Plant Cell Physiol. 43(7): 726–742 (2002)
JSPP © 2002
BP-80 and Homologs are Concentrated on Post-Golgi, Probable Lytic Prevacuolar Compartments
Yu-Bing Li 1, Sally W. Rogers 2, Yu Chung Tse 1, Sze Wan Lo 1, Samuel S. M. Sun 1, Guang-Yuh Jauh 3 and
Liwen Jiang 1, 4
1
Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, U.S.A.
3
Institute of Botany, Academic Sinica, Taiwan
2
;
quently deliver that cargo to the vacuole by fusion with the tonoplast (Bethke and Jones 2000). Based on precedents from
mammalian and yeast systems (Lemmon and Traub 2000), they
are intermediate organelles on the pathways to vacuoles from
both Golgi and from endocytosis of the plasma membrane.
Pathways for endocytosis in plant cells are not well defined
(Battey et al. 1999), so our attention will focus primarily on
Golgi to PVC traffic. PVCs exist to provide a place from which
missorted proteins can be retrieved and sent back to the Golgi
(Lemmon and Traub 2000). Inherent in this model is the concept that delivery of proteins to vacuoles is, in most cases, an
irreversible destination. Thus, PVCs must be defined both
functionally (a site for retrieval) and morphologically (on the
pathways to vacuoles but separate from them) (Robinson et al.
2000).
In yeast and mammalian systems, two different types of
proteins have served as markers for the PVC/endosome. First,
sorting receptors that directed soluble proteins at the transGolgi network (TGN) into transport vesicles destined for the
PVC/endosome were defined. These sorting receptors cycle
between the TGN and PVC (reviewed in Robinson and Hinz
1997). In mammalian cells, the mannose 6-phosphate receptor
has this function but, interestingly, is predominantly concentrated in late endosomes (Griffiths et al. 1988). Second, individual Rab GTPases and syntaxins are associated with each
organelle. In yeast, for example, Pep12p is a syntaxin specifically associated with the PVC/endosome (Pelham 2000).
In contrast to yeast and mammalian cells, plant cells contain two functionally distinct vacuoles: the protein storage
vacuole (PSV) and lytic vacuole (Hoh et al. 1995, Paris et al.
1996, Jauh et al. 1999, Vitale and Raikhel 1999). Additionally,
multiple pathways using distinct transport vesicles are responsible for transporting proteins to the PSV and lytic vacuole in
plant cells (Hara-Nishimura et al. 1998, Hinz et al. 1999, Jiang
and Rogers 1999, Vitale and Raikhel 1999, Jiang and Rogers
2001, Vitale and Galili 2001). There is increasing evidence
suggesting that PVCs exist in plant cells and that they play a
similar role in protein trafficking in the plant secretory pathway (Robinson and Hinz 1999, Bethke and Jones 2000). Identification of the PVCs will enable functional definition of their
roles in the complex plant vacuolar system and the multiple
Prevacuolar compartments (PVCs) are membranebound organelles that mediate protein traffic between Golgi
and vacuoles in the plant secretory pathway. Here we identify and define organelles as the lytic prevacuolar compartments in pea and tobacco cells using confocal immunofluorescence. We use five different antibodies specific for a
vacuolar sorting receptor (VSR) BP-80 and its homologs to
detect the location of VSR proteins. In addition, we use
well-established Golgi-markers to identify Golgi organelles.
We further compare VSR-labeled organelles to Golgi
organelles so that the relative proportion of VSR proteins
in Golgi vs. PVCs can be quantitated. More than 90% of
the BP-80-marked organelles are separate from Golgi
organelles; thus, BP-80 and its homologs are predominantly concentrated on the lytic PVCs. Additionally,
organelles marked by anti-AtPep12p (AtSYP21p) and antiAtELP antibodies are also largely separate from Golgi
apparatus, whereas VSR and AtPep12p (AtSYP21p) were
largely colocalized. We have thus demonstrated in plant
cells that VSR proteins are predominantly present in the
lytic PVCs and have provided additional markers for defining plant PVCs using confocal immunofluorescence. Additionally, our approach will provide a rapid comparison
between markers to quantitate protein distribution among
various organelles.
Keywords: Prevacuolar compartments — Vacuolar sorting
receptor.
Abbreviations: DIP, dark intrinsic protein; Man1, mannosidase I;
PAC, precursor-accumulating vesicle; MVBs, multivesicular bodies;
RMR protein, receptor homology region-transmembrane domain-Ring
H2 motif; PSV, protein storage vacuole; PVCs, prevacuolar compartments; V-PPase, vacuolar pyrophosphatase; VSR, vacuolar sorting
receptor.
Introduction
Prevacuolar compartments (PVCs) are defined as
organelles that receive cargo from transport vesicles and subse4
Corresponding author: E-mail, [email protected]; Fax, +852-2603-5646.
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Plant prevacuolar compartments
vesicular pathways leading to vacuoles (Jiang and Rogers
1999, Vitale and Raikhel 1999, Robinson et al. 2000, Vitale
and Galili 2001).
Several approaches have been used to identify and characterize PVCs in plant cells. In Arabidopsis, AtPep12p (or
AtSYP21, Sanderfoot et al. 2000), a yeast Pep12p homologue
that can functionally complement the yeast pep12 mutant, has
been localized to what was termed a PVC (a late post-Golgi
compartment) by electron microscopic (EM) immunocytochemistry (immunoEM) and subcellular fractionation in Arabidopsis root tip cells (Bassham et al. 1995, Conceição et al.
1997). Another approach utilizes a vacuolar sorting receptor,
BP-80 (Kirsch et al. 1994) that belongs to a member of a novel
family of membrane proteins termed vacuolar sorting receptor
(VSR) proteins (Ahmed et al. 1997, Paris et al. 1997, Shimada
et al. 1997). BP-80 was first purified because it bound to the
vacuolar targeting determinant of proaleurain, a cysteine protease that has served as a functional marker for lytic vacuoles
(Paris et al. 1996, Jauh et al. 1999). It was thus predicted to be
a sorting receptor for the lytic vacuole pathway. Studies of the
traffic of BP-80 were carried out using a chimeric reporter that
was transiently expressed in tobacco suspension culture cell
protoplasts (Jiang and Rogers 1998). The transmembrane
domain and cytoplasmic tail of BP-80 were sufficient and specific for targeting a reporter protein via Golgi to the lytic PVC
where the lumenal domain of the reporter, comprised of the
proenzyme for aleurain, was proteolytically processed into
mature form and separated from its membrane association. Proteolytic processing of proaleurain therefore provided a precise
definition for one function of lytic PVC. As would be expected
from its molecular composition, this chimeric reporter colocalized with endogenous tobacco VSR proteins as demonstrated
by double labeling in confocal immunofluorescence (Jiang and
Rogers 1998). In addition, the functional implication of BP-80
as a vacuolar sorting receptor has been demonstrated in vitro
biochemically (Cao et al. 2000) and in vivo using a yeast
expression system (Humair et al. 2001).
The subcellular localization of VSR proteins has been carried out mainly using two techniques: immunoEM and subcellular fractionation. Studies using these two approaches have
shown that VSR proteins are not present in the vacuolar
membrane or tonoplast (Ahmed et al. 1997, Paris et al. 1997,
Sanderfoot et al. 1998, Hinz et al. 1999, Ahmed et al. 2000).
BP-80 has been localized by immunoEM to the Golgi apparatus and a lytic PVC approximately 250 nm in diameter but not
presented in any vacuolar membrane (tonoplasts) of root tip
and cotyledon cells of pea (Paris et al. 1997). AtELP, a BP-80
homolog from Arabidopsis, has been located to Golgi and
putative PVC ~100 nm in diameter in Arabidopsis root tip cells
(Sanderfoot et al. 1998). Using subcellular fractionation, BP-80
was found to co-fractionate partially with Golgi membrane in
pea cells (Hinz et al. 1999), as did AtELP in both Arabidopsis
(Ahmed et al. 1997, Sanderfoot et al. 1998) and transgenic Arabidopsis plants expressing a mammalian Golgi enzyme alpha-
727
2,6-sialyltransferase (Wee et al. 1998). Additionally, AtELP cofractionated in sucrose density gradients of Arabidopsis root
membrane (Sanderfoot et al. 1998) with AtPep12p (AtSYP21p),
a protein that can functionally complement the yeast pep12
mutant and has been localized to what was termed a PVC (a
late post-Golgi compartment) by immunoEM (Bassham et al.
1995, Conceição et al. 1997, Sanderfoot et al. 2000). These
results together suggest that VSR proteins are localized in both
the Golgi apparatus and a putative post-Golgi PVC in these
cells. However, the natural distribution and functional role of
VSR proteins in PVCs vs. Golgi apparatus remain unclear, and
additional markers that can be used to define PVCs in plant
cells other than Arabidopsis are yet to be identified (Bethke
and Jones 2000).
