Actin Filaments Play a Critical Role in Vacuolar Traffickingat the Golgi Complex in Plant Cells

Hyeran Kim,a Misoon Park,a Soo Jin Kim,b and Inhwan Hwanga,b,1

a Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784, Koreab Center 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 aleurain-

like 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 sialyltrans-

ferase or retrograde Golgi to ER trafficking. Lat B also failed to influence the Golgi to plasma membrane trafficking of Hþ-

ATPase: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 Sudhof, 1999).

Cytoskeletal components such as actin filaments and micro-

tubules 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 compart-

ments (Simon and Pon, 1996; Huang et al., 1999; Nebenfuhr 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 andWelch, 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

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 secre-

tion (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; Nebenfuhr 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

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 thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Inhwan Hwang(ihhwang@postech.ac.kr).Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.104.028829.

The Plant Cell, Vol. 17, 888–902, March 2005, www.plantcell.orgª 2005 American Society of Plant Biologists

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 mecha-

nismbywhich 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 plasmamembrane, 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 in-

tracellular 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, themajority

of actin was detected in the pellet fraction (Figure 1A, top, lanes 1

to 3). In the presence of up to 1mMLat B, themajority 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 mMLat Bmay 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.

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 pro-

teins, namely, GFP-fused sporamin (Spo:GFP) and Arabidopsis

aleurain-likeprotein (AALP:GFP), are targeted to thecentral vacu-

ole in the presence of Lat B. Sporamin is a tuber protein of

sweet potato (Ipomoea batatas), whereas AALP is a Cys pro-

tease 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

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, chloro-

phyll. Bar ¼ 20 mm.

Actin Filaments in Vacuolar Trafficking 889

et al., 1995; Ahmed et al., 2000; Jin et al., 2001; Sohn et al., 2003).

Protoplasts transformed with Spo:GFP or AALP:GFP were in-

cubated 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 trans-

formed 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 pro-

toplasts 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 proto-

plasts 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 cen-

tral 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 proto-

plasts. 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 auto-

fluorescence. 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.

890 The Plant Cell

actin1[D11K] or actin1[D11Q] results in a dominant lethal pheno-

type (Johannes and Gallwitz, 1991). The D11 residue in yeast

actin1 is surface exposed and is thought to interact with divalent

metal ionsor nucleotides (Johannes andGallwitz, 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 endoge-

nous actins. First, we examined the effect of overexpressing

actin2[D13K]:HA, actin2[D13Q]:HA, orwild-typeactin2:HAon 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 fila-

ments in a staining pattern that was nearly identical to that of

protoplasts expressing GFP:talin alone (data not shown). Thus,

overexpression ofwild-type actin2 does not affectmorphology 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] overexpressionmay 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 actin2-

transformed 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 trans-

formation, respectively, which was nearly the same as when the

protoplastswere only transformedwithSpo:GFP (Figures 2Band

3D). However, actin2[D13K] or actin2[D13Q] overexpression pro-

duced 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 possibil-

ity, protoplasts were transformed with actin2[D13K]:HA or ac-

tin2[D13Q]:HA, after which protein extracts were prepared

and fractionated by low-speedcentrifugation into pellet and solu-

ble fractions, which contain the filamentous and soluble forms of

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 appa-

ratus 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) taggedwith 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 Nebenfuhr 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:GFPmay 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 Nebenfuhr et al.,

2002).

To examine the localization further, we compared the locali-

zation 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 sug-

gest that AALP:GFP accumulates at the Golgi complex, and

quite possibly at the TGN. Furthermore, >90% of AtVTI1a-

positive 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 (VSR-

At)/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).

Actin Filaments in Vacuolar Trafficking 891

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

892 The Plant Cell

Surprisingly, the majority of the green punctate stains also

overlapped the red punctate stains of endogenous VSR-

At-(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

(Ahmed et al., 1997; Li et al., 2002). Thus, one possible expla-

nation 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 Golgi-

localizing ST:GFP protein. As shown in Figure 5A, the majority

of the VSR-At–positive speckles overlapped the AtSYP21:HA-

positive 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), respec-

tively.

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 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 VSR-

At, respectively. Protoplasts were transformed with the indicated con-

structs, 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 anti-

rat IgG antibody labeled with Cy3. GFP signals were directly observed in

the fixed protoplasts. Bar ¼ 20 mm.

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.

Actin Filaments in Vacuolar Trafficking 893

can be separated from the nonvacuolar fraction by ultracentri-

fugation 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 ultracentrifuga-

tion. When the Lat B–untreated AALP:GFP-transformed proto-

plasts 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 dif-

ferent 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 ap-

peared 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 proto-

plasts 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

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.

894 The Plant Cell

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 endo-

genous 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 re-

spective 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 exam-

ined 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

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 anti-

aleurain 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.

