Download Actin Filaments Play a Critical Role in Vacuolar

The Plant Cell, Vol. 17, 888–902, March 2005, www.plantcell.org ª 2005 American Society of Plant Biologists
Actin Filaments Play a Critical Role in Vacuolar Trafficking
at the Golgi Complex in Plant Cells
Hyeran Kim,a Misoon Park,a Soo Jin Kim,b and Inhwan Hwanga,b,1
a Division
b Center
of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Korea
for Plant Intracellular Trafficking, Pohang University of Science and Technology, Pohang, 790-784, Korea
Actin filaments are thought to play an important role in intracellular trafficking in various eukaryotic cells. However, their
involvement in intracellular trafficking in plant cells has not been clearly demonstrated. Here, we investigated the roles actin
filaments play in intracellular trafficking in plant cells using latrunculin B (Lat B), an inhibitor of actin filament assembly, or
actin mutants that disrupt actin filaments when overexpressed. Lat B and actin2 mutant overexpression inhibited the
trafficking of two vacuolar reporter proteins, sporamin:green fluorescent protein (GFP) and Arabidopsis thaliana aleurainlike protein:GFP, to the central vacuole; instead, a punctate staining pattern was observed. Colocalization experiments with
various marker proteins indicated that these punctate stains corresponded to the Golgi complex. The A. thaliana vacuolar
sorting receptor VSR-At, which mainly localizes to the prevacuolar compartment, also accumulated at the Golgi complex in
the presence of Lat B. However, Lat B had no effect on the endoplasmic reticulum (ER) to Golgi trafficking of sialyltransferase or retrograde Golgi to ER trafficking. Lat B also failed to influence the Golgi to plasma membrane trafficking of HþATPase:GFP or the secretion of invertase:GFP. Based on these observations, we propose that actin filaments play a critical
role in the trafficking of proteins from the Golgi complex to the central vacuole.
INTRODUCTION
In eukaryotic cells, a large number of proteins are targeted after
translation to specific organelles by a process called intracellular
trafficking. This targeting process is quite complex and involves
many different pathways depending on the organelle involved
(Rothman, 1994; Hawes et al., 1999; Jahn and Südhof, 1999).
Cytoskeletal components such as actin filaments and microtubules play important roles in intracellular trafficking. It is
generally assumed that the cytoskeleton acts like a network of
tracks for the movement of vesicles between cellular compartments (Simon and Pon, 1996; Huang et al., 1999; Nebenführ and
Staehelin, 2001). In addition, reorganization of actin filaments
mediated by small GTPases such as ADP ribosylation factor (Arf)
and Rho is required for the generation of endocytotic vesicles
and the docking at and fusion of secretory granules with the
plasma membrane in animal cells (Wendland et al., 1998; Goode
et al., 2001; Musch et al., 2001; Ridley, 2001). In addition, many
actin binding proteins interact either directly or indirectly with
proteins involved in endocytosis (Jeng and Welch, 2001; Schafer
et al., 2002). For example, the F-actin binding protein Abp1
interacts with dynamin through the SH3 domain (Kessels et al.,
2001), and dynamin 2 interacts directly with cortactin, an actin
1 To
whom correspondence should be addressed. E-mail ihhwang@
postech.ac.kr; fax 82-54-279-8159.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Inhwan Hwang
([email protected]).
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.104.028829.
binding protein, at the plasma membrane (Schafer et al., 2002).
Actin filaments also have been shown to be important in
trafficking through the Golgi apparatus (Godi et al., 1998;
Hirschberg et al., 1998; De Matteis and Morrow, 2000; Wu
et al., 2000; Fucini et al., 2002). In yeast (Saccharomyces
cerevisiae) cells, actin filaments are critical for polarized secretion (Johnston et al., 1991; Govindan et al., 1995; Schott et al.,
1999; Zhang et al., 2001). Similarly, actin filaments are involved in
trafficking of apical and basolateral proteins from the trans-Golgi
network (TGN) in certain animal cell types (Musch et al., 2001).
Expression of a dominant-negative mutant of Cdc24, a member
of the Rho GTP binding protein subfamily and a key modulator of
cortical actin, inhibited the exit of basolateral proteins from the
TGN (Musch et al., 2001). In addition, Valderrama et al. (2001)
reported that in mammalian cells, retrograde trafficking from the
Golgi apparatus to the endoplasmic reticulum (ER) is inhibited by
latrunculin B (Lat B), an inhibitor of actin filament assembly.
The large amount of data described above was largely derived
from yeast and animal cells, but actin filaments also are thought
to be important for vesicle trafficking in plant cells (Mollenhauer
and Morre, 1976; Picton and Steer, 1981, 1983; Nebenführ and
Staehelin, 2001; Waller et al., 2002). One of clearest lines of
evidence for this is the role actin filaments play in pollen tube
growth and root tips (Picton and Steer, 1981, 1983; reviewed in
Taylor and Hepler, 1997; Gibbon et al., 1999; Vidali et al., 2001).
In particular, at the tip of the pollen tube, exocytic vesicles are
thought to travel along actin filaments that run parallel to the
direction of the pollen tube. In addition, cytochalasin D and Lat
B treatment inhibited brefeldin A (BFA)–induced intracellular
PIN1 accumulation as well as its relocalization to the plasma
membrane when BFA was washed out (Geldner et al., 2001;
Baluska et al., 2002). Furthermore, actin filaments play a role in
Actin Filaments in Vacuolar Trafficking
fluid-phase endocytosis or internalization of sterol from the plant
cell plasma membrane (Grebe et al., 2003; Baluska et al., 2004).
Thus, actin filaments appear to be involved in intracellular
trafficking in plant cells. However, the exact molecular mechanism by which this occurs is not clearly understood. Recently, the
Arabidopsis thaliana actin binding protein AtSH3P1 was shown
to colocalize with clathrin at various locations (Lam et al., 2002),
which further supports the idea that actin filaments are involved
in intracellular trafficking.
In this study, we investigated the involvement of actin filaments
in various steps of anterograde trafficking in plant cells. To do
this, we examined the trafficking of newly synthesized marker
proteins to various final destinations in protoplasts, including the
Golgi apparatus, the plasma membrane, and the central vacuole,
in the presence of Lat B. We present evidence that actin
filaments play a critical role in vacuolar trafficking of proteins
from the Golgi complex to the prevacuolar compartment (PVC)
but do not participate in the trafficking of plasma membrane or
secretion of proteins.
RESULTS
Lat B Inhibits Trafficking of Two Reporter Proteins to the
Central Vacuole
To investigate the roles played by actin filaments during intracellular trafficking, we examined the effect of Lat B, an agent
known to disassemble the actin filaments (Spector et al., 1983),
on the trafficking of vacuolar cargo proteins. As an experimental
system, we used protoplasts derived from Arabidopsis leaf
tissues. First, we determined the effective concentration of Lat
B needed to disassemble the actin filaments in these protoplasts
because the Lat B concentrations used in previous studies with
plant cells varied greatly from a few nanomoles per liter to several
hundred micromoles per liter (Vidali et al., 2001; Brandizzi et al.,
2002; Saint-Jore et al., 2002). Thus, protoplasts were treated
with varying Lat B concentrations, after which protein extracts
were prepared and fractionated by low-speed centrifugation into
pellet and soluble fractions, which contain the filamentous and
soluble forms of actin, respectively (Abe and Davies, 1995). The
actin in these fractions was detected by protein gel blot analysis
using an anti-actin antibody. In the absence of Lat B, the majority
of actin was detected in the pellet fraction (Figure 1A, top, lanes 1
to 3). In the presence of up to 1 mM Lat B, the majority of actin was
still detected in the pellet fraction, although the amount of the
soluble form did gradually increase as the Lat B concentration
rose (Figure 1A, top, lanes 4 to 12). By contrast, at 10 mM Lat B,
;50% of the actin was detected in the soluble fraction (Figure
1A, top, lanes 13 to 15), which indicates that 10 mM Lat B may be
the appropriate concentration to use with Arabidopsis leaf
protoplasts. As a control for the fractionation, we detected the
ER-localized chaperonine binding protein (BiP) by protein gel
blot analysis using an anti-BiP antibody. The majority of BiP was
detected in the soluble fraction (Figure 1A, bottom), indicating
that BiP is not pelleted by this low-speed centrifugation, and
this fractionation pattern of BiP was not affected by up to 10 mM
Lat B.