In contrast to PVCs specific to the lytic vacuole pathway,
putative PVCs for the protein storage vacuoles (PSVs) have
been identified and characterized. In developing pea seeds,
multivesicular bodies (MVBs) ranging in size from 0.5 to several mm containing storage proteins have been postulated as
PVCs for PSVs (Robinson et al. 1998). On the other hand, in
developing pumpkin seeds, precursor-accumulating vesicles
(PAC) less than 0.5 mm in size may serve as PVCs for ERderived storage proteins (Hara-Nishimura et al. 1998). Similarly, in developing tobacco seeds and root tip cells, cytosolic
organelles ~0.5–1 mm in size that are labeled by antibodies to
the tonoplast intrinsic protein isoform DIP (for dark intrinsic
protein) (Culianez-Macia and Martin 1993) may serve as PVCs
for both ER- and Golgi-derived proteins leading to PSVs (Jiang
et al. 2000). These DIP organelles contain RMR proteins, receptor-like proteins that traffic through the Golgi to PSVs (Jiang et
al. 2000). The mechanism proposed for PSV formation suggests that the multivesicular bodies (Robinson et al. 1998), precursor-accumulating vesicles (Hara-Nishimura et al. 1998), and
DIP organelles (Jiang et al. 2000, Jiang et al. 2001) fuse with
developing PSVs to deliver their cargo (Robinson et al. 2000).
Here we address the related questions of the relative abundance of VSR proteins in different cell compartments, and their
utility as markers for specific compartments. We take advantage of the availability of one polyclonal anti-peptide antibody
RA3 that is specific to the N-terminal region of pea BP-80, and
four different monoclonal antibodies that recognize BP-80 and
its homologs (Paris et al. 1997, Jiang and Rogers 1998, Cao et
al. 2000). Our approach is based on the principle that labeling
of the same organelle with two or more completely different
antibodies recognizing different parts of the same protein provides strong evidence for the presence of that protein in the
organelle. We therefore use the different anti-BP-80 antibodies
to identify organelles containing VSR proteins in both pea and
tobacco cells and compare their localization in a quantitative
manner to that of three established Golgi markers. Here we
demonstrate that organelles labeled by anti-VSR antibodies are
largely (more than 90%) separate from Golgi organelles, hence,
VSR proteins are predominantly concentrated on the lytic
PVCs. Additionally, organelles marked by anti-AtPep12p
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Plant prevacuolar compartments
(AtSYP21p) and anti-AtELP antibodies are also largely separate from Golgi apparatus, and that VSR and AtPep12p
(AtSYP21p) colocalize. Thus, we demonstrate for the first time
that VSR proteins are predominantly present in the PVCs and
provide additional valuable markers to identify PVCs in cells
other than Arabidopsis, including pea and tobacco, using confocal immunofluorescence.
Results
Anti-BP-80 antibodies colocalized in pea root tip cells
BP-80, a probable vacuolar sorting receptor (VSR) for
sorting protein to the lytic vacuole, was first isolated and
cloned from pea (Kirsch et al. 1994, Paris et al. 1997). Two
antibodies against BP-80 were then prepared: RA3, a rabbit
polyclonal antibody raised against a synthetic peptide corresponding to the N-terminal region of BP-80 that only recognizes the pea BP-80, and a monoclonal antibody (MAb) 14G7
raised against purified BP-80 protein from pea (Paris et al.
1997). These two antibodies colocalized in pea root tip cells in
immunofluorescent double labeling (Paris et al. 1997). Additionally, at the immunoEM level, RA3 antibodies labeled both
Golgi apparatus and what was defined as a prevacuolar compartment (clear vesicle-like structures about 250 nm in diameter that appeared to fuse with larger vacuoles) in pea root tip
cell (Paris et al. 1997). Three additional MAbs, 17F9, 18E7,
and 19F2 that recognize different epitopes on BP-80 were subsequently obtained and characterized (Jiang and Rogers 1998,
Cao et al. 2000). MAb 14G7 recognizes the third EGF repeat or
the Ser/Thr-rich sequence (or both) of BP-80, whereas MAb
19F2 sees the first and second EGF repeat sequences of BP-80,
while epitopes for both 17F9 and 18E7 are contained entirely
within the N-terminal unique domain of BP-80 (Cao et al.
2000). In addition, both 14G7 and 17F9 were shown to immunoprecipitate the same endogenous tobacco VSR proteins
(Jiang and Rogers 1998), as well as to immunoprecipitate VSR
proteins from extracts of tobacco stigma cells (Miller et al.
1999).
Here we want to determine if the different antibodies
against BP-80 recognize the same structure in pea root tip cells
using double labeling and confocal immunofluorescence (Fig. 1).
Consistent with previous results, 14G7 and RA3 detect the
same structures in cytoplasm (Fig. 1, panel 5). When the extent
of colocalization of these two antibodies was quantified in 45
cells, 93±7% of the fluorescent signals detected by these two
antibodies superimposed (Table 1, line 5). In contrast, 14G7
does not colocalize with calnexin, an ER marker (Fig. 1, panel
6; Table 1, line 6). When the four monoclonal antibodies were
Fig. 1 Colocalization of anti-VSR antibodies in pea root-tip cells.
Four monoclonal antibodies (14G7, 17F9, 18E7 and 19F2) and one
polyclonal (RA3) were used for double labeling in confocal immunofluorescence as described (Jiang et al. 2000). Calnexin is a marker for
ER. Shown are images from single optical section of confocal immunofluorescence. Open arrows and arrowheads indicate examples of
colocalization and separation of the two antibodies, respectively. n,
nucleus. Bar = 10 mm.
Plant prevacuolar compartments
Table 1
729
Quantitation of antibodies colocalization in confocal immunofluorescence images
Antibodies compared
VSR markers in pea root-tip cells (Fig. 1)
1. 14G7 : 17F9
2. 17F9 : 14G7
3. 14G7 : 18E7
4. 17F9 : 19F2
5. 14G7 : RA3
6. 14G7 : Calnexin
VSR vs. Golgi in pea cells (Fig. 4)
7. JIM84 : 14G7
8. JIM84 : 17F9
9. JIM84 : 19F2
10. JIM84 : RA3
11. JIM84 : Calnexin
Golgi markers in transgenic BY-2 cells (Fig. 5, 7)
12. GFP : anti-GFP
13. GFP : Calnexin
14. GFP : JIM84
15. GFP : anti-Man1
16. Man1 : JIM84
VSR vs. Golgi in transgenic BY-2 cells (Fig. 8)
17. 14G7 : GFP
18. 17F9 : GFP
19. 14G7 : JIM84
20. 17F9 : JIM84
Pep12/AtELP vs. Golgi in tobacco and tomato cells (Fig. 10)
21. JIM84 : Pep12 (SYP21) (tomato)
22. JIM84 : Pep12 (SYP21) (tobacco)
23. Pep12 (SYP21) : GFP
24. AtELP : GFP
25. JIM84 : AtELP
26. JIM84 : AtELP
VSR vs. Pep12 in pea and tomato cells (Fig. 11)
27. 14G7 : Pep12 (SYP21)
28. 17F9 : Pep12 (SYP21)
29. 14G7 : AtELP
30. 17F9 : AtELP
31. Pep12 (SYP21) : AtELP
Percent colocalization
(mean±SD)
n
92±7
91±5
91±7
89±8
93±7
5±3
43
47
42
41
45
35
7±3
8±4
9±5
8±4
6±4
51
47
37
55
36
96±3
4±4
85±11
93±5
92±5
47
46
52
51
53
8±4
7±4
9±5
8±5
48
52
55
43
9±5
8±6
7±5
8±7
11±7
8±6
48
47
35
53
43
45
82±6
80±4
85±6
80±7
82±7
23
22
23
20
15
Antibody markers for Golgi: JIM84 (a trans-Golgi marker), Man1 (mannosidase I), and GFP (Man1-GFP
fusion expressed in transgenic tobacco BY-2 cells, a cis-Golgi marker. VSR markers: RA3, 14G7, 17F9,
18E7, and 19F2. Calnexin is a marker ER. Pep12 (SYP21) (anti-AtPep12p) is a PVC marker in Arabidopsis
and AtELP is an Arabidopsis homolog identical to VSRAt-1. The antibodies were used to detect proteins in
pea, tomato and tobacco cells. GFP represents the Man1-GFP fusion that is expressed constitutively in transgenic tobacco BY-2 cells and is a marker for cis-Golgi. Quantitation of the extent of colocalization for the
two antibodies (or proteins) was performed from one direction only (e.g. it asked how much of the VSR signal colocalized with AtPep12p (AtSYP21p), not the other way around) as previously described (Jiang and
Rogers 1998). Percent colocalization is expressed as the mean ±SD (standard derivation) for the (n) number
of cells analyzed.
compared directly to each other via double labeling in confocal immunofluorescence in pea root tip cells, again, more than
90% of the signals generated by the two antibodies colocalized
(Fig. 1, panels 1–4 and Table 1, lines 1–4). In addition, the
labeling results with the two monoclonal antibodies were independent of the order in which the two primary antibodies were
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Plant prevacuolar compartments
applied (Fig. 1, panels 1–2). Thus, all these MAbs recognize
the same organelles as does the polyclonal RA3 antibody in
pea root tip cells. The antibodies are therefore reliable markers
for detecting BP-80 (and presumably other VSR proteins for
the MAbs, also see below) in pea cells. For simplicity, we will
henceforth refer to all these monoclonal antibodies as anti-VSR
antibodies.
