Download Characterization of Organelles in the Vacuolar

Plant Cell Physiol. 41(9): 993–1001 (2000)
JSPP © 2000
Characterization of Organelles in the Vacuolar-Sorting Pathway by Visualization with GFP in Tobacco BY-2 Cells
Naoto Mitsuhashi 1, 2, 3, Tomoo Shimada 3, Shoji Mano 1, Mikio Nishimura 1, 2 and Ikuko Hara-Nishimura 3, 4
1
Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan
Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Okazaki, 444-8585 Japan
3
Department of Botany, Graduate School of Science, Kyoto University, Kyoto, 606-8502 Japan
2
;
reported; pumpkin PV72 (Shimada et al. 1997), pea BP-80
(Paris et al. 1997) and Arabidopsis AtELP (Ahmed et al. 2000).
The receptors are a type I integral membrane protein with epidermal growth factor (EGF)-like motifs in the lumenal domain. The lumenal domains of PV72 (our unpublished data)
and BP-80 (Paris et al. 1997) are involved in ligand binding,
while the cytosolic tail of BP-80 is reported to be involved in
efficient recycling of the receptor from prevacuolar compartments to the Golgi complex (Jiang and Rogers 1998).
However, the mechanism responsible for recycling mediated by
the cytosolic tail has not been characterized.
On the other hand, vacuolar-targeting signals on the
polypeptide sequences of soluble vacuolar proteins have been
well characterized. The signals are separated into three classes;
N-terminal propeptides (NTPPs), internal peptides and C-terminal propeptides (CTPPs). An NPIR sequence conserved in
the NTPPs of barley aleurain (Holwerda et al. 1992) and sweet
potato sporamin (Matsuoka and Nakamura 1992) has been
shown to function as a vacuolar-targeting signal and to bind to
the receptors (Cao et al. 2000, Shimada et al. 1997). In contrast to the NTPPs, the CTPPs of barley lectin (Bednarek and
Raikhel 1991) and tobacco chitinase (Neuhaus et al. 1991)
have no significant conserved sequence between them. PV72
has been shown to bind to the C-terminal peptide of 2S albumin, a major storage protein of pumpkin (Shimada et al. 1997).
This raises the question of whether the peptide functions as a
vacuolar-targeting signal of 2S albumin to protein storage vacuoles.
Recently, green fluorescent protein (GFP) is being used in
an increasing number of cell biology studies. GFP makes it
possible for organelles in living cells to be visualized in real
time. Thus, GFP should be a useful tool to investigate the retention or sorting signals of proteins delivered through the secretory pathway and the pathway to the vacuoles in plant cells. In
plants, the modified GFPs have been reported to be localized in
non-acidic vacuoles (Sansebastiano et al. 1998) as well as in
ER (Berger et al. 1995, Saito et al. 1999) and Golgi complex
(Nebenführ et al. 1999, Sansebastiano et al. 1998). GFP fluorescence has not been observed in the acidic and lytic vacuoles
in plants. This raises the second question of whether GFP is de-
We have shown the localization and mobilization of
modified green fluorescent proteins (GFPs) with various
signals in different compartments in a vacuolar-sorting system of tobacco BY-2 cells. In contrast to the efficient secretion of GFP from the transformed cells expressing SP-GFP
composed of a signal peptide and GFP, accumulation of
GFP in the vacuoles was observed in the cells expressing
SP-GFP fused with the C-terminal peptide of pumpkin 2S
albumin. This indicated that this peptide is sufficient for
vacuolar targeting. Interestingly, the fluorescence in the
vacuoles disappeared sharply at 7 d after inoculation of the
cells, but it appeared again after re-inoculation into a new
culture medium. When SP-GFP was fused with the region,
termed PV72C, including a transmembrane domain and a
cytosolic tail of a vacuolar-sorting receptor PV72, GFPPV72C was detected in the Golgi-complex-like small particles. Prolonged culture showed that GFP-PV72C that
reached the prevacuolar compartments was cleaved off the
PV72C region to produce GFP, that arrived at the vacuoles
to be diffused. These findings suggested that the vacuolarsorting receptor might be recycled between the Golgi complex and prevacuolar compartments.
Key words: BY-2 cells — Endoplasmic reticulum — GFP —
Golgi complex — Vacuolar-sorting receptor — Vacuole.
Abbreviations: BCECF, 2,7-bis-(2-carboxyethyl)-5,(6)-carboxyfluorescein; CTPPs, C-terminal propeptides; DEX, dexamethasone;
EGF, an epidermal growth factor; ER, endoplasmic reticulum; EST,
expressed sequence tag; GFP, green fluorescent protein; NTPPs, N-terminal propeptides; PV72, a vacuolar-sorting receptor of pumpkin; TIP,
tonoplast intrinsic protein.