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Figure 1. Effect of Lat B Treatment on the Actin Filaments in Arabidopsis
Protoplasts.
(A) Concentrations of Lat B needed to disassemble the protoplast actin
filaments. Protoplasts obtained from Arabidopsis leaf tissues were
treated with varying concentrations (10 nM to 10 mM) of Lat B for 24 h.
Subsequently, protein extracts were prepared, fractionated into pellet
and soluble fractions by centrifugation, and subjected to protein gel blot
analysis with anti-actin and anti-BiP antibodies. T, total; S, soluble
fraction; P, pellet fraction.
(B) Effect of 10 mM Lat B on actin filament architecture as shown by
GFP:talin fluorescence. Protoplasts transformed with GFP:talin were
incubated in the presence [Lat B (þ); panels c and d] and absence [Lat B
(); panels a and b] of Lat B (10 mM), and the pattern of GFP:talin
fluorescence was examined by fluorescence microscopy. CH, chlorophyll. Bar ¼ 20 mm.
To obtain independent evidence that the actin filaments are
disassembled by 10 mM Lat B, we visualized the actin filaments
by transforming the protoplasts with GFP:talin, which expresses
a chimeric protein consisting of green fluorescent protein (GFP)
and the actin binding domain of talin (Kost et al., 1998).
Fluorescence microscopy of the protoplasts showed that in the
absence of Lat B, GFP:talin revealed numerous networks that
indicate actin networks (Figure 1B, panel a). By contrast, at
10 mM Lat B, GFP:talin showed a diffuse pattern (Figure 1B,
panel c), indicating that the actin filaments are disassembled.
Previous studies have shown that Lat B in a nanomolar
concentration is sufficient to disassemble the actin filaments in
pollen tubes (Vidali et al., 2001), whereas 25 and 265 mM were
used with tobacco (Nicotiana tabacum) leaf and BY-2 cells,
respectively (Brandizzi et al., 2002; Saint-Jore et al., 2002). Thus,
although the Lat B concentration needed to disassemble the
actin filaments in Arabidopsis leaf protoplasts is much higher
than that used with pollen tubes, it is similar to that used with
tobacco leaf cells.
Next, we examined whether two vacuolar reporter proteins, namely, GFP-fused sporamin (Spo:GFP) and Arabidopsis
aleurain-like protein (AALP:GFP), are targeted to the central vacuole in the presence of Lat B. Sporamin is a tuber protein of
sweet potato (Ipomoea batatas), whereas AALP is a Cys protease homologous to barley (Hordeum vulgare) aleurain; both
have been shown to be targeted to the central vacuole in both
their native form and as part of a GFP fusion protein (Matsuoka
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et al., 1995; Ahmed et al., 2000; Jin et al., 2001; Sohn et al., 2003).
Protoplasts transformed with Spo:GFP or AALP:GFP were incubated with Lat B, and the localization of the GFP signal was
examined at various time points after transformation. In the
absence of Lat B, both reporter proteins were targeted to the
central vacuole (Figure 2A, panels a, f, and k). We also observed
Spo:GFP distributed in an ER pattern (Figure 2A, panels e and g),
which suggests that the Spo:GFP in a fraction of the transformed
protoplasts had not yet been transported to the central vacuole
at this time point (Jin et al., 2001; Kim et al., 2001). In the
presence of Lat B, however, the majority of protoplasts transformed with AALP:GFP displayed a novel punctate staining
pattern (Figure 2A, panel b), and the number of protoplasts
with a GFP-positive central vacuole was significantly reduced.
This also was true for the Spo:GFP-transformed protoplasts. In
the presence of Lat B, notably, the Spo:GFP-transformed protoplasts showed three new patterns, namely, a punctate staining
pattern alone (Figure 2A, panel i), a punctate staining pattern
together with the ER pattern (Figure 2A, panel h), and a punctate
staining pattern together with the vacuolar pattern (Figure 2A,
panel j), in addition to the two typical ER and vacuolar patterns.
Furthermore, when we examined the effect of Lat B on the
trafficking of Spo:GFP and AALP:GFP in tobacco leaf cell
protoplasts, we obtained similar results (data not shown), which
indicates that Lat B may have a similar effect on vacuolar
trafficking in other plant cells.
We next determined how many Spo:GFP-transformed protoplasts had vacuolar versus punctate GFP-staining patterns. As
shown in Figure 2B, in the absence of Lat B, 31 and 58% of
the Spo:GFP-transformed protoplasts had GFP-positive central vacuoles at 24 and 48 h, respectively. By contrast, in the
presence of Lat B, 7 and 29% of protoplasts had the vacuolar
GFP staining pattern at 24 and 48 h, respectively, indicating
significantly reduced vacuolar trafficking of Spo:GFP. Moreover,
in the presence of Lat B, 67 and 47% of the protoplasts had
a punctate GFP-staining pattern at 24 and 48 h, respectively,
whereas this staining pattern was absent in the control protoplasts. In the presence of Lat B, the ER-staining pattern of
Spo:GFP was reduced to 29% from 69%. The reason for this
reduction is not clearly understood at the moment. At the least,
this is in part because of the way by which we counted the
protoplasts; a small fraction of protoplasts that has the punctate
staining pattern together with the ER pattern was counted as the
punctate staining pattern. These results strongly suggest that Lat
B inhibits the vacuolar trafficking of Spo:GFP and AALP:GFP.
Trafficking of Vacuolar Reporter Proteins Is Inhibited
by Overexpressing the Actin Mutants actin2[D13K]
and actin2[D13Q]
While the above results suggest that Lat B interferes with
vacuolar trafficking, this method suffers from the possibility that
prolonged exposure to Lat B may have had a side effect on
various cellular processes in protoplasts that indirectly affect the
vacuolar trafficking of cargo proteins. To obtain independent
evidence for actin filament involvement in vacuolar trafficking, we
sought to interfere with the normal function of actin filaments
by overexpressing actin mutants. In yeast cells, expression of
Figure 2. Inhibition of the Vacuolar Trafficking of AALP:GFP and
Spo:GFP by Lat B.
(A) Phenotypic analysis of the effect of Lat B on AALP:GFP and Spo:GFP
localization. Protoplasts transformed with AALP:GFP or Spo:GFP were
incubated in the presence [Lat B (þ); panels b to d and g to k] and
absence [Lat B (); panels a, e, and f] of Lat B (10 mM), and the
localization of AALP:GFP and Spo:GFP was examined 24 and 48 h after
transformation, respectively. The red images show chlorophyll autofluorescence. CH, chloroplasts. Bar ¼ 20 mm.
(B) Quantification of the Lat B–altered distribution patterns of Spo:GFP.
Protoplasts were counted based on the Spo:GFP distribution patterns in
the presence [Lat B (þ)] and absence [Lat B ()] of Lat B (10 mM) 24 and
48 h after transformation. In each experiment, >100 transformed cells
were counted, and three independent experiments were performed to
obtain means and SD. Representatives of the ER (network pattern),
punctate staining, and vacuole patterns of Spo:GFP are shown in panels
g, i, and k of Figure 2A, respectively. Note that a small fraction of
protoplasts showing the punctate staining pattern together with the ER
pattern or the vacuolar pattern was counted as the punctate staining
pattern because the punctate staining pattern was considered to reflect
the inhibition of the vacuolar trafficking of Spo:GFP.