Fig. 2 VSR-marked organelles are separate from vacuoles marked by
V-PPase in pea root tip cells. Double labeling using anti-VSR (14G7)
MAb and anti-V-PPase polyclonal antibodies was performed and
detected using confocal immunofluorescence. Shown are images from
single optical section. V-PPase is a general vacuolar marker in root tip
cells while JIM84 is a marker for Golgi apparatus and sometimes
plasma membrane. Open arrows and arrowheads indicate examples of
colocalization and separation of the two antibodies, respectively. n,
nucleus; v, vacuole. Bar = 10 mm.
VSR-labeled organelles are distinct from vacuoles
Vacuolar pyrophosphatase (V-PPase) has been used as a
general vacuolar marker in root tip cells of many plant species
including pea and tomato (Jiang and Rogers 1999, Jauh et al.
1999, Jiang et al. 2000). The sizes of vacuoles in different cell
types may be varied and the origin of vacuolar tonoplast
remained to be determined, even though with the recent demonstration that VACUOLELESS1, an Arabidopsis ortholog of
yeast Vps 16p, is essential for vacuole formation in Arabidopsis (Rojo et al. 2001).
To further confirm that VSR proteins locate to organelles
(i.e. Golgi and PVC) that are distinct from vacuolar compartments in the plant secretory pathway, we compared VSRmarked organelles to vacuoles that are marked by anti-V-PPase
antibodies in pea root tip cells (Fig. 2). Anti-V-PPase antibodies labeled the tonoplasts of vacuolar compartments of various
sizes in different cell types (Fig. 2, panels 1–3, green). These
V-PPase-labeled vacuolar compartments are mostly separate
from organelles marked by anti-VSR (14G7) antibodies as
indicated by the separation of red color (14G7) from green
color (V-PPase) (panels 1–3). When more than 20 cells of each
cell type were analyzed, less than 5% of the VSR-marked
organelles (red) colocalized with V-PPase-marked vacuoles.
Similar results were obtained from using the other anti-VSR vs.
anti-V-PPase antibodies (date not shown). Thus, essentially the
same results were obtained from different cell types with different sizes of vacuolar compartments. The partial colocalization (as indicated by yellow color and open arrow, Fig. 2,
panels 1–3) between VSR (red) and vacuole (green) might
represent areas where the fusion between PVC and vacuole
occurs. In addition, when JIM84, a monoclonal antibody that
marks Golgi apparatus and sometimes plasma membrane
(Satiat-Jeunemaitre and Hawes 1992, Fitchette et al. 1999),
was used to compare V-PPase-marked vacuoles, the JIM84marked Golgi apparatus and/or plasma membrane (Fig. 2,
panels 4 and 5, red) are also mostly separate from V-PPasemarked vacuoles (panels 4 and 5, green). Thus, both VSRlabeled organelles and JIM-84-marked Golgi apparatus are
mostly separate from the vacuolar compartments of various cell
types with different sizes of vacuoles.
Anti-BP-80 MAbs detect an Arabidopsis BP-80 homolog
BP-80 from pea and its homologs of other plant species
such as Arabidopsis are very closely related with numerous
conserved regions (Hadlington and Denecke 2000). The Arabidopsis BP-80 homolog (VSRAt-1, Paris et al. 1997) is identi-
Plant prevacuolar compartments
731
Fig. 3 Anti-BP-80 antibodies recognize the Arabidopsis BP-80
homolog VSRAt-1. The truncated form (missing its TMD/CT) of VSRAt-1,
which is identical to AtELP, was expressed and secreted in insect cells.
For Western blot detection, affinity-purified recombinant protein
equivalent to 100 ml of culture media was loaded on to each lane, followed by SDS-PAGE and immunoblotting. Each identical strip was
labeled by individual anti-BP-80 antibodies. The arrow indicates the
position of the truncated AtBP-80. The individual antibody used in
Western blot detection is indicated above each lane (1–5). The positions of molecular mass marker are indicated in kilodaltons on the left.
cal to the Arabidopsis AtELP (Ahmed et al. 1997). To further
test the specificity and cross-reactivity of anti-BP-80 MAbs in
other plant species, we expressed the VSRAt-1 in Drosophila S2
cells (Cao et al. 2000) as a truncated protein lacking transmembrane and cytoplasmic domains. Affinity-purified recombinant
protein was then used for Western blot detection using various
anti-BP-80 antibodies. Results were shown in Fig. 3. All four
monoclonal anti-BP-80 antibodies (19F2, 18E7, 14G7 and
17F9) detected the Arabidopsis BP-80 homolog VSRAt-1 specifically as indicated by the appearance of a single band of the
expected size in a Western blot (Fig. 3, lanes 2–5, arrow). In
contrast, RA3, a polyclonal antibody raised against a synthetic
peptide corresponding to the pea BP-80 that is very specific for
the pea BP-80 protein (Paris et al. 1997), did not recognize the
VSRAt-1 (Fig. 3, lane 1), indicating its high specificity for pea
BP-80. RA3 did not recognize the Arabidopsis VSR homologs
in either Western blot detection of protein extracts or confocal
immunofluorescence of Arabidopsis root tip cells (data not
shown). So far at least six BP-80 homologs have been identified from Arabidopsis thaliana, where the positions of the
transmembrane domain and the numerous cysteine residues are
highly conserved within these BP-80 isoforms (Hadlington and
Denecke 2000). The overall sequence homology among BP-80
isoforms is very high, with ~86% amino acid identity between
pea BP-80 and the AtBP-80a, whereas the similarity between
pea BP-80 and the VSRAt-1 (AtBP-80b or AtELP) was ~70%
(Hadlington and Denecke 2000). MAb 14G7 recognizes the
third EGF repeat or the Ser/Thr-rich sequence (or both) of BP80, whereas MAb 19F2 sees the first and second EGF repeat
sequences of BP-80, while epitopes for both 17F9 and 18E7
are contained entirely within the N-terminal unique domain of
BP-80 (Cao et al. 2000). Thus, the different MAbs are useful
markers in detecting other VSR homologs that may be divergent in sequence from BP-80. In addition, since MAbs 14G7
Fig. 4 Prevacuolar organelles labeled by anti-VSR antibodies are
separate from Golgi organelles marked by JIM84 in pea root-tip cells.
Shown are 3D images created from combinations of 15 optical confocal sections (1 mm per section) from the top to bottom of individual
cells. JIM84 is a trans-Golgi marker. The organelles labeled by JIM84
and VSR antibodies are indicated as red and green color, respectively.
Panels on the right present DIC images of single optical collection of
confocal microscope using transmission light. Open arrows indicate
examples of colocalization of the two antibodies. Bar = 10 mm.
and 19F2 detect the EGF repeats of BP-80, it is thus likely
these two MAbs might be useful to see even the more distantly
related members of Arabidopsis BP-80 isoforms. However, we
do not know which Arabidopsis BP-80 isoforms were detected
by these BP-80 MAbs in addition to the Arabidopsis BP-80
homolog VSRAt-1.
BP-80 and its homologs are predominantly concentrated on
post-Golgi, prevacuolar compartments in pea root tip cells
Previous studies demonstrated the presence of BP-80 in
Golgi and organelles with characteristics of PVCs, but there
was no information in those studies as to how the protein was
distributed between the two organelles. The availability of
markers for both cis- and trans-Golgi allowed us to compare
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Plant prevacuolar compartments
the distribution of VSR proteins relative to these Golgi markers. JIM84 is a trans-Golgi marker that recognizes a Lewis Nlinked glycan epitope in plant cells (Satiat-Jeunemaitre and
Hawes 1992, Fitchette et al. 1999). When compared in doublelabeling experiments, more than 90% of the anti-VSR-marked
organelles were separate from JIM84-labeled Golgi organelles
(Fig. 4, panels 1–5; Table 1, lines 7–10). As shown in Fig. 4,
structures that were labeled by anti-VSR antibodies (14G7,
17F9, 18E7, 19F2 and RA3; panels 1–5) do not coincide with
Golgi organelles labeled by anti-JIM84. The degree of separation was the same for all the anti-VSR antibodies tested in pea
root tip cells. When the extent of separation or colocalization
was quantitated in ~250 cells, more than 90% of anti-VSR
marked organelles were separate from JIM-84 labeled Golgi
organelles (Table 1, lines 7–10). In addition, less than 10% of
JIM84-labeled Golgi organelles colocalized with calnexin, an
ER marker (Table 1, line 11). Approximately 50 cells were analyzed for colocalization in each of the double-labeling experiments, and each analysis included up to as many as 2500 Golgi
stacks (assuming that there are at least 50 Golgi stacks per cell
on average based on the images we have examined). The extent
of colocalization for anti-VSR and anti-JIM84 antibodies was
less than 10% in all of the four different double-labeling experiments (Table 1, lines 7–10). These results indicate that VSR
proteins are predominantly localized in PVCs.