Introduction
Protein trafficking toward vacuoles is composed of highly
complex processes in higher plant cells (Neuhaus and Rogers
1998, Okita and Rogers 1996, Robinson and Bäumer 1998,
Rogers 1998). Three vacuolar-sorting receptors have been
4
Corresponding author: E-mail, [email protected]; Fax, +81-75-753-4141.
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GFP in endomembrane systems
graded and/or protonated not to generate fluorescence under
the acidic condition in the vacuole. If not, GFP could be used to
investigate the vacuolar-targeting machinery.
In contrast to the well-characterized soluble proteins, vacuolar sorting of integral membrane proteins is less understood.
The transport pathway for vacuolar membrane proteins has not
been studied with GFP in living cells. Tonoplast intrinsic proteins (TIPs) are typical integral membrane proteins. It has been
shown that -TIP is specific to protein-storage vacuoles and TIP is to vegetative vacuoles. Both types of vacuoles are found
in the same cells of barley roots (Jauh et al. 1999, Okita and
Rogers 1996, Paris et al. 1996) and of maturing pea cotyledons
(Robinson et al. 1995). In these cells, each TIP synthesized on
the ER is sorted and delivered to the respective vacuoles. The
third question is whether different targeting signals for both
TIPs are involved in their sorting.
To answer these questions, we have visualized the compartments in the transport pathway to the vacuoles in plants by
using GFP tagged with various signals to be localized in different organelles. We also investigated the developmental changes in the fluorescent image during the culture of the cells and in
various culture conditions.
Materials and Methods
Plant materials
Suspension-cultured cells of tobacco BY-2 (Nicotiana tabacum
L. cv. Bright Yellow 2) were kindly provided by Dr. K. Nakamura of
Nagoya University. The cells were subcultured in Murashige-Skoog
medium once a week at 26.5C in the dark with an orbital shaker (BioShaker BR-3000LF, Taitec, Koshigaya, Japan). The BY-2 cells were
transformed with each of the following GFP-chimeric genes.
Plasmid construction
sGFP-TYG was kindly provided by Dr. Y. Niwa of University of
Shizuoka (Chiu et al. 1996). pSGFP-BE, that had both HindIII and
BamHI sites in the 5 flanking region, an NcoI site on the starting codon, and both BglII and EcoRI sites on the stop codon of sGFP-TYG,
was generated from sGFP-TYG by Mano et al. (1999). Based on pSGFP-BE, a chimeric gene encoding each of six modified GFPs as
shown in Figure 1 was constructed.
For the chimeric gene encoding SP-GFP, two annealing complementary oligonucleotides, 5-AGCTTGGATCCATGGCCAGACTCACAAGCATCATTGCCCTCTTCGCAGTGGCTCTGCTGGTTGCAG
ATGCGTACGCCTACCGCAC-3 as a sense strand and 5-CATGGTGCGGTAGGCGTACGCATCTGCAACCAGCAGAGCCACTGCGAAG
AGGGCAATGATGCTTGTGAGTCTGGCCATGGATCCA-3 as an
antisense strand, were synthesized and annealed to produce a doublestranded DNA with protruding ends to be ligated with the HindIII and
NcoI sites. The DNA fragment was inserted into the HindIII-NcoI site
of pSGFP-BE to produce pSP-GFP. The chimeric gene encodes a fusion protein composed of the 22-amino-acid signal peptide followed
by a tripeptide YRT sequence of pumpkin prepro2S albumin and GFP.
For the chimeric gene encoding SP-GFP-HDEL, two annealing
complementary oligonucleotides, 5-GATCTCGGGGGGGGGCACCACCACCACCACCACGATGAGCTTTGAG-3 as a sense strand
and 5-AATTCTCAAAGCTCATCGTGBGTGGTGGTGGTGGTGCCCCCCCCCGA-3 as an antisense strand, were synthesized and annealed to produce a double-stranded DNA with protruding ends to be
Fig. 1 Constructs of modified GFPs that were expressed in tobacco
BY-2 cells. Six modified GFPs are schematically represented. SP-GFP
is composed of the signal peptide (SP) of pumpkin 2S albumin followed by GFP. SP-GFP-HDEL is composed of SP-GFP followed by a
12-amino-acid sequence including an ER-retention signal, HDEL. SPGFP-2SC is composed of SP-GFP followed by a linker of GGG and
the C-terminal 18-amino-acid sequence of pumpkin 2S albumin
including a putative vacuolar-targeting signal, NLPS. SP-GFP-PV72C
is composed of SP-GFP followed by the C-terminal 68-amino-acid
sequence of pumpkin PV72, a vacuolar-sorting receptor. The C-terminal sequence includes a transmembrane domain (TMD) and a cytosolic
tail with a tyrosine-based signal, YMPL. TIP-GFP is composed of
pumpkin -TIP, MP28, followed by GFP. TIP-GFP is composed of
Arabidopsis -TIP that is encoded by an EST clone (Genbank accession number x72581) followed by GFP.
ligated with the BglII and EcoRI sites. The DNA fragment was inserted into the BglII-EcoRI site of pSP-GFP. The chimeric gene encodes a
fusion protein composed of SP-GFP followed by a 12-amino-acid sequence including an ER-retention signal, HDEL.