Actin Filaments in Vacuolar Trafficking
actin1[D11K] or actin1[D11Q] results in a dominant lethal phenotype (Johannes and Gallwitz, 1991). The D11 residue in yeast
actin1 is surface exposed and is thought to interact with divalent
metal ions or nucleotides (Johannes and Gallwitz, 1991; Wertman
et al., 1992). Arabidopsis actin2 is the most abundant actin
isoform in Arabidopsis vegetative tissues (Ringli et al., 2002).
Thus, we substituted the Asp 13 (D13) residue of Arabidopsis
actin2 with Lys (K) or Gln (Q) to generate mutants equivalent to
yeast actin1[D11K] or actin1[D11Q]. In addition, the actin2
proteins were tagged with the small epitope hemagglutinin
(HA) to distinguish the introduced molecules from the endogenous actins. First, we examined the effect of overexpressing
actin2[D13K]:HA, actin2[D13Q]:HA, or wild-type actin2:HA on the
morphology of actin filaments in protoplasts. The actin filaments
were visualized by cotransforming the cells with GFP:talin (Kost
et al., 1998). As shown in Figure 3A (panel a), protoplasts
transformed with wild-type actin2 showed numerous actin filaments in a staining pattern that was nearly identical to that of
protoplasts expressing GFP:talin alone (data not shown). Thus,
overexpression of wild-type actin2 does not affect morphology of
actin filaments. By contrast, overexpression of actin2[D13K] or
actin2[D13Q] in the protoplasts resulted in a diffuse staining
pattern of GFP:talin (Figure 3A, panels c and e), which strongly
suggests that transiently expressed actin2[D13K] and
actin2[D13Q] disrupt endogenous actin filaments. Approximately
46% of the protoplasts transformed with actin2[D13K] showed
a diffuse staining pattern of GFP:talin (Figure 3B). The expression
of the introduced actin2 proteins was confirmed by protein gel
blot analysis using a monoclonal anti-HA antibody (Figure 3E).
Thus, actin2[D13K] or actin2[D13Q] overexpression may be used
in place of Lat B to disrupt actin filaments.
We next examined the vacuolar trafficking of Spo:GFP and
AALP:GFP in the presence of actin2[D13K] or actin2[D13Q]
overexpression. As expected, overexpression of wild-type
actin2 did not affect the staining pattern of Spo:GFP and AALP:
GFP (data not shown). The percentage of wild-type actin2transformed protoplasts that showed the vacuolar staining
pattern, which indicates the vacuolar trafficking efficiency of
Spo:GFP, was 22, 46, and 53% at 24, 36, and 48 h after transformation, respectively, which was nearly the same as when the
protoplasts were only transformed with Spo:GFP (Figures 2B and
3D). However, actin2[D13K] or actin2[D13Q] overexpression produced a punctate staining pattern for Spo:GFP and AALP:GFP
(Figure 3C), and the vacuolar trafficking efficiency of Spo:GFP
was greatly reduced (Figure 3D). Furthermore, the degree to
which the actin2 mutants inhibited vacuolar trafficking was
nearly comparable to that of Lat B. This was rather unexpected
because the percentage of protoplasts showing the diffuse actin
pattern in the presence of an actin2 mutant was approximately
half of that observed upon Lat B treatment. One possible
explanation is that actin2 mutants may be incorporated into
actin filaments as actin subunits, and these actin filaments with
the mutants may not be functional, even though they are
morphologically filamentous forms. To examine this possibility, protoplasts were transformed with actin2[D13K]:HA or actin2[D13Q]:HA, after which protein extracts were prepared
and fractionated by low-speed centrifugation into pellet and soluble fractions, which contain the filamentous and soluble forms of
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actin, respectively (Abe and Davies, 1995). The actin2 mutants in
these fractions were detected by protein gel blot analysis using
an anti-HA antibody. Approximately 30% of actin2 mutants was
detected in the pellet fraction (Figure 3F), which indicates that
a portion of actin2 mutants may be incorporated into the actin
filaments. Together, these results further support the notion that
actin filaments play a critical role in the trafficking of vacuolar
proteins in plant cells.
Spo:GFP and AALP:GFP Accumulate in the Golgi Complex
in the Presence of Lat B
Next, we performed colocalization experiments with marker
proteins to identify the organelle in which vacuolar cargo proteins
accumulate in the presence of Lat B. Because vacuolar proteins
are transported to the central vacuole through the Golgi apparatus and the PVC, we reasoned that the punctate staining
pattern may correspond to either of these organelles. First, we
examined the colocalization in Lat B–treated protoplasts of
AALP:GFP and rat sialyltransferase (ST) tagged with HA because
ST has been shown to localize to the Golgi complex in plant cells
(Boevink et al., 1998; Wee et al., 1998; Lee et al., 2002; reviewed
in Nebenführ et al., 2002). The majority of the AALP:GFP in the
punctate stains colocalized with the ST:HA signals at the Golgi
complex (Figure 4A). Interestingly, however, a portion of the
punctate stains of AALP:GFP did not exactly overlap the ST:HA
molecules, rather, they colocalized side by side. This indicates
that in the presence of Lat B, AALP:GFP may localize at the Golgi
complex but at a slightly different location within the Golgi
complex than that of ST:HA, which is thought to mainly localize
at the trans-Golgi (Wee et al., 1998; reviewed in Nebenführ et al.,
2002).
To examine the localization further, we compared the localization in Lat B–treated protoplasts of AALP:GFP with that of
AtVTI1a tagged with HA (AtVTI1a:HA). A previous study showed
that AtVTI1a, a v-SNARE, and its T7-tagged form localize at the
TGN and the PVC and are thought to travel from the TGN to the
PVC together with vesicles (Zheng et al., 1999). Unlike when
ST:HA was coexpressed with AALP:GFP, the green punctate
staining pattern of AALP:GFP more closely overlapped the red
punctate staining pattern of AtVTI1a:HA that was detected by an
anti-HA antibody (Figure 4B). Thus, these results strongly suggest that AALP:GFP accumulates at the Golgi complex, and
quite possibly at the TGN. Furthermore, >90% of AtVTI1apositive punctate stains closely overlapped with AALP:GFP
signals, which was rather unexpected because AtVTI1a is
distributed in equal measure to both the TGN and the PVC
(Zheng et al., 1999). One possible reason for this is that, like
Spo:GFP and AALP:GFP, AtVTI1a:HA may not travel to the PVC
in the presence of Lat B.
To obtain additional evidence for localization of vacuolar
proteins in the Golgi complex in the presence of Lat B, we
compared the localization in Lat B–treated protoplasts of
AALP:GFP and endogenous vacuolar sorting receptor (VSRAt)/A. thaliana epidermal growth factor receptor-like protein 1
At(AtELP1). VSR-At(AtELP1), the vacuolar sorting receptor of
Arabidopsis, localizes to both the TGN and the PVC although its
primary residence is the PVC (Ahmed et al., 1997; Li et al., 2002).
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Figure 3. Actin2 Mutant Overexpression Inhibits the Trafficking of Vacuolar Proteins.
(A) Disruption of actin filaments by actin2 mutant overexpression. GFP:talin was introduced into protoplasts together with actin2 (panels a and b),
actin2[D13K] (panels c and d), or actin2[D13Q] (panels e and f), and the green fluorescence of GFP:talin was observed. Scale bar ¼ 20 mm. Filamentous
patterns ([A], panel a) and diffuse pattern ([A], panels c and e) are shown.
(B) Quantification of the effects of actin2 mutant overexpression on actin morphology. In each experiment, >100 transformed protoplasts were counted
for GFP:talin staining patterns, and three independent experiments were performed to calculate means and SEs.
(C) Punctate pattern of reporter proteins in the presence of overexpressed actin2 mutants. Protoplasts were transformed with the indicated constructs,
and the localization of the reporter proteins was examined 48 h later. Note that the GFP-staining patterns of AALP:GFP (panels a, b, e, and f) and
Spo:GFP (panels c, d, g, and h) in protoplasts overexpressing actin2 mutants were nearly identical to those in the Lat B–treated protoplasts. Only the
typical punctate staining patterns are shown here to simplify the presentation. Scale bar ¼ 20 mm.