To test this hypothesis further, we compared the localization and distribution of VSR proteins between PVCs and Golgi
organelles in tobacco cells, where other established Golgi
markers are available (see below).
JIM84 and Man1-GFP fusion colocalize in transgenic BY-2
cells
To characterize other Golgi markers in tobacco cells, we
first compared anti-JIM84 antibody to other established Golgi
markers and to our newly developed antibodies for a cis-Golgi
protein, the soybean a-1,2 mannosidase (mannosidase I or
Man1) (Nebenführ et al. 1999). When a Man1-GFP (green fluorescent protein) fusion protein was expressed constitutively in
transgenic BY-2 cells, the GFP fusion protein appeared as
many fluorescent spots corresponding to individual Golgi
stacks. This Man1-GFP fusion protein was localized in large
part to the cis-Golgi (Nebenführ et al. 1999). Thus the GFP
fusion protein expressed in these transgenic BY-2 cells is a
marker for cis-Golgi or Golgi stacks. In transgenic BY-2 cells
Fig. 5 JIM84 and the Man1-GFP fusion colocalize in transgenic BY2 cells. (A) Panels 1 and 4 show colocalization of anti-GFP antibodies
and Man1-GFP fusion in transgenic BY-2 cells expressing the Man1GFP fusion. Panel 4 on the right presents a DIC image of a single optical collection of a confocal microscope using transmission light. The
open arrows indicate examples of colocalization (panels 1 and 4) and
close association of two proteins (panel 3). GFP, GFP-Man1 fusion
protein; n, nucleus. Bar = 10 mm. (B) Shows an example of nine serial
optical sections (1 mm per section) of a single cell in which the GFPMan1 fusion (green) is compared to JIM84 (red). Solid arrows (panels
1–3), open arrowheads (panels 4–7) and open arrows (panels 8 and 9)
indicate examples of close association of the two proteins at various
optical sections.
Plant prevacuolar compartments
733
Fig. 6 Characterization of anti-Man1 antibodies. Western blot detection of mannosidase I (Man1) in tissue extracts of membrane fractions
from Arabidopsis roots (lanes 1), pea roots (lane 2), tobacco roots
(lane 3), and BY-2 suspension culture cells (lane 4). The arrow indicates the position of Man1, while the arrowhead indicates the minor
band detected by the antibodies in extracts of BY-2 cell and Arabidopsis. M indicates molecular mass markers in kilodaltons.
expressing the Man1-GFP fusion protein, organelles detected
by anti-GFP antibodies colocalized with the GFP fusion protein
as indicated by the appearance of yellow color in the merged
images (Fig. 5A, open arrow, panels 1 and 4). In contrast, the
GFP fusion protein did not localize to ER, as detected by calnexin antibodies (Fig. 5A, panel 2). When JIM84 was compared directly to GFP in transgenic BY-2 cells expressing the
Man1-GFP fusion, these two Golgi markers closely overlapped
(Fig. 5A, panel 3, arrow) as indicated by the coincidence of
JIM-84 labeled organelles (red) with the GFP (green). These
results are consistent with the known localization of JIM84 and
the Man1-GFP fusion protein, being localized to trans- and cisGolgi, respectively (Nebenführ et al. 1999). When a series of
optical sections was reviewed, JIM84-marked Golgi organelles
and the Man1-GFP fusion protein were frequently associated
with one another (Fig. 5B, panels 1–9; solid arrow, open arrow,
and open arrowhead indicate examples of individual Golgi
stack containing two markers). We thus conclude that both
JIM84 and the Man1-GFP fusion protein can be used as reliable markers for detecting Golgi stacks in BY-2 cells.
Characterization of anti-Man1 antibodies
To generate more markers for the plant Golgi apparatus,
we expressed a truncated form of Man1 that lacks its transmembrane domain and cytoplasmic tail in E. coli and used the
purified recombinant protein to generate rabbit antibodies
against the soybean Man1. When tested by Western blot analysis, the affinity-purified anti-Man1 antibodies identified a single major band of about 63 kDa (Fig. 6, arrow) in extracts from
Arabidopsis roots (Fig. 6, lanes 1), pea roots (lane 2), tobacco
roots (lane 3), and BY-2 cells (lane 4). The ~63 kDa band
detected by the anti-Man1 antibodies was of the size expected
based on the sequence of the soybean mannosidase I cDNA
(Nebenführ et al. 1999). In addition, these anti-Man1 antibodies also detected the truncated recombinant protein (data not
shown). These results strongly suggest that anti-Man1 antibodies are specific for detecting Man1 protein in extracts of Arabi-
Fig. 7 Colocalization of three different Golgi markers in transgenic
BY-2 cells. Stable transformed BY2 cells expressing the Man1-GFP
fusion (green) were double labeled using anti-JIM84 (red) and antiMan1 (blue) antibodies. The arrows and arrowheads indicate examples of colocalization or separation of the three proteins. n, nucleus.
Bar = 10 mm.
dopsis, tobacco, pea, and BY-2 cells.
Three Golgi markers colocalize in tobacco BY-2 cells
To find out if the polyclonal anti-Man1 antibodies could
be used as a specific Golgi marker in confocal immunofluorescence, we performed double labeling using anti-Man1 and antiJIM84 antibodies in transgenic BY-2 cells expressing the
Man1-GFP fusion. All three proteins were detected using confocal immunofluorescence (Fig. 7). Approximately 90% of the
734
Plant prevacuolar compartments
Fig. 8 The anti-VSR-marked prevacuolar organelles separate from
Golgi organelles in tobacco cells. (A) VSR (red) and the Man1-GFP
fusion (green) are largely separate in transgenic BY-2 cells. Six optical sections (1 mm per section) of a single cell were shown. The arrows
and arrowheads indicate examples of colocalization and separation of
two proteins, respectively. n, nucleus. Bar = 10 mm. (B) VSR (green)
and JIM84 (red) are separate in tobacco root-tip cells. The arrows and
arrowheads indicate examples of colocalization and separation of the
two antibodies, respectively. n, nucleus. Bar = 10 mm.
Golgi stacks delineated by the Man1-GFP fusion (green, panel
2) contains both anti-JIM84-marked Golgi (red, panel 1) and
anti-Man1-labeled Golgi organelles (blue, panel 3), as indicated by the colocalization of these three-color signals (arrow
indicates an representative example) (Fig. 7, panel 4, merged).
These results strongly argue that the polyclonal anti-Man1 antibodies specifically detect Golgi-localized tobacco Man1 proteins. Thus, the anti-JIM84, anti-Man1 and the Man1-GFP
fusion are three specific markers that can be used individually
Fig. 9 Characterization of anti-AtPep12p (AtSYP21) and anti-AtELP
polyclonal antibodies for their cross-reactivity and specificity. Total
proteins were extracted from roots of tomato, tobacco, pea, Arabidopsis and BY-2 suspension culture cells and separated by SDS-PAGE,
followed by Western blot detection using either anti-AtPep12p
(AtSYP21p) antibodies (A) or anti-AtELP antibodies (B). Arrows
indicate positions of Pep12p (SYP21) protein (~32 kDa) in 8A and
positions of AtELP in 9B, respectively. The extra band ~80 kDa
detected by anti-AtPep12p antibodies in Fig. 8A is indicated by an
arrowhead. M indicates molecular weight markers in kilodaltons.
to mark Golgi stacks in tobacco cells, and the two antibodies
may be used interchangeably as a marker for Golgi stacks, in
order to compare Golgi with other organelles using confocal
immunofluorescence.
VSR-marked PVCs are separated from Golgi organelles in
tobacco cells
As shown previously in Fig. 4, in pea root tip cells, BP80/VSR-marked organelles (PVCs) are predominantly separated from JIM84-marked Golgi stacks. We then compared the
distribution of VSR proteins between PVCs and Golgi
organelles in tobacco cells using established Golgi markers
characterized above (Fig. 5–7). We first compared VSR-labeled
PVC organelles (red) to Golgi organelles marked by the Man1-
Plant prevacuolar compartments
735
GFP fusion (green) in transgenic BY-2 cells (Fig. 8A). Consistent with results obtained from pea root tip cells, anti-VSR(14G7) labeled organelles (red) are largely separate from the
Man1-GFP fusion (green) in transgenic BY-2 cells (Fig. 8A,
panels 1–6). The extent of colocalization between anti-VSR
antibodies and Man1-GFP fusion in transgenic BY-2 cells was
less than 10% (Table 1, lines 17–18). Similarly, more than 90%
of the PVC organelles (green) labeled by anti-VSR antibodies
(14G7 and 17F9) are separate from JIM84-marked Golgi
organelles (red) when the two antibodies were compared
directly in tobacco root tip cells (Fig. 8B, panels 1–2; Table 1,
lines 19–20). BY-2 cells, tobacco root tip cells and pea root tip
cells all demonstrate the separation of PVCs from the Golgi
markers. These results are consistent with our hypothesis that
VSR proteins are predominantly concentrated on the lytic prevacuolar compartments.
