The Plant Cell, Vol. 12, 2201–2217, November 2000, www.plantcell.org © 2000 American Society of Plant Physiologists

A Rab1 GTPase Is Required for Transport between the Endoplasmic Reticulum and Golgi Apparatus and for Normal Golgi Movement in Plants

Henri Batoko,

a

Huan-Quan Zheng,

b

Chris Hawes,

b

and Ian Moore

a,1

a

Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom

b

Research School of Biological and Molecular Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom

We describe a green fluorescent protein (GFP)–based assay for investigating membrane traffic on the secretory path-way in plants. Expression of

At

Rab1b(N121I), predicted to be a dominant inhibitory mutant of the Arabidopsis Rab GTP-ase

At

Rab1b, resulted in accumulation of a secreted GFP marker in an intracellular reticulate compartment reminiscentof the endoplasmic reticulum. This accumulation was alleviated by coexpressing wild-type

At

Rab1b but not

At

Rab8c.When a Golgi-targeted and

N

-glycosylated variant of GFP was coexpressed with

At

Rab1b(N121I), the variant also accu-mulated in a reticulate network and an endoglycosidase H–sensitive population appeared. Unexpectedly, expression of

At

Rab1b(N121I), but not of the wild-type

At

Rab1b, resulted in a reduction or cessation of vectorial Golgi movement, aneffect that was reversed by coexpression of the wild type. We conclude that

At

Rab1b function is required for transportfrom the endoplasmic reticulum to the Golgi apparatus and suggest that this process may be coupled to the control ofGolgi movement.

INTRODUCTION

The structure and biosynthetic activities of the endoplas-mic reticulum (ER) and Golgi apparatus in higher plants arewell described, but the mechanisms that promote and reg-ulate membrane traffic between these compartments arenot well understood (reviewed in Robinson and Kristen,1982; Staehelin and Moore, 1995; Dupree and Sherrier,1998; Vitale and Denecke, 1999). The Golgi apparatus inhigher plants exists as a large number of independentGolgi stacks distributed throughout the cytoplasm. EachGolgi stack typically comprises five to eight flattened cis-ternae, whose protein and polysaccharide compositionmay vary in a

cis

to

trans

direction across the stack (Zhangand Staehelin, 1992; Wee et al., 1998; Nebenführ et al.,1999). Analysis of tobacco cells in which the Golgi stackswere labeled with green fluorescent protein (GFP) revealed

that each stack can travel as much as 2 to 4

m

m sec

2

1

onan actin network that is coextensive with the dynamic ERnetwork (Boevink et al., 1998; Nebenführ et al., 1999).Consequently, despite the dynamic organization of boththe ER and Golgi, both organelles remain in close associa-

tion. The movement of individual Golgi stacks appears toalternate between two modes: a directed vectorial mode,probably dependent on myosin motors, and a nonvectorialmode involving apparently random oscillations reminiscentof Brownian motion at particular points in the cytoplasm(Nebenführ et al., 1999). Video microscopy of GFP-labeledGolgi stacks in cultured tobacco cells suggests that thecontrol of Golgi movement is devolved to the individualstacks because adjacent stacks can enter independentlyinto phases of vectorial movement, and a stack undergo-ing vectorial movement can travel past another that isexhibiting the nondirected oscillatory type of motion(Nebenführ et al., 1999).

In contrast, the mammalian Golgi apparatus in generalforms a single interconnected cluster of membranes at themicrotubule organizing center. ER-derived COPII transportvesicles arising at peripheral sites fuse to form vesiculo-tubular clusters, which are transported along microtubulesto the central Golgi cluster (Presley et al., 1997; Scales etal., 1997; Burkhardt, 1998; Infante et al., 1999). As theclusters are transported, they mature by the selective re-moval of noncargo molecules in COPI vesicles, which aretargeted back to the ER (Letourneur et al., 1994; Martínez-Menárguez et al., 1999).