(D) Vacuolar trafficking efficiency in the presence of actin2[D13K] and actin2[D13Q]. To estimate the vacuolar trafficking efficiency, protoplasts with the
Actin Filaments in Vacuolar Trafficking
Figure 4. AALP:GFP Accumulates in the Golgi Complex in the Presence
of Lat B.
(A) to (C) Colocalization of AALP:GFP with ST:HA, AtVTI1a:HA, and VSRAt, respectively. Protoplasts were transformed with the indicated constructs, and the localization of these proteins was examined in the
presence of Lat B (10 mM) 24 h after transformation. To detect
endogenous VSR-At, fixed protoplasts were stained with an anti-VSR
antibody followed by a Cy3-labeled anti-rabbit IgG antibody. ST:HA and
AtVTI1a:HA were detected with an anti-HA antibody followed by an antirat IgG antibody labeled with Cy3. GFP signals were directly observed in
the fixed protoplasts. Bar ¼ 20 mm.
Surprisingly, the majority of the green punctate stains also
overlapped the red punctate stains of endogenous VSRAt-(AtELP1) that was detected with an anti-VSR-At antibody
(Figure 4C). That AALP:GFP colocalizes with both ST:HA and
VSR-At(AtELP1) is rather contradictory because ST:HA localizes
to the Golgi complex (Boevink et al., 1998; Wee et al., 1998; Lee
et al., 2002), whereas VSR-At localizes primarily to the PVC (Li
et al., 2002). VSR is thought to travel to the PVC from the TGN
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(Ahmed et al., 1997; Li et al., 2002). Thus, one possible explanation for this is that, like AtVTI1a:HA, VSR-At(AtELP1) may not
travel to the PVC from the TGN in the presence of Lat B.
To address this possibility, we first examined the distribution
of VSR-At in Arabidopsis leaf protoplasts in the absence of Lat
B. We used AtSYP21 (AtPEP12p) as another PVC marker (da
Silva Conceicao et al., 1997) and compared the localization of
VSR-At with HA-tagged AtSYP21 (AtPEP12p) or the Golgilocalizing ST:GFP protein. As shown in Figure 5A, the majority
of the VSR-At–positive speckles overlapped the AtSYP21:HApositive punctate stains (panel b), whereas a minor portion
overlapped the ST:GFP-positive punctate stains (panel f). When
these colocalization patterns were quantified, ;70% of the
VSR-At–positive speckles overlapped AtSYP21:HA and 30%
overlapped with ST:GFP. Thus, the majority of VSR-At normally
localizes at the PVC, similar to what has been observed in root
cells, albeit to a lesser degree (Li et al., 2002). Next, we
examined the degree to which ST:GFP and VSR-At overlap in
the presence of Lat B. As shown in Figure 5B, the majority of
VSR-At colocalized with ST:GFP, with only a minor portion of
VSR-At–positive speckles failing to colocalize with ST:GFP.
When the overlap was quantified, 66% of the VSR-At–positive
speckles closely overlapped the ST:GFP speckles in the
presence of Lat B, in contrast with 30% in the absence of Lat
B. Thus, in the presence of Lat B, the overlap between ST:GFP
and VSR-At increased by approximately twofold, indicating
that Lat B also affects the localization of VSR-At. These results
suggest that in the presence of Lat B, VSR-At may not be able
to leave the TGN, similar to AtVTI1a:HA, which in turn results in
the accumulation of both VSR-At and AALP:GFP in the TGN.
To confirm the specificity of the antibodies we used to detect
endogenous VSR-At and transiently expressed AtSYP21:HA, we
performed protein gel blot analysis using proteins extracts
prepared from AtSYP21:HA-transformed protoplasts. As shown
in Figure 5C, the anti-VSR and anti-HA antibodies were highly
specific for VSR-At and AtSYP21:HA (AtPEP12p:HA), respectively.
Vacuolar Reporter Proteins Cofractionate with VSR-At in
the Presence of Lat B
To obtain additional evidence for the inhibition of vacuolar
trafficking by Lat B, we analyzed the localization of the reporter
proteins after fractionation of the transformed and Lat B–
treated cells. A previous study demonstrated that vacuoles
Figure 3. (continued).
vacuolar staining pattern were counted 24, 36, and 48 h after transformation. In each experiment, >100 transformed cells were counted, and three
independent experiments were performed to calculate means and SEs.
(E) Protein gel blot analysis of actin expression. Protein extracts were prepared from protoplasts transformed with the indicated constructs and
analyzed by protein gel blot analysis using a monoclonal anti-HA antibody. Un, untransformed protoplasts; WT, wild-type actin; [D13K], actin2[D13K];
[D13Q], actin2[D13Q].
(F) Subcellular fractionation of actin2 mutants. Protoplasts were transformed with actin2[D13K]:HA or actin2[D13Q]:HA. Subsequently, protein extracts
were prepared, fractionated into pellet and soluble fractions by low-speed centrifugation, and subjected to protein gel blot analysis with anti-HA and
anti-BiP antibodies. T, total; S, soluble fraction; P, pellet fraction.
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Figure 5. Effect of Lat B on the Distribution of VSR-At(AtELP1).
(A) and (B) Localization patterns of VSR-At in the presence and absence of Lat B, respectively, are shown. Protoplasts were transformed with the
indicated constructs, incubated in the absence (A) and presence (B) of Lat B (10 mM), and fixed with paraformaldehyde 24 h after transformation. To
detect endogenous VSR-At, the fixed protoplasts were stained with an anti-AtVSR antibody followed by a Cy3-labeled anti-rabbit IgG antibody. To
detect AtSYP21:HA, fixed cells were stained with an anti-HA antibody followed by a fluorescein isothiocyanate–labeled anti-rat IgG antibody. ST:GFP
was directly observed through its green fluorescence. The arrows and arrowheads indicate an overlap and nonoverlap, respectively, between AtSYP21
and VSR-At. To quantify the overlapping signals, the number of specks that indicated colocalization of VSR-At with ST:GFP or VSR-At with AtSYP21:HA
was counted. More than 40 protoplasts were counted in each experiment, and at least three independent experiments were performed to obtain means
and SE. Bar ¼ 20 mm.
(C) Specificity of antibodies. Protein extracts were prepared from protoplasts transformed with AtSYP21:HA and subjected to protein gel blot analysis
with an anti-VSR antibody and an anti-HA antibody. C, control serum; A, anti-VSR-At antibody; U, untransformed protoplasts; T, protoplasts
transformed with AtSYP21:HA.
can be separated from the nonvacuolar fraction by ultracentrifugation using a Ficoll step gradient (Jiang and Rogers, 1998).
Thus, Lat B–treated and untreated AALP:GFP-transformed
protoplasts were loaded on top of a step gradient consisting
of 4, 12, and 15% Ficoll and fractionated by ultracentrifugation. When the Lat B–untreated AALP:GFP-transformed protoplasts were fractionated, the proteolytically processed form of
AALP:GFP, which is thought to be generated in the Golgi or
post-Golgi compartments (Sohn et al., 2003), was detected in
the vacuole-containing top fraction, whereas the unprocessed
form was present in the pellet fraction (data not shown).
However, Lat B–treated AALP:GFP-transformed protoplasts
gave the same fractionation pattern (data not shown), although
Lat B–treated AALP:GFP-transformed protoplasts showed different GFP staining patterns from that of untreated samples.
This suggests that the Ficoll step gradient cannot separate
AALP:GFP-positive Lat B–induced speckles from the vacuoles.
Because the AALP:GFP punctate-staining compartment appeared to cofractionate with the vacuoles in the top fraction, we
modified the gradient to include 1, 2, and 3% Ficoll layers on
top of the 4% layer. The Lat B–treated and untreated protoplasts were loaded on top of the modified gradient and
fractionated by ultracentrifugation. Using a hemocytometer,
we determined the number of vacuoles in each fraction and
found that ;50% of the vacuoles were present in the top
fraction, with this percentage gradually decreasing with
Actin Filaments in Vacuolar Trafficking
Figure 6. Fractionation of Protoplasts on a Ficoll Gradient Confirms the
Inhibition of Vacuolar Trafficking by Lat B.