For the chimeric gene encoding SP-GFP-2SC, two annealing
complementary oligonucleotides, 5-GATCTCGGGGGGGGGAAGGCTAGGAACTTGCCTTCCATGTGCGGAATCCGCCCACAGCGAT
GCGACTTCTGAG-3 as a sense strand and 5-AATTCTCAGAAGTCGCATCGCTGTGGGCGGATTCCGCACATGGAAGGCAAGTTCC
TAGCCTTCCCCCCCCCGA-3 as an antisense strand, were synthesized and annealed to produce a double-stranded DNA with protruding ends to be ligated with the BglII and EcoRI sites. The DNA fragment was inserted into the BglII-EcoRI site of pSP-GFP. The chimeric
gene encodes a fusion protein composed of SP-GFP followed by a
tripeptide, GGG, and the C-terminal 18-amino-acid sequence of pumpkin prepro2S albumin including a putative vacuolar-targeting signal,
NLPS.
For the chimeric gene encoding SP-GFP-PV72C, the DNA fragment, that was produced by PCR-amplification using PV72 cDNA as a
template and a set of the oligonucleotide primers, 5-AGTAGATCTCGGTAACATTGGGAGCACT-3 and 5-TATGAATTCTCATACGCCCCCACGGGC-3, was inserted into the BglII-EcoRI site
of pSP-GFP. The chimeric gene encodes a fusion protein composed of
SP-GFP followed by the C-terminal 68-amino-acid sequence of pumpkin PV72, a vacuolar-sorting receptor. The C-terminal sequence includes a transmembrane domain and a cytosolic tail with a tyrosinebased signal, YMPL.
For the chimeric gene encoding TIP-GFP, the DNA fragment,
that was produced by PCR-amplification using a cDNA for pumpkin
GFP in endomembrane systems
MP28 (-TIP) as a template and a set of the oligonucleotide primers,
5-AAGCTTATGCCGCCGAGACGATATGCC-3 and 5-CCATGGAGCCGCCGCCGTAATCTTCCGGAGCTAAAGG-3, was inserted
into the HindIII-NcoI site of pSGFP-BE. The chimeric gene encodes a
fusion protein composed of -TIP and GFP.
For the chimeric gene encoding TIP-GFP, the DNA fragment,
that was produced by PCR-amplification using an expressed sequence
tag (EST) clone for Arabidopsis -TIP (Genbank accession number
x72581) as a template and a set of the oligonucleotide primers, 5AAGCTTATGCCGATCAGAAACATCGCC-3 and 5-CCATGGAGCCGCCGCCGTAGTCGGTGGTTGGGAGCTG-3, was inserted into
the HindIII-NcoI site of pSGFP-BE. The chimeric gene encodes a fusion protein composed of -TIP and GFP.
The chimeric genes encoding SP-GFP, SP-GFP-HDEL, SP-GFP2SC and SP-GFP-PV72C were inserted into pBI121. Both chimeric
genes encoding TIP-GFP and TIP-GFP were inserted into
pMAT037. The gene encoding TIP-GFP was also inserted into
pTA7002 to use a glucocorticoid dexamethasone (DEX)-inducible system (Aoyama et al. 1995). These plasmids were then introduced into
Agrobacterium tumefaciens (strain EHA101; Hood et al. 1986) by
electroporation. BY-2 cells were transformed with each of the chimeric genes via A. tumefaciens according to the method of Matsuoka and
Nakamura (1991).
Extraction from the transformant cells and immunoblot analysis
The transformed BY-2 cells were gently filtered to be packed.
Each 1 g of the packed cells was homogenized in 2 ml of the following buffers. The buffer of 10 mM Tris-HCl, pH 7.5, including proteinase inhibitors (Complete Mini, Boehringer Mannheim, Tokyo, Japan)
was used for SP-GFP/BY2, SP-GFP-HDEL/BY2 and SP-GFP-2SC/
BY2 that accumulated soluble GFPs. The other buffer of 10 mM TrisHCl, pH 7.5, including the proteinase inhibitors and 0.1% SDS was
used for SP-GFP-PV72C/BY2, TIP-GFP/BY2, TIP-GFP/BY2 that
accumulated GFPs in the cellular membranes. The homogenates were
centrifuged at 15,000g and 4C for 20 min to obtain the cellular extracts as supernatant solutions.
To analyze secreted proteins, the culture medium of the transformed BY-2 cells at 4 d after inoculation into a new medium was used
as described by Saito et al. (1999). The medium was filtered through
four layers of cheesecloth to remove the cells. The filtrate was centrifuged at 5,000g and 4C for 15 min. The supernatant was treated with
70% (w/v) ammonium sulfate for 1 h followed by centrifugation. The
precipitate was suspended with 5 mM Tris-HCl, pH 7.0, and then dialyzed against 5 mM Tris-HCl for 15 h to obtain a solution containing
secreted proteins.