The GFP studies have led to formulation of two modelsto account for the membrane traffic between the ER andGolgi apparatus in higher plants. Both models invoke the

1

To whom correspondence should be addressed. E-mail ian.moore@plant-sciences.ox.ac.uk; fax 44-01865-275-074.

2202 The Plant Cell

existence of a vesicular intermediate, although candidatevesicles have yet to be unambiguously identified (Movafeghiet al., 1999) and direct tubular connections have been ob-served in some plant cell types (Satiat-Jeunemaitre andHawes, 1996).

In the “vacuum cleaner” model (Boevink et al., 1998),derived from observations of leaf epidermal cells, Golgistacks moving along the actin network are thought to en-counter and fuse with relatively static transport vesiclesreleased from the associated ER. In this model, transportvesicles can be produced at any point on the ER/actinnetwork, and Golgi movement is considered essential forefficient targeting and consumption of transport vesicles.These vesicles may remain associated with the ER/actinnetwork until they encounter and fuse with an itinerantGolgi stack, or the release of nascent vesicles from the ERmay be coupled to an interaction with a passing Golgistack.

The second model (Nebenführ et al., 1999) attaches par-ticular significance to the alternation between vectorial andoscillatory modes of Golgi movement in cultured tobacco

BY-2

cells and to the observation that particular regions ofthe cytoplasm can be identified at which successive Golgistacks appear to cease vectorial movement and enter oscil-latory motion (Nebenführ et al., 1999). Although the ER wasnot visible, this model proposes that vesicle exchange oc-curs during the periods of nonvectorial oscillatory move-ment, when Golgi stacks are probably positioned adjacentto particular sites on the ER or plasma membrane that arehypothesized to be specialized for vesicle exchange. Vecto-rial movement is thus proposed to allow the Golgi to movefrom sites of vesicle acquisition to sites of secretion to pro-mote the distribution of Golgi-derived secretory vesiclesaround the cell. This model predicts that vesicle productionis restricted to particular ER sites and that membrane ex-change depends on the controlled arrest of vectorial Golgimovement at these sites.

Although these models are not mutually exclusive, thefirst model attaches no particular significance to the periodsof nonvectorial oscillatory movement that may arise triviallywhen a Golgi stack encounters a discontinuity in the actinnetwork and is temporarily arrested until the network is re-engaged and vectorial movement resumes (Nebenführ et al.,1999).

Investigation of the validity of each of these models ne-cessitates identifying the sites and mechanisms of vesicleproduction and exchange in membrane traffic. However,work to date has failed to identify a single molecule with ademonstrable function in directing vesicle traffic betweenthe ER and Golgi. Work on yeasts and mammalian cellshas identified many molecular components of a conservedmechanism that promotes efficiency and accuracy in vesi-cle transport between compartments of the endomem-brane system. Some of the key components of thismechanism are encoded by multigene families in which in-dividual members act to promote specific trafficking

events in the cell. One important family is the Rab GTPasefamily, which is subdivided into several functionally distinctsubclasses. Although Rab GTPases do not themselves en-code specificity in vesicle targeting (reviewed in Pfeffer,1999), they have been implicated in regulating the activityof important classes of molecules, including the tetheringfactors and the soluble

N

-ethylmaleimide-sensitive fusionattachment protein receptors (or SNAREs), which betweenthem promote accurate vesicle targeting and vesicle fusion(although their detailed modes of action are still controver-sial [Mellman and Warren, 2000]). Some members of theRab GTPase family have also been implicated in interac-tions between the vesicle transport machinery and the cy-toskeleton (Schmitt et al., 1986; Bush et al., 1993; Valentijnet al., 2000).

All eukaryotes appear to have a Rab GTPase family, butmammals, brewer’s yeast, and plants each possess a differ-ent complement of Rab GTPase subclasses, which may re-flect the trafficking requirements of each organism. The 11Rab GTPases in Saccharomyces are grouped into justseven functional subclasses, but

.

30 different subclassesare recognized in mammals (Lazar et al., 1997; Novick andZerial, 1997). Sequencing expressed sequence tags and ge-nomes suggests that species such as Arabidopsis encode

.