Characterization of anti-AtPep12p (AtSYP21p) and anti-AtELP
polyclonal antibodies for their cross-reactivity and specificity
In yeast, Pep12p is a syntaxin specifically associated
with the PVC endosome (Pelham 2000). The AtPep12p
(AtSYP21p), an Arabidopsis protein that can functionally complement the yeast pep12p mutant (Bassham et al. 1995), has
been localized to a late post-Golgi, prevacuolar compartment in
Arabidopsis root tip cells via immunoEM (Conceição et al.
1997). Additionally, AtELP, an Arabidopsis BP-80 homolog,
has been localized to Golgi and PVC ~100 nm in diameter in
Arabidopsis root tip cells (Sanderfoot et al. 1998). To further
test the above conclusion that VSR proteins are predominantly
concentrated on post-Golgi prevacuolar compartments, we
compared the localization between AtPep12p (AtSYP21p) /
AtELP homolog proteins and Golgi markers in tomato,
tobacco, and transgenic BY-2 cells expressing the Man1-GFP
fusion.
We first determined the specificity and cross-reactivity of
anti-AtPep12p (AtSYP21p) and anti-AtELP antibodies
(Conceição et al. 1997, Sanderfoot et al. 1998) in other plant
cells via Western blot detection. As shown in Fig. 9A, antiAtPep12p antibodies detected a major band ~31 kDa in size
from protein extracts of tomato (lane 1), pea (lane 3) and BY-2
suspension culture cells (lane 4) but a less intense band from
tobacco root tip cells (lane 2), which was similar in size to the
Arabidopsis Pep12p (AtSYP21p) (lane 5, arrow). Additionally,
a minor band ~80 kDa was detected from all extracts (lanes 1–
5, arrowhead). Similarly, anti-AtELP detected a major band
~80 kDa in protein extracts from tomato, tobacco, Arabidopsis,
pea, and BY-2 cells (Fig. 9B, lanes 1–5, arrow). In addition, the
antisera cross-react with some minor proteins in all the tissues
tested. These results indicate that both anti-AtPep12p (AtSYP21p)
and anti-AtELP antibodies may be used to detect their homolog
proteins in these plant cells, in addition to Arabidopsis.
Fig. 10 AtPep12p- and At-ELP-marked organelles are separate from
the Golgi apparatus in tomato and tobacco cells. AtPep12p (AtSYP21)
(panels 1 and 2) or AtELP (panels 5 and 6) was compared to JIM84 in
tomato and tobacco root-tip cells via double labeling in confocal
immunofluorescence. AtPep12p (AtSYP21) (panel 3) and AtELP
(panel 4) were also compared to the Man1-GFP fusion in transgenic
tobacco BY-2 cells. Shown are images from either single optical section (panels 3 and 6) or 3D images of multiple optical sections (panels
1–2 and 4–5). The open arrows and arrowheads indicate examples of
colocalization and separation of the two antibodies, respectively. n,
nucleus. Bar = 10 mm.
736
Plant prevacuolar compartments
AtPep12p (AtSYP21p)-marked organelles are separate from the
Golgi apparatus in tomato and tobacco cells
To determine the relative distributions between AtPep12p
(AtSYP21p) or At-ELP-marked organelles and Golgi apparatus, we performed double labeling and compared protein localization using confocal immunofluorescence. Both antiAtPep12p (AtSYP21p) and anti-AtELP antibodies resulted in
typical punctuate patterns of labeled organelles (Fig. 10). As
shown in Fig. 10, AtPep12p (AtSYP21p)-marked organelles in
tomato and tobacco cells (panels 1–2, green) were separate
from JIM84-marked Golgi apparati (red, panels 1–2). When
approximately 47 cells were counted and quantitated, less than
10±5% of the AtPep12 (AtSYP21p)-marked organelles colocalized with the JIM84-marked Golgi apparati (Table 1, lines
21–22). Similarly, AtELP-marked organelles were also separate from JIM84-marked Golgi apparati in these cells, with less
than 11% of these two signals overlapping (Fig. 10, panels 5–6;
Table 1, lines 25–26). Additionally, in transgenic BY-2 cells
expressing the Man1-GFP fusion protein, neither AtPep12p
(AtSYP21p) nor AtELP (red, panels 3–4) colocalized with the
GFP-marked Golgi apparati (green, panels 3–4). Again, less
than 10% of either AtPep12p (AtSYP21p)-marked or AtELPmarked organelles colocalized with the Man1-GFP -marked
Golgi apparati in transgenic BY-2 cells (Table 1, lines 23–24).
These results support the previous conclusion that VSRmarked organelles were mostly separate from Golgi apparati
and concentrated on post-Golgi, prevacuolar compartments.
VSR-marked organelle colocalized with AtPep12p-marked
compartments in pea and tomato cells
AtPep12p was localized to a late post-Golgi compartment
that also contained AtELP in Arabidopsis root tip cells
(Conceição et al. 1997, Sanderfoot et al. 1998). Since both
AtPep12p- and AtELP-marked organelles were also separate
from Golgi apparatus as demonstrated for VSR-marked
organelles in confocal immunofluorescence, we wanted to
determine if organelles marked by VSR and AtPep12p were the
same compartments. Double labeling was thus performed to
compare the degree of colocalization of the two antibodies in
both pea and tomato root tip cells (Fig. 11; Table 1, lines 27–
30). Consistent with previous results, anti-VSR, anti-AtPep12p
and anti-AtELP antibodies all resulted in typical punctuate patterns of labeled organelles (Fig. 11). In addition, VSR-marked
organelles (either 14G7 or 17F9 antibodies) were largely colocalized with either AtPep12p-marked organelles (Fig. 11, panels 1–3; Table 1, lines 27–28) or AtELP-marked organelles
(Fig. 11, panels 4–5; Table 1, lines 29). When more than 20
cells were analyzed to quantitate the percentage of colocalization between the two antibodies (either VSR/AtPep12p or
VSR/AtELP) in one direction (i.e. by asking how many VSR
signals colocalize with AtPep12, but not the reverse), more
than 80% of the signals detected by anti-VSR antibodies colocalized with signals detected by anti-AtPep12p or anti-AtELP
in both pea and tomato cells (Table 1, lines 27–30). However,
Fig. 11 VSR-marked organelle colocalized with AtPep12p-marked
compartments in pea and tomato cells. Anti-VSR monoclonal antibodies (14G7 and 17F9) were compared to either anti-AtPep12p
(AtSYP21) or anti-AtELP polyclonal antibodies via double labeling of
confocal immunofluorescence in root-tip cells derived from pea (panels 1, 2, 4) and tomato (panels 3 and 4). The open arrows and arrowheads indicate examples of colocalization and separation of the two
antibodies, respectively. n, nucleus. Bar = 10 mm.
there were some organelles labeled by anti-AtPep12p or antiAtELP that did not also label with the VSR (14G7 or 17F9)
(Fig. 11; Table 1). Since the majority of proteins detected by
anti-VSR antibodies colocalized with anti-AtPep12p, both
VSR and AtPep12p thus recognize the same PVCs that are
mainly separate from the Golgi apparatus. Again, these results
strongly support the conclusion that VSR are predominantly
concentrated on the lytic PVCs. When anti-AtPep12p and anti-
Plant prevacuolar compartments
Fig. 12 VSR is concentrated in PVCs. Tobacco protoplasts were
used to isolate vacuole (V) and pellet (P) fractions, followed by SDSPAGE and Western blot detection using various antibodies (Abs) as
indicated. Lanes 1–2 represent fractions prepared from tobacco cells
transiently expressed soluble proaleurain as previously described
(Jiang and Rogers 1998) and lanes 13–14 indicate fractions prepared
from transgenic BY-2 cells expressing the Man1-GFP fusion. Numbers to the left in each panel indicate positions of molecular weight
markers in kilodaltons. Abs, antibodies; Aleu, aleurain; DIP, dark
intrinsic protein; VSR, vacuolar sorting receptor; CNX, calnexin;
Man1, mannosidase I; GFP, green fluorescent protein. Asterisks indicate the proper positions of proteins detected by the specific antibodies.
AtELP were compared directly with double labeling of confocal immnufluorescence, about 80% of the signals detected by
these two antibodies colocalized (Table 1, lines 31). These
results are consistent with those obtained from comparing VSR
to AtELP or AtPep12p.