Immunoblot analysis was performed essentially as described previously (Inoue et al. 1995). The above sample solutions were subjected to SDS-PAGE and the separated proteins on gels were transferred
electrophoretically to a GVHP membrane (0.22 m; Nihon Millipore,
Tokyo, Japan). The membrane blot was incubated with specific antibodies against GFP (diluted 1,000-fold, Clontech, Palo Alto, CA,
U.S.A.) for 1 h. Horseradish peroxidase-conjugated antibodies raised
in donkey against rabbit IgG (Amersham Japan, Tokyo, Japan) were
diluted 10,000-fold and used as second antibodies. Immunodetection
was performed with an enhanced chemiluminescence kit (an ECL system, Amersham Japan) according to the manufacturer’s directions.
Fluorescent microscopy and laser-scanning confocal microscopy
The transformed BY-2 cells were inspected with a fluorescence
microscope (Axiophot 2, Carl Zeiss, Jena, Germany) using a filter set
(an excitation filter; BP450–490, a dichroic mirror; FT510, a barrier
filter; BP515–565, Carl Zeiss), a CCD camera (CoolSNAP, RS Photometrics, Chiba, Japan), and a light source (Arc HBO 100W, Atto, To-
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kyo, Japan). The lytic vacuoles of non-transformed BY-2 cells that
were stained with 2,7-bis-(2-carboxyethyl)-5,(6)-carboxyfluorescein
(BCECF) were also inspected with the fluorescent microscope. The
transformed BY-2 cells were also examined with a laser-scanning confocal microscope (LSM510, Carl Zeiss) equipped with a krypton-argon laser and a filter set for GFP.
Results
Secretion of GFP from the SP-GFP/BY2 cells and accumulation
of GFP-HDEL in the ER of the SP-GFP-HDEL/BY2 cells
GFP itself is known to be localized in cytosol and/or nuclear matrix in the plant cells transformed with a GFP gene. To
visualize the compartments involved in the transport of vacuolar proteins, we constructed the modified GFPs with various
signals to be localized in different organelles, as shown in Figure 1.
BY-2 cells were transformed with a chimeric gene encoding SP-GFP composed of the signal peptide of pumpkin 2S albumin and GFP to generate a transformant SP-GFP/BY2. Figure 2A and 2B shows that fluorescence was detected on the
network and nuclear envelope of the SP-GFP/BY2 cells. An
immunoblot of the extract of the cells with GFP-specific antibodies revealed that a 28-kDa protein was accumulated in the
cells (Fig. 2F, lane 1). The molecular mass of 28-kDa was consistent with the calculated mass for GFP itself, 27,919 Da. An
18-kDa degradation product was detected in the culture medium as well as a slight amount of the 28-kDa GFP (Fig. 2F, lane
2). These findings indicated that the co-translational cleavage
of the signal peptide of SP-GFP produces GFP that is secreted
into the medium to be degraded.
SP-GFP-HDEL was composed of SP-GFP followed by an
ER-retention signal, HDEL. The transformant SP-GFP-HDEL/
BY2 cells show a strong and stable fluorescence on the network within the cells (Fig. 2C, D, E). The fluorescent image on
the network shows the ER-network. An immunoblot of the cellular extract showed that a 30-kDa protein was accumulated in
the ER (Fig. 2F, lane 3). The molecular mass of 30 kDa was
consistent with 29,264 Da of the calculated mass for GFPHDEL. The 30-kDa GFP-HDEL was retained within the ER,
but not secreted into the culture medium (Fig. 2F, lane 4).
A C-terminal peptide of 2S albumin as a vacuolar-targeting
signal
To elucidate the vacuolar-targeting signal of 2S albumin, a
major storage protein, we constructed SP-GFP-2SC composed
of SP-GFP followed by the C-terminal 18-amino-acid peptide
(2SC) of pumpkin 2S albumin (Fig. 1). The peptide could bind
to PV72, a vacuolar-sorting receptor of developing pumpkin
seeds (Shimada et al. 1997).
The SP-GFP-2SC/BY2 callus cells were inspected with a
laser-scanning confocal microscope. Figure 3A shows that fluorescence was observed in the large compartments as well as
the ER-network and/or nuclear envelope. All the compart-
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GFP in endomembrane systems
Fig. 3 Sorting of GFP-2SC to vacuoles in the transformant SP-GFP2SC/BY2 cells. BY-2 cells were transformed with a p35S::sp-gfp-2sc
gene to produce a transformant, SP-GFP-2SC/BY2. The callus cells
were inspected with a laser-scanning confocal microscope. Comparison of the confocal image (A) with that of the stainable vacuoles with
BCECF of non-transformed cells (B) shows that GFP fluorescence was
observed in both the ER-networks and vacuoles of the transformant.