50 Rab GTPases. However, plant Rab GTPases are usu-ally assigned to just eight subclasses (Bischoff et al., 1999),with each plant Rab subclass being more diverse andheterogeneous than any mammalian subclass. In plants,these subclasses are based almost entirely on sequencecomparisons, and whether they represent functional groupsis not clear. In the absence of effective in vivo trafficking as-says, information on plant Rab GTPase function has beenderived from complementation and expression studies inyeast (Palme et al., 1992; Bednarek et al., 1994; Ueda et al.,2000), immunolocalization data (Ueda et al., 1996), and ex-pression studies (Terryn et al., 1993; Nagano et al., 1995;Moore et al., 1997). Transgenic approaches suggest thatdisrupting Rab function produces various developmentaland cellular anomalies, but they have not identified the invivo trafficking roles of any plant Rab GTPase (Cheon et al.,1993; Bischoff et al., 1999).

A GFP fusion translocated into the lumen of the ER re-portedly fails to accumulate in a fluorescent form unlessits transport to a post-Golgi compartment is prevented(Boevink et al., 1996, 1999). This has been achieved bothby providing the GFP with targeting information thatcauses it to accumulate in either the ER or Golgi apparatusand by treating the cells with brefeldin A (BFA) (Boevink etal., 1996, 1999; Nebenführ et al., 1999). In these cases, afluorescent form of the protein accumulates in either theER or the Golgi apparatus, allowing these organelles to bereadily visualized by fluorescence microscopy. We haveused this observation to develop a visual and biochemicalassay for in vivo transport in plant cells and have investi-gated the function of a member of the Arabidopsis Rab1subclass in ER–Golgi traffic.

Rab1 GTPase Function in Plants 2203

RESULTS

Rapid Membrane Trafficking Assay Is Based on Differential Fluorescence of Apoplastic andIntracellular GFP

To develop a versatile in vivo membrane trafficking assaybased on the differential fluorescence of secreted and re-tained GFP, we constructed a series of vectors for Agrobac-terium-mediated transient expression (Rossi et al., 1993;Kapila et al., 1997) of secreted and retained GFP fusions intobacco leaves (Figure 1). The coding sequence of the ER-localized GFP variant mGFP5-ER, a thermotolerant deriva-tive of mGFP4-ER (Haseloff et al., 1997), was modified suchthat its C-terminal ER retrieval signal was removed and re-placed by a sequence encoding the c-Myc epitope tag. Be-cause the N-terminal ER transit peptide of mGFP5-ER wasleft unaltered, this new GFP variant, secGFP, was expectedto be translocated into the lumen of the ER and then trans-ported by a default trafficking pathway (Wieland et al., 1987;Denecke et al., 1990; Martínez-Menárguez et al., 1999; Vitaleand Denecke, 1999) to the apoplast. secGFP and its pro-genitor mGFP5-ER were cloned in a binary transformationvector pVKH18 (I. Moore, unpublished results) behind an en-hanced cauliflower mosaic virus (CaMV) 35S promoter andtobacco mosaic virus–

V

translation enhancer (Mitsuhara etal., 1996) to generate pVKH-secGFP and pVKH-GFP-HDEL,respectively.

Agrobacterium-mediated transient expression of T-DNA–encoded genes in tobacco leaves varies linearly over several

orders of magnitude as the density of the bacterial inoculumis varied (Rossi et al., 1993). Tobacco leaves were inocu-lated with serial dilutions of Agrobacterium strains contain-ing either pVKH-secGFP or pVKH-GFP-HDEL; 48 hr later,the abaxial epidermis was imaged by confocal laser scan-ning microscopy. As shown in Figure 2A, both strains di-rected readily detectable transient expression of GFP wheninfiltrated at OD

600

>

0.1, although the secGFP fluorescencewas usually weaker than that of GFP-HDEL. However, withinocula of OD