VSR is concentrated in PVCs
Subcellular fractionation was also used to characterize
organelles containing VSR proteins in tobacco culture cells.
We used a simple protocol where vacuoles and PVCs, because
of their low buoyant density, remain at the top interface of a
Ficoll cushion when centrifuged at 170,000´g for 2 h, whereas
ER and Golgi pellet to the bottom of the tube. The specificity
of such a fractionation procedure had been demonstrated previously using tobacco protoplasts expressing soluble proaleurain
(Jiang and Rogers 1998), where 90% of proaleurain was localized in the pellet fraction whereas 93% of mature aleurain was
in the vacuole fraction (Jiang and Rogers 1998). Here we used
the same approach to determine organelles that contain VSR
proteins in tobacco suspension culture cells and similar results
were obtained (Fig. 12). Consistent with previous results, most
(more than 95%) proaleurain remained in the pellet (P) fraction whereas the majority of mature aleurain (more than 90%)
737
was detected in the vacuole (V) fraction from cells that transiently express the soluble prolaeurain (Fig. 12, lanes 1–2). In
addition, DIP, a marker for cytosolic organelles of probable
PVCs for protein storage vacuole (Jiang et al. 2000), was concentrated (more than 95%) in the V fraction (Fig. 12, lane 1).
These results demonstrated purity of different fractions and
indicated that the vacuole fraction contained a mixture of prevacuolar and vacuolar compartments. Similarly, more than 85%
of VSR proteins (lane 5) remained in the vacuole fraction after
subcellular fractionation of protoplasts. In contrast, an ER
marker calnexin (CNX) was predominantly concentrated on the
pellet fraction with little in the vacuole fraction (lanes 7–8). In
addition, AtPep12, a PVC marker in Arabidopsis, is similarly
predominantly presented (more than 70%) in the vacuole fraction but much less in the pellet fraction (lanes 9–10). In contrast, two Golgi-localized proteins, Man1 and Man1-GFP
fusion, were predominantly detected (more than 90%) in the
pellet fraction but not in the vacuole fraction (lanes 11–14).
Since both VSR proteins and Pep12 proteins cannot be found
in tonoplasts, these fractionation results, in combination with
those obtained from confocal immunofluorescence studies,
strongly support the hypothesis that VSR proteins are predominantly concentrated in PVCs.
Discussion
In spite of the important role of PVCs in mediating protein
traffic to vacuoles in the plant secretory pathway, identification and characterization of plant PVCs, either functionally or
morphologically, have been challenging due to the complexity
of the plant vacuolar systems and the existence of multiple
pathways of vacuolar targeting (Robinson et al. 2000, Jiang and
Rogers 2001). Our present studies contribute to an understanding of the nature of the plant prevacuolar compartments by
identification and characterization of PVCs in pea and tobacco
cells using confocal immunofluorescence and specific antibodies for proteins in the plant secretory pathway.
In this study, we first determined the specificity of various anti-BP-80 antibodies in pea root tip cells using confocal
immunofluorescence. Using the polyclonal antipeptide antibodies RA3, specific for the pea BP-80 (Paris et al. 1997), and four
monoclonal antibodies that recognize different epitopes on BP80 (Paris et al. 1997, Jiang and Rogers 1998, Cao et al. 2000),
we have shown that different combinations for all five antibodies in double-labeling experiments colocalized in pea root tip
cells, making these antibodies reliable markers for detecting
VSR proteins in pea cells. Additionally, two of these monoclonal antibodies, 14G7 and 17F9, have been demonstrated to
detect specifically tobacco VSR proteins, using both immunofluorescence and immunoprecipitation (Jiang and Rogers
1998 and this study). All four monoclonal antibodies for BP-80
also detected a recombinant Arabidopsis BP-80 homolog
VSRAt-1 that is identical to AtELP (Fig. 3). It is thus reliable to
use these monoclonal antibodies to detect VSR proteins in
738
Plant prevacuolar compartments
both pea and tobacco cells using confocal immunofluorescence. For identifying Golgi organelles, we used three different markers: JIM84, a rat monoclonal antibody directed against
Lewis a-containing N-glycans locating to trans-Golgi (SatiatJeunemaitre and Hawes 1992, Fitchette et al. 1999); a Man1GFP fusion that has been shown to localize to cis-Golgi in
transgenic tobacco BY-2 cells (Nebenführ et al. 1999); as well
as a newly characterized rabbit polyclonal antibody from this
study raised against an E. coli expressed recombinant protein
representing a truncated version of the soybean Golgi enzyme
a-1,2 mannosidase I (Nebenführ et al. 1999). We also compared VSR to AtPep12p (AtSYP21p), a syntaxin that marked a
late post-Golgi, prevacuolar compartment in Arabidopsis cells
(Conceição et al. 1997).
BP-80 was previously shown by both immunofluorescence and immunoEM to locate to Golgi and a lytic PVC but
was not present in either vacuole or vacuolar membrane of pea
cells (Paris et al. 1997, Hinz et al. 1999). Our immunofluorescent approach has allowed us to compare the localization of
VSR proteins to that of established Golgi markers and to quantitate the distribution of VSR proteins between Golgi and PVCs
in hundreds of cells. We first demonstrated that VSR-labeled
organelles are largely separate from ER, Golgi apparatus and
vacuolar compartments, which suggests that VSR proteins
must be concentrated on PVCs. We used multiple specific antibodies to detect VSR proteins and compared them to Golgi
stacks specifically labeled by multiple antibodies or marked by
the GFP fusion protein in transgenic BY-2 cells. Essentially the
same results were obtained using various combinations of antibodies in double-labeling experiments. Similar results were
also obtained in two different cell systems, where these
organelles were marked by multiple specific markers. Additionally, our immunofluorescent approach allowed us to screen
and quantitate a much larger sample of cells than previous
immunoEM experiments; e.g. hundreds of cells (assuming that
each cell contains at least 50 Golgi stacks and PVCs) were
screened and quantitated to compare VSR-labeled organelles to
Golgi organelles in this study.
When VSR-labeled organelles were compared directly to
Golgi using double-labeling experiments, labeling overlapped
in only approximately 10% of these two organelles. This result
is consistent with the previous localization of BP-80, where
both Golgi and PVC organelles were shown to contain VSR
proteins in immunofluorescent, immunoEM and subcellular
fractionation studies (Paris et al. 1997, Jiang and Rogers 1998,
Hinz et al. 1999). However, because 90% of the VSR-labeled
organelles were separate from Golgi, we conclude that BP-80
and its homologs are predominantly concentrated on PVCs.
Such a conclusion was largely supported by the labeling results
derived from using anti-AtPep12p (AtSYP21p) and anti-AtELP
antibodies because first, VSRs and AtPep12p (AtSYP21p)
were largely colocalized and second, organelles labeled by
these two antibodies were also separate from Golgi apparatus.
Such a highly concentrated localization of VSR proteins to
PVCs leads to the hypothesis that VSR proteins only recycle
back to Golgi briefly for selection of transit cargo molecules
and then return to the PVCs for cargo delivery. This distribution of receptor molecules is not unique to plant cells. In addition to their minor Golgi and plasma membrane localization in
mammalian cells, most mannose 6-phosphate receptor (M6P)
molecules within a cell are found in the late endosome (a PVC
equivalent in yeast and plant cells), where the protein is concentrated almost exclusively on the internal membranes (Griffiths et al. 1988). In addition, both M6P in mammalian cells
and the receptor for carboxypeptidase Y (CPY) in yeast are not
present in either the lysosome or vacuole (Kornfeld and Mellman 1989, Cereghino et al. 1995); rather, both receptors are
associated with the formation of Golgi-derived clathrin-coated
vesicles (CCVs) and traffic to a prevacuolar compartment
where their ligand molecules are released before the receptors
recycle back to the Golgi (for a review see Robinson et al.
2000). Similarly, the yeast Pep12p is also found to be concentrated in the prevacuolar compartment and is required for PVC
fusion (Becherer et al. 1996, Gerrard et al. 2000a, Gerrard et al.
2000b).
It is clear that VSR-marked organelles are mostly separate from Golgi apparatus based on our results presented here,
which strongly support that these VSR-marked organelles are
PVCs. However, there seems to be two outstanding questions.
First, are these VSR-marked organelles lytic PVCs? Second,
can the possibility that these compartments are TGN be
excluded? To address the first question, we need to consider the
following studies. BP-80 was localized to both Golgi and PVCs
but not in vacuoles in pea root tip cells (Paris et al. 1997).
When a reporter fusion composed of the mutated proaleurain
and the BP-80 TMD/CT was expressed in tobacco suspension
culture cells, the reporter processed into mature aleurain (Jiang
and Rogers 1998). The processing of proaleurain into mature
aleurain occurred at post-Golgi compartment in tobacco suspension culture cells (Holwerda et al. 1990, Holwerda et al.