Actin Filaments in Vacuolar Trafficking 895

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 PlasmaMembrane

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; Nebenfuhr 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 theGolgi or from theGolgi 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 pres-

ence 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 local-

ization of Hþ-ATPase:GFP. More than 90% of the Hþ-ATPase:

GFP–transformedprotoplasts showedGFP 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 plasmamembrane in leaf protoplasts.

Lat B Does Not Affect the Extracellular Secretion of

Invertase Tagged with GFP

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 (Tymowska-

Lalanne 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 coex-

pressing 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

Figure 7. Lat B Does Not Affect the Trafficking of ST:GFP or Hþ-

ATPase:GFP.

(A) Localization of ST:GFP in the presence of Lat B. Protoplasts trans-

formed with ST:GFP plus BiP:RFPwere 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 trans-

formation. 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.

896 The Plant Cell

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 fromprotoplasts and the incubationmedium

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

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 accumu-

lated 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 continu-

ously 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 sug-

gest 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 proto-

plasts. 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 domi-

nant 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 (equiva-

lent 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 actin2mutants inhibited the trafficking

of vacuolar cargo proteins.

In the presence of Lat B or overexpressed actin2mutants, both

vacuolar reporter proteins examined, AALP:GFP and Spo:GFP,

presented a punctate staining pattern. AALP:GFP also colocal-

ized closelywith AtVTI1a:HA andVSR-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-Atmainly 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.

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 trans-

formation.

(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 pre-

pared 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.

Actin Filaments in Vacuolar Trafficking 897

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 propep-

tide 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 theGolgi 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, asso-

ciates 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 clathrin-

coated 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 andMcMahon, 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 Hþ-

ATPase:GFP and invertase:GFP, which served as plasma mem-

brane and secretory pathway reporter proteins, respectively,

was unaffected by the presence of Lat B in Arabidopsis proto-

plasts. 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 filament-

dependentand-independentpathways thatoperate inplantcells.

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 fromplant 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 path-

ways in plant cells. In addition, it is not known whether the actin

filaments participate in the secretion of the carbohydrate pre-

cursors 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).

898 The Plant Cell

METHODS

Growth of Plants

Arabidopsis thaliana (ecotype Columbia) and tobacco (Nicotiana taba-

cum) plants were grown on B5 plates (Sigma-Aldrich, St. Louis, MO) in

a growth chamber. Leaf tissueswere harvested from the plants (10- to 14-

d-old Arabidopsis, 3- to 4-week-old tobacco) and immediately used for

protoplast isolation as described previously (Jin et al., 2001).

Construction of Plasmids

Actin2 cDNA (accession number NM_112764) was PCR amplified from

a cDNA library using the primers Act-5 (59-CATATGATGGCTGAGGCT-

GATGATATTCA-39) and Act-3 (59-CTCGAGTTAGAAACATTTTCTGT-

GAACGATT-39). Actin2[D13K] and actin2[D13Q] also were generated

by PCR using the specific primers 59-ATTCAACCAATCGTGTGTAAG-

AATGGTACC-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 pri-

mers 59-CACAAAGAGTAAAGAAGAACAATGAGTGACGCGTTTGATG-39

and 59-GGATCCCTTGGTGAGTTTGAAGTACAA-39. The nucleotide se-

quence 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.

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 15min. 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 pre-

cipitated by centrifugation at 10,000g at 48C for 5 min. The protein

aggregateswere 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).

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 pre-

pared from the protoplasts at various time points and analyzed by protein

gel blot analysis using anti-GFP andanti-aleurain antibodies. The turnover

rate of endogenous aleurain was determined bymeasuring the amount of

endogenous aleurain detected by the anti-aleurain antibody in the

presenceof cyclohexamide. The inhibitionof translation bycycloheximide

was confirmed by measuring the amount of newly synthesized GFP.

Fractionation of Protoplasts by a Ficoll Step Gradient

The central vacuole was purified according to the method described by

Jiang andRogers (1998). Briefly, the transformedprotoplastswere 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 endomem-

brane 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, andproteins from these

fractionswere concentrated by TCAprecipitation and analyzed byprotein

gel blot analysis using anti-GFP (Clontech, Palo Alto, CA), anti-VSR (Li

et al., 2002), and anti-aleurain antibodies (Jiang and Rogers, 1998).

Transient Expression and in Vivo Targeting of

Reporter Cargo Proteins

Plasmids were introduced by polyethylene glycol–mediated transforma-

tion (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; emit-

ter, 605DF50), and XF137 (exciter, 540AF30; dichroic, 570DRLP; emitter,

585ALP; Omega, Brattleboro, VT) for GFP, RFP, and chlorophyll auto-

fluorescence, 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 micro-

scope. 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 plusGolgi pattern, and aGolgi plus vacuolar pattern.When traffick-

ing 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

Actin Filaments in Vacuolar Trafficking 899

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 mono-

clonal 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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