600

0.01 to 0.033, little or no secGFP fluores-cence was detectable at low magnification, but fluores-cence of GFP-HDEL was still clearly detectable in mostcells. Confocal imaging at greater resolution indicated thatGFP-HDEL expression resulted in a dynamic reticulate pat-tern of GFP fluorescence typical of the ER of tobacco epi-dermal cells (Figure 2B) (Boevink et al., 1996, 1999). NoGFP-HDEL fluorescence was visible in the apoplast, even athigh inoculum densities. secGFP fluorescence, on the otherhand, was observed principally in the apoplast (Figure 2B),and at the highest inoculum densities, faint fluorescencewas also detectable in an intracellular reticulum typical ofthe ER (data not shown). At OD

600

0.01, secGFP fluores-cence was just barely imaged, even in the apoplast, but withhigh laser power, faint apoplastic GFP fluorescence was vis-ible in many cells with no detectable intracellular accumula-tion (Figure 2B). The apoplastic location of this signal wasconfirmed by labeling the adjacent plasma membranes withFM4-64 dye (Figure 2B, inset). Fluorescence from both GFPfusions peaked at

z

48 hr, but secGFP fluorescence de-creased to undetectable levels over a period of 5 days,whereas GFP-HDEL was still fluorescent in most cells aslong as 7 days later (data not shown). This is consistent withdecreased stability of fluorescent GFP secretion to the apo-plast. In all subsequent experiments, strains containing GFPconstructs were inoculated at the discriminating OD

600

valueof 0.01 unless otherwise stated.

Export of secGFP from the ER Results in Proteolysis and Degradation in a Downstream Compartment

To test the interpretation that the reduced accumulation offluorescent secGFP was associated with its transport to theapoplast, we incubated sections of infiltrated leaf either inwater or, to inhibit export from the ER, in 10

m

g/mL BFA(Boevink et al., 1999). The resulting marked intracellular ac-cumulation of fluorescent secGFP was apparent after 6 hrand reached a maximum after 15 to 24 hr. As shown in Fig-ure 3A, in the presence of BFA, secGFP fluorescence wasobserved in a network of tubules and planar structures typi-cal of tobacco epidermal ER. If leaf pieces were incubatedfor 8 hr in BFA and then were washed and transferred to wa-ter without BFA for 7 hr, the accumulated secGFP fluores-cence was lost (Figure 3A); in contrast, control samples thatwere returned to 10

m

g/mL BFA after being washed showedno loss of secGFP accumulation during the 7-hr incubation.

Figure 1. Constructs for Transient Expression.

secGFP is derived from the ER-targeted GFP-HDEL by replacing theC-terminal tetrapeptide with a c-Myc epitope tag. N-secGFP, N-GFP-HDEL, and N-ST-GFP are derived from secGFP, GFP-HDEL, andST-GFP (Boevink et al., 1998), respectively, by addition of an N-ter-minal peptide containing an N-glycosylation site. The steady statelocation and the fluorescence characteristics of each GFP in this lo-cation are indicated at right. All constructs were inserted under con-trol of an enhanced CaMV promoter in an Agrobacterium binaryvector (Hawes et al., 2000). NGT, single-letter amino acid code; ST,sialyltransferase.

2204 The Plant Cell

Figure 2.

Agrobacterium-Mediated Transient Expression of secGFP and GFP-HDEL.

(A)

Differential fluorescence signals from tobacco epidermal cells after transient expression of secGFP and GFP-HDEL using Agrobacteriumstrains infiltrated at various OD

600

values, as indicated above each pair of images. Individual epidermal cells exhibiting GFP fluorescence in theapoplast (secGFP) and cortical cytoplasm (GFP-HDEL) can be seen. (Because the epidermal cells possess a large central vacuole with a thin

Rab1 GTPase Function in Plants 2205

Meanwhile, the intensity of GFP-HDEL fluorescence wasunaltered by incubation in BFA or by BFA washout (Figure3A), although the use of BFA

led to the appearance of smallpunctate structures closely associated with the ER network.Similar structures were observed to a lesser extent withsecGFP in the presence of BFA. These structures, whichwere specific for BFA-treated cells, were clearly smaller andless mobile than GFP-labeled Golgi stacks and appeared toexclude Golgi markers (H.Q. Zheng, C. Saint-Jore, I. Moore,and C. Hawes, unpublished observations), but their identityremains to be established.