(A) Protein gel blot analysis of Ficoll gradient fractions. Protoplasts were
left untransformed or were transformed with AALP:GFP or Spo:GFP and
then incubated in the presence [Lat B (þ)] and absence [Lat B ()] of Lat
B (10 mM). They were loaded on a Ficoll step gradient consisting of 1, 2,
3, 4, 12, and 15% Ficoll 36 h after transformation and fractionated by
ultracentrifugation. The fractions were collected from each step of the
gradient as well as from the top and the pellet, and protein gel blot
analysis was performed to determine the location of AALP:GFP and
Spo:GFP in these fractions using an anti-GFP antibody. In addition, the
location of endogenous AALP and the vacuolar sorting receptor VSR-At
in the fractions of untransformed protoplasts was determined using antialeurain and anti-VSR antibodies, respectively.
(B) Quantification of band intensity. The intensity of the protein bands
indicated was measured using image analysis software. In addition, the
number of vacuoles in each fraction was determined by counting with
a hemocytometer to determine the degree of fractionation. At least three
independent experiments were performed to obtain means and SEs.
895
increasing Ficoll concentrations (Figure 6B). Lat B treatment
did not affect the distribution of the vacuoles in the gradient
(data not shown). Next, we examined the distribution of endogenous VSR-At in Lat B–treated and untreated untransformed
protoplasts. In the absence of Lat B, VSR-At was broadly
distributed from the top to the 3% fraction, with a slightly higher
amount in the 2% fraction (Figures 6A and 6B). However, the
distribution pattern of VSR-At in the presence of Lat B was
quite different because 60% of the VSR-At had accumulated in
the 1% fraction (Figures 6A and 6B). This result confirms that
Lat B alters the subcellular distribution of VSR-At. Furthermore,
these results suggest that the 1% fraction may contain the Lat
B–induced speckles.
We then used protein gel blot analysis to assess the respective presence of AALP:GFP and Spo:GFP in the
AALP:GFP- and Spo:GFP-transformed protoplast fractions
that were obtained using the modified gradient. As shown in
Figures 6A and 6B, in the absence of Lat B, 42 and 46% of the
processed forms of AALP:GFP and Spo:GFP, respectively,
were present in the top fraction and gradually decreased as the
Ficoll concentration increased, indicating that these reporter
proteins were transported to the central vacuole. By contrast, in
the presence of Lat B, the amounts of the proteolytically
processed forms of AALP:GFP and Spo:GFP in the top fraction
were reduced to 35 and 30%, respectively, whereas the 1%
fraction contained 53% of the AALP:GFP and 64% of the
Spo:GFP, suggesting that Lat B inhibits the transport of these
proteins to the central vacuole.
Next, we examined the localization of endogenous AALP in
untransformed Lat B–treated and untreated protoplasts. In the
absence of Lat B, 43% of the endogenous AALP was found in
the top fraction, and this amount gradually decreased as the
Ficoll concentration increased (Figures 6A and 6B), as was
observed with the GFP reporter proteins. By contrast, in the
presence of Lat B, the highest amount (45%) of AALP was
found in the 1% fraction but not in the top fraction, which
indicates that Lat B also affects the trafficking of endogenous
AALP. However, the fact that the amount of AALP present in
the 1% fraction is higher than that present in the vacuole was
rather surprising because endogenous AALP is likely present in
the vacuole even before Lat B treatment. To explain the lower
level of AALP in the top fraction than that in the 1% fraction
upon Lat B treatment, we postulated that the amount of AALP
in the vacuole is rapidly reduced without continuous supply of
newly synthesized AALP. To address this question, we examined the turnover rate of endogenous AALP by measuring the
endogenous AALP levels in the presence and absence of
cycloheximide, an inhibitor of protein synthesis. After treating
protoplasts with cycloheximide for 24 h, the amount of AALP
was reduced to 30% of that in the control protoplasts (data not
shown), which indicates that AALP has to be continually
supplied to the vacuole to maintain its levels there. These
results clearly demonstrate that in the presence of Lat B, the
majority of vacuolar proteins are not transported to the central
vacuole; rather, they accumulate in the 1% fraction, even
though they are processed into smaller forms. In addition,
these results demonstrate that in the presence of Lat B,
proteins destined to go to the central vacuole cofractionate
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The Plant Cell
with the vacuolar sorting receptor VSR-At in the gradient, which
further confirms the immunohistochemistry result that showed
the majority of VSR-At colocalizes with Spo:GFP in a punctate
staining pattern.
Lat B Does Not Affect the Trafficking of Reporter Proteins
from the ER to the Golgi or from the Golgi Apparatus
to the Plasma Membrane
We examined the effects of Lat B on other trafficking pathways
using ST:GFP and Hþ-ATPase:GFP as reporter proteins for the
trafficking to the Golgi and plasma membrane, respectively
(Boevink et al., 1998; Wee et al., 1998; Jin et al., 2001; Kim
et al., 2001). As shown in Figure 7A, ST:GFP appeared in
a punctate staining pattern in the presence (panel f) and absence
(panel b) of Lat B, indicating that Lat B does not affect its
trafficking from the ER to the Golgi apparatus. To unequivocally
demonstrate that this punctate staining pattern represents the
Golgi apparatus, the protoplasts displaying this pattern were
treated with BFA, a chemical agent known to disrupt the Golgi
apparatus (Driouich et al., 1993; Boevink et al., 1999; Lee et al.,
2002; Nebenführ et al., 2002). As shown in Figure 7A, both in the
presence (panel h) and absence (panel d) of Lat B, BFA treatment
generated a network pattern of the green fluorescent signal of
ST:GFP that closely overlapped the red fluorescent signal of
BiP:red fluorescent protein (RFP), a marker protein that localizes
to the ER (Kim et al., 2001). This result indicates that BFA induces
the relocation of ST:GFP to the ER both in the presence and
absence of Lat B. This is consistent with the results from
a previous study that showed that trafficking of cargo proteins
from the ER to the Golgi or from the Golgi to the ER is not inhibited
by Lat B (Brandizzi et al., 2002; Saint-Jore et al., 2002). In
addition, because BFA treatment can induce the relocation of
ST:GFP from the Golgi apparatus to the ER, actin filaments may
not be required for retrograde trafficking in plant cells. This
contrasts with what has been observed in animal cells, namely,
that Lat B inhibits retrograde trafficking from the Golgi apparatus
to the ER (Valderrama et al., 2001).
Next, we investigated the trafficking of Hþ-ATPase:GFP to the
plasma membrane in the presence of Lat B. Hþ-ATPase:GFP is
thought to be targeted to the plasma membrane through the
Golgi apparatus because BFA treatment inhibits this targeting
(Lee et al., 2002; Lefebvre et al., 2004). As shown in Figure 7B, the
Hþ-ATPase:GFP distribution pattern was the same in the presence or absence of Lat B, which indicates that Lat B treatment
does not affect the trafficking of Hþ-ATPase:GFP to the plasma
membrane. To confirm this, we estimated the percentage of
transformed protoplasts showing the plasma membrane localization of Hþ-ATPase:GFP. More than 90% of the Hþ-ATPase:
GFP–transformed protoplasts showed GFP signals at the plasma
membrane in the presence or absence of Lat B, confirming
that Lat B does not affect this trafficking (data not shown).
Together, these results suggest that actin filaments may not play
a role in the trafficking from the ER to the Golgi apparatus or from
the Golgi apparatus to the plasma membrane in leaf protoplasts.
Lat B Does Not Affect the Extracellular Secretion of
Invertase Tagged with GFP
Figure 7. Lat B Does Not Affect the Trafficking of ST:GFP or HþATPase:GFP.