1992). In addition, this reporter colocalized with the tobacco
endogenous VSR proteins that were detected by the same antiVSR antibodies used in this study (Jiang and Rogers 1998),
with a typical punctuating staining pattern as observed here in
this study. Since this reporter colocalized with VSR proteins
that were largely separate from Golgi organelles, processing of
the reporter into mature form most likely occurred in PVC.
Because aleurain is a marker for lytic vacuole (Paris et al.
1996), the processing of proaleurain into mature aleurain in
PVC would thus functionally define the lytic PVC. On the
other hand, we used both Man1-GFP and JIM84 as makers for
Golgi apparatus where Man1-GFP is a cis-Golgi marker and
JIM84 is a trans-Golgi marker and demonstrated that VSRmarked organelles were separate from both JIM84 and Man1GFP. However, unlike mammalian cells where markers for
TGN are available, no TGN markers have been identified in
plants. Thus, even though with strong substantial evidence
obtained from both biochemical and immunocytochemical
Plant prevacuolar compartments
studies that support the identity of VSR-marked PVCs, our
results do not rule out possible TGN localization of VSR proteins. Further study is needed to address such possibility by
identifying plant TGN markers or via detailed immunoEM
studies.
Golgi, VSR-marked and AtPep12p-marked organelles
have similar punctuate staining patterns in cytoplasm of pea
and tobacco cells in confocal immunofluorescence (e.g. Fig. 1,
2, 4, 7–10), even though the sizes of the structures/organelles
may different from one to another. In this study, we clearly
demonstrate that the Golgi structures are distinct and can be
separated from the PVCs marked by VSR and AtPep12p
(AtSYP21p) proteins in confocal immunofluorescence in these
cell types, supporting our hypothesis that VSR proteins are
concentrated on PVCs. Thus, a punctuate staining pattern cannot be used to define the Golgi or PVC organelles in plant
cells, unless the labeling is compared to those of specific
known markers using double labeling in immunofluorescence
or immunoEM. These findings are consistent with our previous results that a chimeric reporter (construct 491 or BP-80
reporter), when expressed in transgenic tobacco suspension
culture cells, colocalized with the endogenous tobacco VSR
proteins (detected by anti-VSR antibodies) in confocal immunofluorescence (Jiang and Rogers 1998). This chimeric reporter,
containing the sequences of BP-80 transmembrane domain
(TMD) and cytoplasmic tail (CT), marked the lytic PVCs in
tobacco suspension culture (Jiang and Rogers 1998) and in
Arabidopsis (Kim et al. 2001). Similarly, a GFP fusion containing the TMD/CT of a pumpkin BP-80 homolog was localized
to the Golgi complex and PVCs in tobacco BY-2 cells (Mitsuhashi et al. 2000). It will be of interest to find out if this GFP
fusion also localizes to an organelle separate from the Golgi
apparatus and is concentrated on PVCs of transgenic BY-2
cells.
In this study, we used four different monoclonal antibodies specific for the pea BP-80 to specifically detect BP-80 and
its homologs in root tip cells of pea, tomato, tobacco and BY-2
cells. We demonstrated all these four anti-VSR MAbs specifically recognize the recombinant Arabidopsis homolog VSRAT-1,
which is identical to AtELP. Based on the analysis of the whole
Arabidopsis genomic sequences data, seven BP-80 homologs
have been identified from A. thaliana, where the positions of
the transmembrane domain and the numerous cysteine residues are highly conserved within these BP-80 isoforms (Hadlington and Denecke 2000, Li and Jiang data not presented).
The overall sequence homology among BP-80 isoforms is very
high, with ~86% amino acid identity between pea BP-80 and
the AtBP-80a, whereas the similarity between pea BP-80 and
the VSRAt-1 (AtBP-80b or AtELP) was ~70% (Hadlington and
Denecke 2000). MAb 14G7 recognizes the third EGF repeat or
the Ser/Thr-rich sequence (or both) of BP-80, whereas MAb
19F2 recognizes the first and second EGF repeat sequences of
BP-80. Epitopes for both 17F9 and 18E7 are contained entirely
within the N-terminal unique domain of BP-80 (Cao et al.
739
2000). Thus, the different MAbs are useful markers in detecting other VSR homologs that may be divergent in sequence
from BP-80. In addition, since MAbs 14G7 and 19F2 detect the
EGF repeats of BP-80, it is thus likely these two MAbs might
be useful to see even the more distantly related members of
Arabidopsis BP-80 isoforms. However, we do not know which
Arabidopsis BP-80 isoforms were detected by these BP-80
MAbs in addition to the Arabidopsis BP-80 homolog VSRAt-1.
In this regard, these MAbs might also label organelle populations containing more than one type of PVC, because it has
been demonstrated that different PVCs exist in different cell
types, for example MVBs in pea cotyledons have been speculated to be PVCs for the PSV while DIP organelles have been
speculated to be the PVCs leading to the PSV in tobacco cells
(Robinson et al. 2000, Jiang et al. 2000). However, our recent
results indicated that DIP organelles might be separate from
BP-80-labeled PVCs in certain cell types (unpublished results).
When anti-VSR MAbs were compared in double labeling
with confocal immunofluorescence, about 90% of signals
detected by the two antibodies colocalized (e.g. Table 1, lines
1–5). However, when anti-VSR MAbs were compared with
AtPep12p (AtSYP21p) or AtELP using the same approach,
only about 80% of the signals detected by the two antibodies
colocalized (e.g. Table 1, lines 27–28). In fact, there were some
organelles detected by anti-AtPep12p (AtSYP21p) or antiAtELP that did not colocalize with the VSR (14G7 or 17F9)
(Fig. 11). In addition, only about 80% of the two signals colocalized when comparing AtPep12p (AtSYP21p) with AtELP
(Table 1, line 31). What would cause such a difference when
comparing among anti-VSR or between anti-VSR and
AtPep12p? There would seem to be several possibilities. First,
AtPep12p (AtSYP21p) and AtELP could also be present in
another organelle that does not contain proteins recognized by
anti-VSR monoclonal antibodies; second, the minor proteins
recognized by the antisera in the Western blot might contribute
to detection of background proteins in confocal immunofluorescence; third, anti-VSR MAbs or AtPep12p (AtSYP21p)
might detect heterogeneous populations of PVCs. However,
since the majority (more than 80%) of proteins detected by
anti-VSR antibodies colocalized with anti-AtPep12p
(AtSYP21p), both VSR and AtPep12p (AtSYP21p) are recognizing mainly the same PVC populations that are also separate
from the Golgi apparatus. Therefore, the 10% difference in
colocalization does not rule out our conclusion that VSR are
predominantly concentrated on the lytic PVCs.
Why study PVCs in plant cells? It has been well documented that PVCs play important roles in mediating protein
traffic from the late Golgi to lysosome or vacuole, a lytic compartment in both mammalian and yeast cells (Braulke 1996,
Conibear and Stevens 1998). In mammalian cells, the M6P
receptors mediate recruitment of the lysosomal hydrolases to
the TGN, from which clathrin-coated carrier vesicles deliver
the M6P receptor–hyrolase complex to the endosome, a prevacuolar compartment (Puertollano et al. 2001), where the
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Plant prevacuolar compartments
hydrolases are released from the M6P receptor before they are
transported to the lysosome, while the M6P receptor returns to
the TGN for additional rounds of sorting. In addition, M6P
receptor sorting at the TGN is mediated by interaction between
the VHS (VPS27, Hrs, and STAM) domain of GGA (Golgilocalized, g-ear-containing, ARF-binding protein) protein and
the cytoplasmic tail of M6P receptor (Puertollano et al. 2001,
Zhu et al. 2001). Similarly, a prevacuolar compartment mediates protein transport between TGN and vacuole in yeast
(Gerrard et al. 2000a, Gerrard et al. 2000b). In plant cells, BP80/AtELP may function in a similar sorting mechanism (Jiang
and Rogers 1998, Sanderfoot et al. 1998). However, the system for protein sorting to vacuoles in plant cells is more complicated because of the probable existence of: (1) two vacuolar
types; (2) multiple vesicular transport pathways leading to distinct vacuoles; (3) multiple transport vesicles responsible for
Golgi to vacuole sorting. For example, protein sorting to the
protein storage vacuole is unique to plant cells and may involve
previously undescribed transport vesicles (Hara-Nishimura et
al. 1998, Hinz et al. 1999, Jiang et al. 2000). It is thus possible
that a unique PVC may be responsible for PSV sorting (Robinson et al. 2000, Jiang and Rogers 2001, Jiang et al. 2001) and
that distinct PVCs may be involved in protein transport leading to lytic vacuole and PSV, respectively. Such a hypothesis is
currently being tested in our laboratory using both molecular
and biochemical approaches.