Bars = 20 m.
Fig. 2 Secretion of GFP from the transformant SP-GFP/BY2 cells,
and accumulation of GFP-HDEL in the ER of the transformant SPGFP-HDEL/BY2 cells. BY-2 cells were transformed with each of a
p35S::sp-gfp gene and a p35S::sp-gfp-hdel gene to produce transformants, SP-GFP/BY2 (left) and SP-GFP-HDEL/BY2 (right), respectively.
(A–D) The 3-day-old transformant cells were inspected with a fluorescent (FL) or differential-interference-contrast (DIC) microscope. Fluorescent images (A, C) show the localization of the modified GFPs
within the cells and DIC micrographs (B, D) show the cellular structures of the respective fields. (E) The fluorescent ER networks were
observed in the SP-GFP-HDEL/BY2 cells with a higher magnification. Bars = 20 m. (F) Both the cellular extract (c) and the culture
medium (m) from each transformant of SP-GFP/BY2 and SP-GFPHDEL/BY2 were subjected to SDS-PAGE and subsequent immunoblot
analysis with GFP-specific antibodies. The 28-kDa GFP was synthesized on the ER of the SP-GFP/BY2 cells (lane 1) and then secreted
into the culture medium to be degraded to form the 18-kDa product
(lane 2). The 30-kDa GFP-HDEL was synthesized to be accumulated
in the ER of the SP-GFP-HDEL/BY2 cells (lane 3) and no GFPrelated proteins were detected in the medium of the cells (lane 4).
ments were stainable with BCECF (Fig. 3B), that is known to
give the fluoresced vacuoles of the BY-2 cells (Matsuoka et al.
1997). This indicated that GFP-2SC was transported and accumulated in the vegetative vacuoles. The confocal image
showed that the vacuoles had a complicated structure rather
than a single central vacuole (discussed below). The detailed
confocal image of the SP-GFP-2SC/BY2 cells showed that fluorescence was also detected in small particles with a similar
size to that of Golgi complex (discussed below). Such small
particles were not detected in the SP-GFP-HDEL/BY2 cells.
These findings indicated that the C-terminal peptide functions
as a targeting signal for vacuoles via Golgi complex.
Cyclical change in the fluorescent image and the accumulation
level of GFP during the growth of SP-GFP-2SC/BY2 cells
Although the SP-GFP-2SC/BY2 cells exhibited fluorescent vacuoles (Fig. 3A), the fluorescent image was not stable in
contrast to the stable fluorescent image throughout the growth
of the SP-GFP/BY2 and SP-GFP-HDEL/BY2 cells. The fluorescent pattern of the SP-GFP-2SC/BY2 cyclically changed
during the cell culture (Fig. 4A).
The 9- to 10-day-old cells in a stationary phase gave faint
fluorescence only around the nuclear envelope. The cells with a
large central vacuole were inoculated into a new medium to be
cultured for another cycle of 9 d. The 2-day-old cells were proliferated and divided into the smaller cells. The fluorescent intensity around the nuclear envelope and the ER-network increased and reached the maximum in the 3-day-old cells with
GFP in endomembrane systems
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Fig. 4 Cyclical changes in the fluorescent images and the level of the expressed GFP during the growth of the transformant SP-GFP-2SC/BY2
cells. (A) The 10-day-old transformant cells were transferred to a new medium and were cultured for another 9 d. During the growth, the cells
were inspected in the same field with a fluorescent (FL) or differential-interference-contrast (DIC) microscope. The numbers at the bottom represent days after inoculation of the transformant cells into fresh medium. Bars = 20 m. (B) The transformant cells at each day after inoculation as
in A were collected. Each cellular extract containing 10 g total proteins was subjected to SDS-PAGE and subsequent immunoblot analysis with
GFP-specific antibodies. The numbers represent days after inoculation as in A.
the smallest size. Fluorescence was also detected in small particles within the cytosol of the cells (discussed below). The fluorescent vacuoles were found in the 4-day-old cells and the fluorescence increased up to 6 d after inoculation. It should be
noted that the fluorescence rapidly disappeared in 7-day-old
cells and only a slight fluorescence was detectable around the
nuclear envelope in the 8- to 9-day-old cells. Interestingly,
when the 10-day-old cells were transferred into fresh medium,
the change in the above fluorescent patterns was repeated cyclically (data not shown).
To clarify the change in the accumulation level of GFP
during the cell growth, immunoblot analysis of the extract from
the SP-GFP-2SC/BY2 cells was performed with GFP-specific
antibodies (Fig. 4B). The molecular mass of the accumulated
protein was the same as that of 28-kDa GFP, indicating that
GFP itself was accumulated in the vacuoles after removal of
2SC peptide. The accumulation level of the GFP protein increased and reached the maximum at 6 d after inoculation of
the cells and then dropped at 7 d after inoculation. The change
in the GFP level during the growth was associated with the
change in the fluorescent intensity found in the vacuoles (Fig.