(A) Localization of ST:GFP in the presence of Lat B. Protoplasts transformed with ST:GFP plus BiP:RFP were incubated in the presence (Lat B;
panels e to h) and absence (DMSO; panels a to d) of Lat B (10 mM), and
the localization of the reporter proteins was examined 24 h after transformation. The localization of ST:GFP also was examined in the presence
(BFA; panels c, d, g, and h) and absence (DMSO; panels a, b, e, and f) of
30 mg/mL BFA. Bar ¼ 20 mm.
(B) Localization of Hþ-ATPase:GFP in the presence of Lat B. Protoplasts
transformed with Hþ-ATPase:GFP were incubated in the presence [Lat B
(þ); panels c and d] and absence [Lat B (); panels a and b] of Lat B
(10 mM), and the localization of the reporter proteins was examined 24 h
after transformation. CH, chlorophyll. Bar ¼ 20 mm.
To further understand the roles actin filaments play in intracellular
trafficking, we examined the effect of Lat B and actin2[D13K] on
the secretory pathway. As a reporter protein for the secretory
pathway, we selected a secreted form of invertase (TymowskaLalanne and Kreis, 1998) and tagged it with GFP at its C terminus
(Sohn et al., 2003). Protoplasts were transformed with invertase:
GFP together with RFP, a cytosolic protein that was included as
a control for the secretion. The localization of the green and red
fluorescent signals was examined at various time points after
transformation. In both the presence and absence of Lat B, the
transformed protoplasts did not show detectable levels of green
fluorescent signals, although strong red fluorescent signals were
readily observed (Figure 8A). However, the green fluorescent
signals of invertase:GFP were readily detected in the protoplasts
when the trafficking of invertase:GFP was inhibited by coexpressing AtArf11[S31N], a dominant-negative mutant of AtArf1
that is known to inhibit anterograde trafficking from the ER to the
Golgi complex (Figure 8A, panel d; Lee et al., 2002; Takeuchi
Actin Filaments in Vacuolar Trafficking
Figure 8. Lat B and Actin2[D13K] Overexpression Do Not Affect the
Secretion of Invertase:GFP.
(A) Phenotype of protoplasts expressing invertase:GFP in the presence
of Lat B (10 mM) and actin2[D13K]. Protoplasts were transformed with
the indicated constructs. To examine the effect of Lat B, transformed
protoplasts were incubated in the presence of Lat B (10 mM) for 24 h. The
localization of RFP or invertase:GFP was examined 24 h after transformation.
(B) Secretion of invertase:GFP in the presence of Lat B and actin2[D13K].
Protoplasts transformed with invertase:GFP and RFP were incubated in
the presence [Lat B (þ)] and absence [Lat B ()] of Lat B. To examine the
effect of actin2[D13K] on the secretion of invertase:GFP, protoplasts
were transformed with three plasmids encoding invertase:GFP, RFP, and
actin2[D13K]. Protein extracts were prepared from protoplasts as well
as from the incubation medium 24 and 48 h after transformation and
analyzed for the presence of invertase:GFP, actin2[D13K], and RFP by
protein gel blot analysis using anti-GFP (panel a), anti-HA (panel b), and
anti-RFP (panel c) antibodies, respectively. Protein gel blots were prepared using equal volumes of total protoplast proteins (;20 mg) and total
medium proteins (;2 mg). To examine the presence of invertase:GFP in
protoplasts, a protein gel blot prepared using proteins obtained 24 h after
transformation was greatly overexposed. p, proteins obtained from
protoplasts; m, proteins from the incubation medium.
et al., 2002). Thus, the lack of green fluorescent signals is likely to
be as a result of the efficient secretion of invertase:GFP into the
medium (Sohn et al., 2003). To confirm this, proteins were
obtained separately from protoplasts and the incubation medium
at different time points after transformation and analyzed by
protein gel blot analysis with anti-GFP and anti-RFP antibodies.
In the control Lat B–untreated protoplasts, invertase:GFP was
mainly detected in the medium proteins and not in the protoplast
proteins (Figure 8B, panel a, lanes 2 and 4). Indeed, to detect any
897
invertase:GFP signals in the protoplast protein preparation, an
identical protein gel blot had to be greatly overexposed (Figure
8B, panel d). By contrast, RFP was detected only in the proteins
prepared from the protoplasts (Figure 8B, panel c). Thus, the
localization of invertase:GFP in the medium is likely as a result of
secretion rather than breakage of protoplasts.
Next, we examined the effect of Lat B and actin2[D13K] on the
secretion of invertase:GFP. The expression of actin2[D13K] was
confirmed by protein gel blot analysis using an anti-actin
antibody (Figure 8B, panel b). In the presence of both Lat B
and actin2[D13K], the amount of invertase:GFP that accumulated in the medium was nearly the same as that obtained in the
absence of either treatment at both time points (Figure 8B, panel
a, lanes 6, 8, 10, and 12). This indicates that the secretion of
invertase:GFP in the presence of Lat B occurs as efficiently as in
the absence of Lat B. Furthermore, even in the presence of Lat B
and actin2[D13K], the amount of RFP in the protoplast proteins
and the amount of invertase:GFP in the medium were continuously increased up to 48 h after transformation. Moreover, the
levels to which these proteins increased were nearly the same as
those in the absence of Lat B or actin2[D13K] (Figure 8B, panel a),
which strongly suggests that protoplasts in the presence of
both Lat B and actin2[D13K] are still active in the translation
of proteins as well as their secretion. These results strongly suggest that actin filaments may not be required for the secretion of
invertase:GFP.
DISCUSSION
In this study, we demonstrated that actin filaments play a critical
role in vacuolar trafficking of proteins in Arabidopsis leaf protoplasts. This was shown by disrupting the actin filaments in two
ways, namely, by treating the protoplasts with Lat B, which is an
inhibitor of actin filament assembly, or by overexpressing
actin2[D13K] and actin2[D13Q]. In yeast cells, the equivalent
actin mutants actin1[D11K] and actin1[D11Q] result in a dominant lethal phenotype (Johannes and Gallwitz, 1991). It is not yet
clear how actin2[D13K] or actin2[D13Q] overexpression disrupts
actin filaments, although the D11 residue in yeast actin1 (equivalent to D13 in Arabidopsis actin2) is thought to be involved in
the formation of a hydrophilic pocket that either accommodates
the calcium ion that binds to the b- and g-phosphates of the
bound ATP or binds bivalent metal ions or nucleotides (Johannes
and Gallwitz, 1991; Wertman et al., 1992). Both Lat B treatment
and the overexpression of actin2 mutants inhibited the trafficking
of vacuolar cargo proteins.
In the presence of Lat B or overexpressed actin2 mutants, both
vacuolar reporter proteins examined, AALP:GFP and Spo:GFP,
presented a punctate staining pattern. AALP:GFP also colocalized closely with AtVTI1a:HA and VSR-At in this punctate staining
pattern. Both AtVTI1a and VSR-At localize at both the TGN and
the PVC, although the degree of their distribution to these two
organelles differs; AtVTI1a is distributed in equal measure to the
TGN and the PVC, whereas VSR-At mainly localizes to the PVC in
Arabidopsis leaf protoplasts (in this study) and root cells (Ahmed
et al., 1997; Li et al., 2002). Interestingly, the distribution of both
AtVTI1a and VSR-At was also altered in the presence of Lat B.
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The Plant Cell
For example, although ;30% of the VSR-At–positive punctate
stains colocalized with the Golgi marker ST:GFP in Arabidopsis
leaf protoplasts in the absence of Lat B, 66% overlapped with
ST:GFP in the presence of Lat B. VSR is thought to be involved in
the sorting of soluble vacuolar proteins with N-terminal propeptide sequences such as the NPIR motif, and it travels from the
TGN to the PVC together with vacuolar cargo proteins, releases
the cargo proteins to the PVC, and then returns to the TGN
(Ahmed et al., 1997; Humair et al., 2001; Li et al., 2002). Lat B
treatment also altered the distribution of AtVTI1a:HA, which is
thought to travel from the TGN to the PVC together with vesicles,
to a predominant TGN localization. Thus, it is possible that both
VSR-At and AtVTI1a:HA may not leave the TGN in the presence
of Lat B. This is consistent with the observed accumulation of
AALP:GFP and Spo:GFP in the Golgi complex in the presence of
Lat B. The Ficoll gradient fractionation experiments that showed
cofractionation upon Lat B treatment of vacuolar cargo proteins
and VSR-At in the 1% fraction but not in the vacuole fraction
further support this possibility.
