248, 453-463 |
22. |
Okamoto, T.,
Yuki, A.,
Mitsuhashi, N.,
and Minamikawa, T.
(1999)
Eur. J. Biochem.
264,
223-232 |
23. | Hirano, H., Komatsu, S., Takakura, H., Sakiyama, F., and Tsunasawa, S. (1992) J. Biochem. (Tokyo) 111, 754-757[Abstract] |
24. | Suzuki, Y., and Minamikawa, T. (1985) Plant Physiol 79, 327-331 |
25. | Tomura, H., Koshiba, T., and Minamikawa, T. (1981) Plant Physiol. 79, 935-938 |
26. | Kalinski, A. J., Rowley, D. L., Loer, D. S., Foley, C., Butta, G., and Herman, E. M. (1994) Planta 195, 611-621 |
27. |
Hara-Nishimura, I.,
Shimada, T.,
Hatano, K.,
and Nishimura, M.
(1998)
Plant Cell
10,
825-836 |
28. | Okamoto, T., and Minamikawa, T. (1999) Plant Mol. Biol. 39, 63-73[CrossRef][Medline] [Order article via Infotrieve] |
29. | von Heijne, G. (1983) Eur. J. Biochem. 133, 17-21[Abstract] |
30. | Yu, W. J., and Greenwood, J. S. (1994) J. Exp. Bot. 271, 261-268 |
31. | Yu, W. J., and Greenwood, J. S. (1996) Plant Physiol. PGR. 112, 862 |
32. |
Vernet, T.,
Berti, P. J.,
deMontigny, C.,
Musil, R.,
Tessier, D. C.,
Menard, R.,
Magny, M. C.,
Storer, A. C.,
and Thomas, D. Y.
(1995)
J. Biol. Chem.
270,
10838-10846 |
33. | Chrispeels, M. J., and Raikhel, N. V. (1992) Cell 68, 613-618[Medline] [Order article via Infotrieve] |
34. | James, M. N. G., and Sielecki, A. R. (1986) Nature 319, 33-38[Medline] [Order article via Infotrieve] |
35. |
Vernet, T.,
Khouri, H. E.,
Laflamme, P.,
Tessier, D. C.,
Musil, R.,
Gour-Salin, B. J.,
Storer, A. C.,
and Thomas, D. Y.
(1991)
J. Biol. Chem.
266,
21451-21457 |
36. | Carmona, E., Dufour, E., Plouffe, C., Takabe, S., Manson, P., Mort, J. S., and Menard, R. (1996) Biochemistry 35, 8149-8157[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Smith, S. M.,
and Gottesman, M. M.
(1989)
J. Biol. Chem.
264,
20487-20495 |
38. | Nomura, T., and Fujisawa, Y. (1997) Biochem. Biophys. Res. Commun. 230, 143-146[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Mach, L.,
Mort, J. S.,
and Glössl, J.
(1994)
J. Biol. Chem.
269,
13030-13035 |
40. | Coulombe, R., Grochulaki, P., Sivaraman, J., Menard, R., Mort, J. S., and Cygler, M. (1996) EMBO J. 15, 5492-5503[Abstract] |
41. |
Jerala, R.,
Zerovnik, E.,
Kidric, J.,
and Turk, V.
(1998)
J. Biol. Chem.
273,
11498-11504 |
42. | McIntyre, G. F., and Erikson, A. H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10588-10592[Abstract] |
43. |
McIntyre, G. F.,
Godbold, G. D.,
and Erikson, A. H.
(1994)
J. Biol. Chem.
269,
567-572 |
44. | Herman, E. M., Melroy, D. L., and Buckhout, T. J. (1990) Plant Physiol. 94, 341-349 |
45. |
Kalinski, A. J.,
Weisemann, J. M.,
Matthews, B. F.,
and Herman, E. M.
(1990)
J. Biol. Chem.
265,
13843-13848 |
46. |
Kalinski, A. J.,
Melroy, D. L.,
Dwivedi, R. S.,
and Herman, E. M.
(1992)
J. Biol. Chem.
267,
12068-12076 |
47. | Saitou, N., and Nei, M. (1987) Mol. Biol. Evol. 4, 406-425[Abstract] |
48. | Felsenstein, J. (1985) Evolution 39, 783-791 |