4A).
A C-terminal region including the transmembrane domain and
the cytosolic tail of a vacuolar-sorting receptor, PV72,
functions in recycling of the receptor to Golgi complex
To clarify the mechanism for recycling of a vacuolar-sorting receptor, we focused on the C-terminal region of pumpkin
PV72. We constructed SP-GFP-PV72C composed of SP-GFP
fused with the C-terminal region (PV72C) that included the
transmembrane domain and the cytosolic tail with a tyrosinebased motif, YMPL (Fig. 1). Figure 5A shows that the fluores-
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GFP in endomembrane systems
cence was localized in the small particles in the 3-day-old cells.
A similar fluorescent image of the small particles in the cytosol was observed in the SP-GFP-2SC/BY2 cells (Fig. 3A,
4A). The image is consistent with fluoresced Golgi complex as
reported by Nebenführ et al. (1999). These findings suggested
that the C-terminal region including the transmembrane domain and the cytosolic tail of a vacuolar-sorting receptor is responsible for the presence of the receptor in Golgi complex. An
immunoblot of the cellular extract showed that the 36-kDa protein was accumulated (Fig. 5C, lane 1). The molecular mass of
36 kDa was consistent with the calculated mass of 35,325 Da
for GFP-PV72C.
In contrast to the distinct localization of GFP-PV72C in
Golgi complex of the suspension cultured cells, the fluorescence was observed within the vacuoles of the 10-day-old cells
of the SP-GFP-PV72C/BY2 callus (Fig. 5B). The fluorescence
within the vacuoles indicated that GFP-PV72C with a transmembrane domain was converted to soluble and diffusible
GFP. An immunoblot of the cellular extract showed that the
vacuoles accumulated the 28-kDa protein with the same molecular mass of GFP itself (Fig. 5C, lanes 2, 3). The proteolysis of
GFP-PV72C into GFP might occur in the prevacuolar compartments of the 10-day-old callus cells. It has been shown that
BY-2 cells under such conditions give a high proteolytic activity (Moriyasu and Ohsumi 1996). By the high proteolytic activity, GFP-PV72C could be cleaved off the PV72C region to produce GFP that was diffused within the vacuoles and caused the
fluoresced vacuoles.
Both TIP-GFP and TIP-GFP are similarly targeted to the
vacuoles of BY-2 cells
The next issue to be resolved concerned a selective sorting for protein-storage vacuoles and vegetative vacuoles. To
answer the question of whether different targeting signals for
-TIP and -TIP are involved in their sorting to the respective
type of the vacuoles, we constructed TIP-GFP and TIP-GFP
to be expressed in the BY-2 cells. Figure 6 shows that both
TIP-GFP (Fig. 6A, B) and TIP-GFP (Fig. 6C, D) were localized on the vacuolar membranes of the 3-day-old cells in a similar pattern. This indicated that both the TIP-GFP fusion targeted to the same vacuoles in the BY-2 cells. One possibility that
could not be excluded was that the free C-terminal region of
each TIP functions as a selective targeting signal for the respective vacuole. To examine this possibility, we constructed
the GFP-TIP and GFP-TIP fusion proteins to make the Cterminal regions of TIPs free as the authentic TIPs. These GFPTIP fusion proteins gave a fluorescent pattern on the vacuolar
membrane of the transformed BY-2 cells similar to that of the
TIP-GFP and TIP-GFP fusion proteins (data not shown).
In the cells within several weeks after transformation, the
localization of TIP-GFP on the ER network was found (Fig.
6F). The transformant cells with a DEX-inducible system also
showed the localization of TIP-GFP on the ER-network with-
Fig. 5 Developmental changes in fluorescent images and localization of the expressed GFP during the growth of the transformant SPGFP-PV72C/BY2 cells. BY-2 cells were transformed with a p35S::spgfp-pv72c to produce a transformant, SP-GFP-PV72C/BY2. The 3day-old suspension cells (A) and the 10-day-old callus (B) of the transformant were inspected with a confocal and fluorescent microscope,
respectively. Fluorescent images of the transformant were changed
from a Golgi-complex type (indicated by arrowheads) into a vacuole
type (indicated by asterisks) during the growth of the cells. Bars =
20 m. (C) Each cellular extract containing 10 g total proteins from
the 3-day-old cells and 10-day-old callus was subjected to SDS-PAGE
and subsequent immunoblot analysis with GFP-specific antibodies.
The 3-day-old cells accumulated a 36-kDa GFP-PV72C in the Golgi
complex (lane 1) and the 10-day-old cells accumulated the 28-kDa
GFP, but not GFP-PV72C, in the vacuoles (lane 2). The 28-kDa GFP
from the SP-GFP/BY2 cells is also shown as a marker (lane 3).
in a few days after treatment with DEX (Fig. 6G). These finding suggested that both TIP-GFP and TIP-GFP are first localized on the ER and then transported to vacuolar membranes.