The exact mechanism by which actin filaments affect the
vacuolar trafficking at the Golgi complex is not presently known.
There are several possible scenarios that can be postulated
based on the results shown here along with the known roles of
actin filaments in various cellular processes. One possibility is
that actin filaments may function as tracks for vesicles derived
from the TGN that are destined to go to the central vacuole. Actin
filaments play such a role during pollen tube growth (Taylor and
Hepler, 1997; Gibbon et al., 1999; Vidali et al., 2001). In plant cells
such as root tip cells and the pollen tube, cytochalasin D inhibits
vesicle transport from the Golgi complex and causes vesicle
accumulation around the Golgi complex (Mollenhauer and
Morre, 1976; Picton and Steer, 1981, 1983), which leads to
suggestion that exocytic vesicles carrying materials facilitating
the polar growth of the pollen tube tip travel along actin filaments.
However, our study here using Arabidopsis protoplasts does not
support this possibility because vacuolar proteins accumulated
in the Golgi complex in the presence of Lat B, as evidenced by
their colocalization with AtVTI1a:HA and VSR-At, which indicates
that vacuolar proteins cannot leave the Golgi complex in the
presence of Lat B. Another possibility, which we favor, is that
actin filaments may play a role in the recruitment of proteins
involved in vesicle formation and/or release at the Golgi complex.
Supporting this notion is an early observation made by Shannon
et al. (1984) showing that vesicle formation at the Golgi complex
is a cytochalasin D–sensitive process. At present, it is not clear
which proteins are involved in this process at the Golgi complex
in plant cells. One of these may be the Arabidopsis dynamin-like
protein DRP 2A (ADL6), which we have shown to play a critical
role in the trafficking of Spo:GFP at the TGN in plant cells (Jin
et al., 2001). In animal cells, dynamin that is involved in vesicle
formation at the plasma membrane and the TGN binds to actin
filaments (Schafer et al., 2002; Sever, 2002). Actin filaments may
guide dynamin to specific target membranes. Cortactin, an actin
binding protein that contains a well-defined SH3 domain, associates with the Pro-rich domain of dynamin through its SH3
domain (McNiven et al., 2000). In plant cells, AtSH3P3, an
Arabidopsis protein with an SH3 domain, binds to DRP2A
(ADL6) through its Pro-rich domain (Lam et al., 2002), although
it is not known whether AtSH3P3 binds to actin filaments as well.
In addition, actin and HiP1R are necessary for the clathrincoated vesicle budding from the TGN needed for lysosome
biogenesis in animal cells (Carreno et al., 2004). It is likely that
many more proteins are involved in vesicle formation and/or
release from the TGN, as observed at the plasma membrane
during endocytosis in animal cells (Higgins and McMahon, 2002;
Merrifield et al., 2002). In the absence of actin filaments, some of
these proteins needed for vesicle formation and/or release at the
TGN may not be efficiently recruited to the TGN, which in turn
results in the accumulation of vacuolar cargo proteins as well as
the cargo receptor and v-SNARE in the Golgi complex, as is
observed in the presence of Lat B.
In contrast with vacuolar trafficking, trafficking of transiently
expressed ST:GFP between the ER and the Golgi complex does
not appear to involve actin filaments, as has been observed
previously in tobacco leaf cells or BY-2 cells (Brandizzi et al.,
2002; Saint-Jore et al., 2002). In addition, the trafficking of HþATPase:GFP and invertase:GFP, which served as plasma membrane and secretory pathway reporter proteins, respectively,
was unaffected by the presence of Lat B in Arabidopsis protoplasts. These results suggest that actin filaments may not be
involved in these pathways in leaf cell protoplasts. However, we
cannot completely rule out the possibility that a requirement for
actin filaments in these pathways in protoplasts of leaf cells is
circumvented by the use of alternative pathways. The failure of
Lat B to disrupt the trafficking of Hþ-ATPase:GFP to the plasma
membrane contrasts with previous studies showing that actin
filaments are involved in the trafficking of PIN and Hþ-ATPase to
the plasma membrane in plant root cells (Geldner et al., 2001;
Muday and DeLong, 2001; Baluska et al., 2002). One way to
explain this is that in polarized cells or cells undergoing polarized
cell elongation, trafficking of proteins to the polar apex or the cell
poles may depend on the actin filaments (Vidali and Hepler,
2001; Waller et al., 2002), whereas the trafficking of proteins such
as Hþ-ATPase:GFP and invertase:GFP in leaf protoplasts (in this
study) and PMA4-GFP in BY2 cells (Lefebvre et al., 2004) occurs
in a nonpolarized fashion and thus may not require the presence
of intact actin filaments. However, it remains possible that certain
proteins may be transported to the plasma membrane in an
actin-dependent fashion even in protoplasts. Further studies
are required to elucidate in greater detail the actin filamentdependent and -independent pathways that operate in plant cells.
In conclusion, we have shown that actin filaments play a critical
role in the trafficking of soluble cargo proteins to the central (lytic)
vacuole in protoplasts obtained from plant leaf cells. By contrast,
actin filaments may not be necessary or their role may be limited
in the trafficking that occurs between the ER and the Golgi
complex and between the Golgi complex and the plasma
membrane in leaf protoplasts. This also appears to be true for
the secretion of proteins by leaf protoplasts. However, further
studies are needed before we can fully understand the exact
roles actin filaments play in the various protein trafficking pathways in plant cells. In addition, it is not known whether the actin
filaments participate in the secretion of the carbohydrate precursors of the cell wall that are known to be produced in plant
cells by the Golgi complex (Moore et al., 1991; Gibeaut and
Carpita, 1994).
Actin Filaments in Vacuolar Trafficking
METHODS
899
Arabidopsis thaliana (ecotype Columbia) and tobacco (Nicotiana tabacum) plants were grown on B5 plates (Sigma-Aldrich, St. Louis, MO) in
a growth chamber. Leaf tissues were harvested from the plants (10- to 14d-old Arabidopsis, 3- to 4-week-old tobacco) and immediately used for
protoplast isolation as described previously (Jin et al., 2001).
To determine the turnover rate of endogenous aleurain, protoplasts
transformed with GFP were incubated in the presence of cycloheximide
(100 mg/mL), a protein translation inhibitor. Protein extracts were prepared from the protoplasts at various time points and analyzed by protein
gel blot analysis using anti-GFP and anti-aleurain antibodies. The turnover
rate of endogenous aleurain was determined by measuring the amount of
endogenous aleurain detected by the anti-aleurain antibody in the
presence of cyclohexamide. The inhibition of translation by cycloheximide
was confirmed by measuring the amount of newly synthesized GFP.
Construction of Plasmids
Fractionation of Protoplasts by a Ficoll Step Gradient
Actin2 cDNA (accession number NM_112764) was PCR amplified from
a cDNA library using the primers Act-5 (59-CATATGATGGCTGAGGCTGATGATATTCA-39) and Act-3 (59-CTCGAGTTAGAAACATTTTCTGTGAACGATT-39). Actin2[D13K] and actin2[D13Q] also were generated
by PCR using the specific primers 59-ATTCAACCAATCGTGTGTAAGAATGGTACC-39 and 59-ATTCAACCAATCGTGTGTCAGAATGGTACC-39,
respectively. To tag the wild-type and mutant forms of actin2 with HA at
the C terminus, the PCR products were digested with SalI and BamH1
and ligated into a pUC-based HA-tagging vector digested with SalI and
BamH1. The pUC-based HA-tagging vector consists of the 35S promoter
of Cauliflower mosaic virus, a multicloning site, the HA epitope, and the
nos terminator. To construct AtVTI1a:HA, AtVTI1a (accession number
NM_123313) was PCR amplified from a cDNA library using the primers 59-CACAAAGAGTAAAGAAGAACAATGAGTGACGCGTTTGATG-39
and 59-GGATCCCTTGGTGAGTTTGAAGTACAA-39. The nucleotide sequence was confirmed and ligated into the HA-tagging vector. GFP:talin
was constructed exactly as described previously (Kost et al., 1998). To
construct AtSYP21 (AtPEP12p) tagged with HA, AtSYP21 was PCR
amplified from an Arabidopsis cDNA library using two specific primers,
59-TCGAGAGAAGAGACGATG-39 and 59-GACCAAGACAACGATGAT-39.