Functional characterization of PVCs in plant cells requires
markers for identifying PVCs. As a first step toward the goal of
defining both structure and function of PVCs, we have identified and characterized PVCs in pea and tobacco cells with confocal immunofluorescence using specific antibodies. We have
demonstrated, for the first time in plant cells, that VSR proteins are predominantly present in the PVCs. These results will
in turn allow us to use these anti-VSR antibodies as additional
markers to identify PVCs in pea, tobacco, tomato, and BY-2
cells. Our approach using double labeling with confocal
immunofluorescence will provide a rapid and reliable method
for quantitating protein distribution among various organelles
in plant cells. Many questions about PVCs in the plant cell system remain to be answered. For example, are there two functionally distinct types of PVCs in plant cells? What is their
nature? What biochemical markers can be used to identify
them? What are the molecular components of distinct PVCs?
What makes them unique and distinct from other compartment
in the secretory pathway? What is the relationship between
Golgi and the different types of PVCs? Our current research is
addressing some of these questions.
Materials and Methods
General methods for construction of recombinant plasmids, characterization of cloned inserts, maintenance of tobacco culture cells,
expression and purification of recombinant proteins from Drosophila
S2 cells, and preparation and characterization of antibodies have been
previously described (Rogers et al. 1997, Jiang and Rogers 1998, Cao
et al. 2000, Jiang et al. 2000, Jiang et al. 2001). Transgenic tobacco
BY-2 cells were maintained in both liquid and solid culture as
described (Nebenführ et al. 1999). BY-2 cells were cultured on MS0
media containing cytokinin and auxin so that cells with small vacuoles could be obtained (Jiang and Rogers 1998).
Antibodies
A recombinant protein representing the lumenal portion of a soybean a-1,2 mannosidase I cDNA (GenBank accession number
AF126550) (Nebenführ et al. 1999) (residues 183–733) was subcloned in frame into the pQE 30 expression vector (Qiagen, Chatsworth, CA, U.S.A.) via Asp718 and HindIII sites with six His residues
at the COOH terminus and expressed in E. coli. The recombinant protein was purified according to manufacturer’s protocol and dialyzed
overnight against PBS with 0.1% SDS prior use for immunization of
two rabbits. Antibodies were prepared by Alpha Diagnostics (San
Antonio, TX, U.S.A.) and titred by serial dilution on dot blots of the
recombinant protein, versus pre-immune serum. Antibodies were further purified by affinity chromatography using a column made with
recombinant protein coupled to CNBr-activated Sepharose (Sigma, St.
Louis, MO, U.S.A.) as previously described (Rogers et al. 1997).
Affinity-purified polyclonal rabbit antibodies (RA3) raised
against a synthetic peptide that represents the N-terminal amino acids
26–41 of pea BP-80 protein and two monoclonal mouse antibodies
(14G7 and 17F9) against BP-80 have been characterized (Paris et al.
1997, Jiang and Rogers 1998). The other monoclonal antibodies to BP80, 18E7 and 19F2, were prepared in a similar manner (Cao et al.
2000). Anti-castor bean calnexin antiserum was generously provided
by S. Coughlan (Pioneer HiBred, Johnson, IA, U.S.A.). The JIM84 rat
mAb directed against Lewis a-containing N-glycans (Satiat-Jeunemaitre and Hawes 1992, Fitchette et al. 1999) was kindly provided by Dr.
C. Hawes (Oxford Brookes University, Oxford, U.K.). The polyclonal
anti-AtPep12p (AtSYP21p) and anti-AtELP antibodies (Conceição et
al. 1997, Sanderfoot et al. 1998, Sanderfoot et al. 2000) were kindly
provided by Dr. N. V. Raikhel (Michigan State University, East Lansing, MI, U.S.A.). The sources for anti-V-PPase antibodies have been
described (Jauh et al. 1999, Jiang et al. 2000). Various anti-GFP antibodies were purchased from Molecular Probes Inc. (Eugene, OR,
U.S.A.). Secondary Cy5- or lissamine rhodamine- or FITC-conjugated affinity-purified anti-rabbit or -mouse antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA,
U.S.A.). For Western blot detection, anti-Pep12p (AtSYP21p) and
anti-AtELP were used at 1 : 1,000 dilution, whereas anti-Man1 and
anti-GFP antibodies were used at 10 mg ml–1.
Confocal immunofluorescence studies
Fixation and preparation of cells from root tips and tobacco BY2
cells, and their labeling and analysis by epifluorescence and confocal
immunofluorescence have been described previously (Jauh et al. 1999,
Jiang and Rogers 1998, Jiang et al. 2000, Paris et al. 1996). The settings for collecting confocal images within the linear range were as
described (Jiang and Rogers 1998). For immune double labeling, polyclonal rabbit and mouse monoclonal antibodies were incubated
together or in order at 4°C overnight at the following working concentrations: 4 mg ml–1 for anti-RA3 and anti-Man1, 10 mg ml–1 for antiVSR monoclonal (14G7, 17F9, 18E7 or 19F2), 1 : 100 dilution for
anti-calnexin, 1 : 1 dilution for anti-JIM84, 1 : 100 dilution for both
anti-AtPep12p and anti-AtELP. For double labeling using two monoclonal antibodies, the Rhod-conjugated Fab fragment was used at a
dilution of 1 : 20. All confocal fluorescence images were collected
using a Bio-Rad MRC 1024 system (Hercules, CA, U.S.A.). 3D
images were also created by combining multiple serial optical sections
Plant prevacuolar compartments
using the OS2 system and Lasersharp program provided by the BioRad confocal system. Images were processed using Adobe PhotoShop
software (San Jose, CA, U.S.A.).
The extent of colocalization of two antibodies in confocal
immunofluorescence images from both root tip cells (pea and tobacco)
and BY2 cells was quantitated as described previously (Jiang and
Rogers 1998, Jiang et al. 2000). In addition, comparison between VSR
and AtPep12p was made in one direction only, i.e. by asking how
many VSR signals also contain AtPepe1p2 signals in confocal immunofluorescence. Controls to ascertain the specificity of double-labeling
experiments were performed as previously described (Jauh et al. 1999,
Jiang et al. 2000). Briefly, for labeling using either two monoclonal or
two polyclonal antibodies, first primary antibodies were incubated at
4°C overnight (in PBST + 1% BSA) followed by washing in PBST.
Rhod-conjugated Fab fragment was then added and incubated at RT
for 4 h prior to a second wash, followed by addition of the second primary antibodies. For double labeling using one monoclonal and one
polyclonal antibody, duplicate labeling of samples was performed in
which each primary antibody was added individually, followed by a
wash and addition of the second primary antibodies, to make sure that
either order of antibody application resulted in the same labeling
results. Additionally, we confirmed that the labeling pattern for an
antibody used individually matched the pattern obtained with the same
antibody when used in double labeling experiments (Jiang et al. 2000).
Subcellular fractionation and vacuole isolation
Tobacco protoplasts were prepared as described from suspension
culture cells (Jiang and Rogers 1998), followed by resuspension in
buffer and layered on a step gradient of 12 and 15% Ficoll in 0.6 M
mannitol and 20 mM HEPES, pH 7.7. After centrifugation at
42,000 rpm (170,000´g) for 2 h at 4°C in an SW 50.1 rotor, the V fraction and P fractions were removed and resuspended in buffer, followed by the addition of SDS to each fraction to a final concentration
of 1%. Proteins from V and P fractions were separated via SDS-PAGE,
followed by Western blot detection using various antibodies as
described (Jiang et al. 2001).
Acknowledgments
We are grateful to Dr. J.C. Rogers (Washington State University,
Pullman, U.S.A.) for valuable discussion during the course of the
experiment and useful comments on this article. We thank Dr. A.
Nebenführ (University of Tennessee, Knoxville, TN, U.S.A.) for providing the transgenic BY2 cells that express the Man1-GFP fusion
protein and the cDNA clone for soybean Man1. We also thank Dr. C.
Hawes (Oxford Brooks University, Oxford, U.K.) for providing antibody JIM84 and Dr. N. V. Raikhel (University of California, Riverside, CA, U.S.A.) for sharing the anti-AtPep12p (AtSYP21p) and antiAtELP antibodies, and Dr. N. Paris (University of Rouen, CNRS,
France) for making available the truncated VSRAt-1 construct. This work
was supported in part by grants from the Chinese University of Hong
Kong (direct grants (projects code 2030238 and 2030262) and a special grant for conducting research abroad in summer of 2001) and from
the Research Grants Council of the Hong Kong Special Administrative Region of China (Project No. CUHK4156/01M) and Germany/
Hong Kong Joint Research Scheme (project code 2900102) to L.
Jiang. S.S.M. Sun was supported by grants from the RGC (CUHK194/
96M), UGC AoE scheme, and Industry Department (AF/31/98) of
Hong Kong. Work by S.W. Rogers was supported by grants from NSF
and DOE to J.C. Rogers. A portion of this work has been presented in
abstract form for a poster (American Society of Plant Biologists
Annual Meeting 2001, http://www.rycomusa.com/aspp2001/public/P22/
0167.html).
741
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(Received February 1, 2002; Accepted April 15, 2002)