GFP in endomembrane systems
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Fig. 6 Sorting of a fusion protein of a vacuolar membrane protein followed by GFP in the transformant TIP-GFP and TIP-GFP cells. BY-2
cells were transformed with each of a p35S::tip-gfp and a p35S::tip-gfp to produce transformants, TIP-GFP/BY2 and TIP-GFP/BY2, respectively. (A-D) The 3-day-old cells of both transformants were inspected in the same field with a fluorescent (FL; A, C) or differential-interferencecontrast (DIC; B, D) microscope. Similar accumulation patterns of TIP-GFP (A, B) and TIP-GFP (C, D) on the vacuolar membranes were
observed. (E) A confocal image of the 3-day-old TIP-GFP/BY2 cells shows the distinct localization of TIP-GFP on the vacuolar membranes of
the cells. (F) A fluorescent image of the TIP-GFP/BY2 cells within several weeks after transformation shows the predominant localization of
TIP-GFP on the ER-network (indicated by arrowheads) rather than on the vacuolar membrane. (G) BY-2 cells were transformed with a fusion
gene of tip-gfp under the control of a DEX-inducible promoter. A confocal image of the transformant cells within a few days after induction with
DEX shows the localization of TIP-GFP both on the ER-network (indicated by arrowheads) and on the vacuolar membranes. (H) A three-dimensional structure of vacuoles in the TIP-GFP/BY2 cells was reconstituted with sequential confocal images taken along the optical z-axis through a
complete cell. Bars = 20 m.
This reflects the transport pathway of the integral membrane
proteins to vacuolar membranes.
The fluorescent images of TIP-GFPs clearly showed a
complex structure of vacuoles. The three-dimensional structure of the vacuoles of the =TIP-GFP/BY2 cells was reconstituted with sequential confocal images (Fig. 6H). The three-dimensional image of vacuoles revealed that several large central
vacuoles were folded within a cell. This reflects the several
compartments found as vacuoles in the thin focal plane (Fig.
3).
Discussion
Visualization of various compartments in a vacuolar-sorting
pathway of the living cells of tobacco BY-2
The ER-networks was visualized with GFP in the SPGFP-HDEL/BY2 cells, as expected because of the ER-reten-
tion signal HDEL (Fig. 1E). The strong and stable fluorescent
image of SP-GFP-HDEL/BY2 can be the best marker of ER in
the living cells. SP-GFP-HDEL has also been reported to be
stably expressed in the transformed Arabidopsis plants
(Haseloff et al. 1997). In addition, the ER-network was also
fluoresced transiently in the transformed cells expressing the
modified GFPs that were synthesized on the ER to be secreted
or transported to vacuoles. The SP-GFP/BY2 cells that secreted GFP into the culture medium exhibited a similar fluorescent
image, but the intensity was much lower than that of the SPGFP-HDEL/BY2 cells (Fig. 1A). The SP-GFP-2SC/BY2 and
TIPs-GFP/BY2 cells gave a transient fluorescence on the ERnetwork, where both the soluble and membrane proteins are
synthesized to be transported to vacuoles (Fig. 4A).
On the other hand, the fluorescence on the ER network in
the SP-GFP-PV72C cells was very weak, because of the low
expression level of the GFP fusion. The 36-kDa GFP-PV72C
1000
GFP in endomembrane systems
protein was found to be localized in small particles of the cytosol. The particles moved along with cytoplasmic streaming in
the transvacuolar strands of the cells (data not shown). The particles were less than 1 m in diameter (Fig. 5A). Similar structures in BY-2 cells have been reported by Nebenführ et al.
(1999). They expressed a fusion protein GmMan1::GFP composed of a Golgi-complex resident protein, -1,2 mannosidase
I (GmMan1), fused with N terminus of GFP. They confirmed
the localization of GmMan1::GFP in Golgi complex by an immunoelectron microscopic analysis. Thus, the GFP-PV72Ccontaining particles are identical to the Golgi complex. This is
also supported by the evidence that BP-80 (Hinz et al. 1999)
and AtELP (Sanderfoot et al. 1998) are localized in the Golgi
complex.
A similar fluorescence on the small particles was also
found in the GFP-2SC/BY2 cells (Fig. 4A), an indication that
GFP-2SC passed through the Golgi complex, together with a
vacuolar-sorting receptor. In contrast, TIP-GFP fusions did not
give such fluoresced small particles at all, in spite of giving the
fluoresced ER-networks (Fig. 6). The results suggested that integral membrane proteins such as TIPs might be transported to
the vacuoles bypassing the Golgi complex. This is consistent
with the report that TIP is sorted to vacuoles in a Golgi-independent manner (Jiang and Rogers 1998).
Figure 3 shows that GFP-2SC was finally transported to
the vacuoles. This indicated that the C-terminal-18-amino-acid
is responsible for targeting to vacuoles. The SP-GFP-2SC/BY2
cells can be used to investigate the molecular mechanism in the
protein-trafficking to plant vacuoles.