The PCR product was confirmed and ligated to the HA tagging vector.
The central vacuole was purified according to the method described by
Jiang and Rogers (1998). Briefly, the transformed protoplasts were loaded
onto a Ficoll step gradient consisting of 4, 12, and 15% Ficoll and
subjected to ultracentrifugation at 150,000g for 2 h. The top and pellet
fractions, which contain the intact vacuoles and nonvacuolar endomembrane compartments, respectively, were collected separately from the
gradient. In addition, protoplasts were fractionated using a much finer
step gradient consisting of 1, 2, 3, 4, 12, and 15% Ficoll. Fractions (1 mL
each) were separately collected from these steps, and proteins from these
fractions were concentrated by TCA precipitation and analyzed by protein
gel blot analysis using anti-GFP (Clontech, Palo Alto, CA), anti-VSR (Li
et al., 2002), and anti-aleurain antibodies (Jiang and Rogers, 1998).
Growth of Plants
Fractionation of Filamentous and Soluble Actins from
Protoplast Extracts
Protoplasts were treated with varying concentrations of Lat B for 24 h and
then lysed gently in a buffer (5 mM Hepes, pH 7.0, 100 mM KCl, 400 mM
KI, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium
vanadate, and 1 mg/mL leupeptin) by repeated freeze and thaw cycles
(Abe and Davies, 1995). The lysates were then fractionated into soluble
and pellet fractions by centrifugation at 2500g at 48C for 15 min. The pellet
fractions were resuspended to the original volume of the lysis buffer.
These soluble and pellet fractions were analyzed by protein gel blot using
anti-actin (ICN, Aurora, OH) and anti-BiP antibodies.
Protein Preparation and Protein Gel Blot Analysis
To prepare cell extracts from protoplasts, transformed protoplasts were
subjected to repeated freeze and thaw cycles in lysis buffer (150 mM
NaCl, 20 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 3 mM MgCl2,
0.1 mg/mL antipain, 2 mg/mL aprotinins, 0.1 mg/mL E-64, 0.1 mg/mL
leupeptin, 10 mg/mL pepstain, and 1 mM phenylmethylsulfonyl fluoride)
and then centrifuged at 7000g at 48C for 5 min in a microfuge (Jin et al.,
2001). To obtain the proteins in the culture medium, cold TCA (100 mL)
was added to the medium (1 mL), and protein aggregates were precipitated by centrifugation at 10,000g at 48C for 5 min. The protein
aggregates were dissolved in the lysis buffer in a volume that corresponds
to the volume used to prepare total protoplast proteins. Protein gel blot
analysis was performed using anti-RFP (a gift of E. Kim, Korea Advanced
Institute of Science and Technology, Daejeon, Korea) and anti-HA (Roche
Diagnostics, Mannheim, Germany) antibodies as described previously
(Jin et al., 2001).
Transient Expression and in Vivo Targeting of
Reporter Cargo Proteins
Plasmids were introduced by polyethylene glycol–mediated transformation (Jin et al., 2001; Lee et al., 2002) of Arabidopsis protoplasts that had
been prepared from leaf tissues. To observe the effects of various
chemicals on trafficking, they were added to the incubation medium
immediately after transformation except when indicated otherwise. The
expression of the fusion constructs was monitored at various time points
after transformation, and images were captured with a cooled CCD
camera and a Zeiss Axioplan fluorescence microscope (Jena, Germany).
The filter sets used were XF116 (exciter, 474AF20; dichroic, 500DRLP;
emitter, 510AF23), XF33/E (exciter, 535DF35; dichroic, 570DRLP; emitter, 605DF50), and XF137 (exciter, 540AF30; dichroic, 570DRLP; emitter,
585ALP; Omega, Brattleboro, VT) for GFP, RFP, and chlorophyll autofluorescence, respectively. The data were then processed using Adobe
Photoshop software (Mountain View, CA), and the images were rendered
in pseudocolor (Jin et al., 2001).
Scoring of Localization in Protoplasts Expressing
Spo:GFP or AALP:GFP
The images of transformed protoplasts were randomly selected and
captured on a cooled CCD camera equipped with a fluorescent microscope. The images stored in the computer were later displayed on the
computer screen and scored on the basis of the GFP patterns generated
by the reporter proteins in the transformed protoplasts. Spo:GFP gave
two patterns, a network pattern (ER pattern) and a vacuolar pattern, in
the absence of inhibitor (Jin et al., 2001; Kim et al., 2001). However, in
the presence of Lat B or overexpressed actin mutants, Spo:GFP gave
three additional patterns: a punctate staining pattern (Golgi pattern) alone,
an ER plus Golgi pattern, and a Golgi plus vacuolar pattern. When trafficking efficiency of Spo:GFP to the central vacuole was estimated in the
presence of Lat B or overexpressed actin2 mutants, both the ER plus
Golgi pattern and the Golgi plus vacuole pattern, which were a minor
portion (<10%) of transformed protoplasts, were included with the Golgi
pattern. AALP:GFP produced the Golgi pattern alone, the Golgi plus
vacuolar patterns, and the vacuolar pattern alone in the presence of Lat B
or overexpressed actin mutants. Again, the Golgi plus vacuolar pattern
900
The Plant Cell
was included with the Golgi pattern when the staining patterns of
AALP:GFP were scored.
Immunohistochemistry
For immunohistochemistry, transformed protoplasts were placed onto
poly-L-Lys–coated glass slides and fixed with 3% paraformaldehyde in
a fixing buffer (10 mM Hepes, pH 7.2, 154 mM NaCl, 125 mM CaCl2,
2.5 mM maltose, 5 mM KCl) for 1 h at room temperature. The fixed cells
were incubated with appropriate primary antibodies such as rat monoclonal anti-HA (Roche Diagnostics) or rabbit anti-VSR antibodies (Li et al.,
2002) in TSW buffer (10 mM Tris-HCl, pH7.4, 0.9% NaCl, 0.25% gelatin,
0.02% SDS, 0.1% Triton X-100) at 48C overnight and then washed with
TSW buffer three times. Subsequently, the cells were incubated with
Cy3-conjugated goat anti-rabbit IgG (Sigma-Aldrich) or Cy3-conjugated
anti-rat IgG (Zymed, San Francisco, CA) secondary antibodies in TSW
buffer and then washed three times with TSW buffer. The images were
captured using a CCD camera and a Zeiss Axioplan fluorescence
microscope as described above.
ACKNOWLEDGMENTS
We thank John Rogers (Washington State University, Pullman, WA),
Liwen Jiang (Chinese University of Hong Kong, Hong Kong, China), and
Eunjeun Kim (Korea Advanced Institute of Science and Technology,
Dajeon, Korea) for anti-aleurain, anti-VSR, and anti-RFP antibodies,
respectively. This work was supported by a Grant 1ST0222601 from the
Creative Research Initiative Program of the Ministry of Science and
Technology (Korea).
Received October 24, 2004; accepted December 8, 2004.
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Actin Filaments Play a Critical Role in Vacuolar Trafficking at the Golgi Complex in Plant Cells
Hyeran Kim, Misoon Park, Soo Jin Kim and Inhwan Hwang
Plant Cell 2005;17;888-902; originally published online February 18, 2005;
DOI 10.1105/tpc.104.028829
This information is current as of June 18, 2017
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