Cyclical change in fluorescent pattern of GFP depend on the
cell growth
The SP-GFP-2SC/BY2 cells show a cyclical change in
fluorescent image and the accumulation level of GFP during
the cell culture (Fig. 4). Such cyclical change has not been reported in the stable transformants, although a time-dependent
transition of fluorescent images was observed in the tobacco
cells with a transient expression of GFP fused with the C-terminal peptide of tobacco chitinase (Sansebastiano et al. 1998).
The chimeric gene driven by CaMV 35S promoter should
generate a constitutive expression of GFP-2SC in the SP-GFP2SC cells. Thus, the cyclical change in the GFP level in the
vacuoles might be reflected by the cyclic ups and downs of the
accumulation of proteinases in the vacuoles. We observed the
highest activity of vacuolar processing enzyme in the 7- to 9day-old cells and the lowest activity in the 3-day-old cells (data
not shown). Vacuolar processing enzyme have been suggested
to be responsible for the maturation and activation of vacuolar
proteinases (Kinoshita et al. 1999). Proteolytic activity of the
vacuoles might be the highest in the 7-day-old cells. The rapid
disappearance of GFP in the 7-day-old cells might be caused
by the proteolytic degradation within the vacuoles.
It has been also shown that the BY-2 cells under sucrose
starvation conditions accumulated proteolytic enzymes in the
cells to degrade cellular components (Moriyasu and Ohsumi
1996). The soluble GFP fusions incorporated into the vacuoles
could be degraded in transformed BY-2 cells as found in the
GFP-2SC cells. This might be one of the reasons why GFP fluorescence has not been observed in the acidic and lytic vacuoles in plants.
Recycling of a vacuolar-sorting receptor between the Golgi
complex and prevacuolar compartments
The SP-GFP-PV72C/BY2 cells showed that the fluorescence of the 36-kDa membrane protein of GFP-PV72C in the
Golgi complex disappeared followed by the appearance of fluorescence of the 28-kDa soluble GFP within the vacuoles (Fig.
5). This suggested that PV72 might be recycled between the
Golgi complex and prevacuolar compartments, as pea BP-80
(Jiang and Rogers 1998). We have reported that PV72 has an
affinity with the C-terminal peptide (2SC) of pumpkin 2S albumin (Shimada et al. 1997). It is likely that PV72 in the Golgi
complex transport pro2S albumin to the prevacuolar compartments with itself and is retrieved to the Golgi complex after releasing the ligand in the compartments. Thus, we could visualize the recycling process visible in the living SP-GFP-PV72C
cells.
Prolonged culture reduced the fluorescence of GFP in the
vacuoles of the SP-GFP-PV72C/BY2 cells (data not shown), as
in the vacuoles of the SP-GFP-2SC cells (Fig. 4). This might
be also caused by the proteolytic degradation of GFP in the
cells. The non-specific degradation occurred in the vacuoles of
the cells. In contrast, the disappearance of the fluorescence of
GFP within the vacuoles, the fluorescence on the vacuolar
membranes was extremely stable during the growth of the TIPGFP/BY2 cells, since the GFP moiety was exposed to the cytosol but not to the vacuolar interior.
Sorting of both types of TIPs to the same vacuoles
-TIP and -TIP are useful markers for protein-storage
vacuoles and lytic vacuoles, respectively (Paris et al. 1996).
Both TIPs are localized in different vacuoles in the same cells
of barley roots (Okita and Rogers 1996, Paris et al. 1996) and
of maturing pea cotyledons (Robinson et al. 1995). This suggested that these TIPs are delivered to their respective vacuoles according to each targeting signal on their polypeptide
chains. However, unexpectedly, both TIP-GFP and TIP-GFP
fusion proteins targeted to the same vacuole in the transformed
BY-2 cells (Fig. 6). Within several weeks after transformation
or within a few days after DEX-induction, TIP-GFP and
TIP-GFP were detected on the ER, but not on the Golgi complex. These findings suggested that both TIP-GFP and TIPGFP are transported to vacuolar membranes via the same pathway that might be bypassed or quickly passed through the
Golgi complex. It remains to be solved how these TIPs are
sorted and delivered to the respective type of vacuole in the
plant cell.
GFP in endomembrane systems
Acknowledgements
We are grateful to Dr. Niwa of University of Shizuoka for his
kind donation of the modified GFP gene with a strong fluorescence
and both Dr. Aoyama of Kyoto University and Dr. Chua of Rockefeller University for their kind donation of pTA7002 to generate a glucocorticoid-inducible system. This work was supported by Grants-inAids for ‘Research for the Future’ Program (JSPS-RFTF96L60407)
from the Japan Society for the Promotion of Science and for Scientific
Research (nos. 10440244 and 10182102) from the Ministry of Education, Science, Sports and Culture of Japan.
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(Received July 7, 2000; Accepted July 28, 2000)