To investigate the molecular basis of the differential fluo-rescence of secGFP and GFP-HDEL, we analyzed proteinextracts of inoculated tissues by SDS-PAGE, immunoblot-ting with anti-GFP antibodies. As indicated in Figure 3B(lane 1), GFP-HDEL accumulated predominantly as a singleband of slightly less than 30 kD, as expected, although afaint band apparently 2 kD smaller was also detected insome experiments. The abundance of this latter band wasnot detectably altered by treatment with BFA or after BFAwashout (lanes 2 and 3). In contrast, secGFP accumulatedas two distinct bands, the upper of which had an apparentmolecular mass consistent with our expectation (

z

1 kDlarger than GFP-HDEL because of the C-terminal c-Mycepitope tag; lane 1), whereas the lower band appeared tomigrate at the same position as the minor band in the GFP-HDEL extracts. Both bands were considerably less intensethan the major GFP-HDEL band, and in contrast to GFP-HDEL, the two bands in secGFP extracts were either similarin intensity or the lower band was more abundant. Strikingly,in the presence of BFA, the upper band accumulated at theexpense of the lower band, an effect that was reversedwhen BFA was washed out (lanes 2 and 3). Thus, the de-creased fluorescence observed from secGFP may reflect inpart reduced protein accumulation within the cell, whereastransport out of the ER appears to result in proteolysis,which may also in part account for the fact that secGFP isless fluorescent than GFP-HDEL (but see Discussion).

These observations suggested that the accumulation offluorescent secGFP might provide a simple, rapid assay forthe ability of diverse inhibitors, mutants, and transgenes toperturb membrane traffic between the ER and the plasmamembrane in plant cells. Furthermore, identifying the en-

domembrane organelles in which GFP accumulates wouldprovide an initial indication of which membrane traffickingstep is disrupted.

A Dominant Inhibitory Mutant of

At

RAB1b Inhibits the Transport of secGFP to the Apoplast

To determine whether the assay described above could beused to investigate the membrane trafficking functions ofthe plant Rab GTPase family, we chose to study a memberof the Arabidopsis Rab1 subclass

At

Rab1b (Bischoff et al.,1999). Members of this subclass in yeasts and mammalshave been implicated in vesicular traffic in the early steps ofthe secretory pathway (Schmitt et al., 1988; Bacon et al.,1989; Plutner et al., 1991; Tisdale et al., 1992; Jedd et al.,1995). To investigate the trafficking function of

At

Rab1b, wegenerated a dominant inhibitory

AtRAB1b

mutant contain-ing a single amino acid substitution, asparagine to isoleu-cine, in the conserved GTP binding motif GNKxD. On thebasis of structure–function analyses of the Ras-like GTPasesuperfamily (Bourne et al., 1990, 1991), we predicted thatthe resulting allele,

AtRAB1b

(N121I), would be antimorphic,or dominant negative, when expressed in plant cells, inhibit-ing the activity of the wild-type GTPase. The equivalent as-paragine-to-isoleucine substitution in many members of theRas superfamily has provided much useful informationabout the cellular functions of the respective wild-type GTP-ases (Schmitt et al., 1988; Bucci et al., 1992, 2000; Tisdaleet al., 1992). To investigate the effect of

At

Rab1b(N121I) onsecGFP transport in tobacco, we cloned both the mutantand wild-type cDNAs, under control of an enhanced CaMV35S promoter in a binary T-DNA vector. To achieve coex-pression of secGFP and

At

Rab1b derivatives, we grew Agro-bacterium strains containing the appropriate T-DNA vectorand mixed them before leaf infiltration. Unless specified, theAgrobacterium mixtures contained the secGFP strain at a fi-nal OD

600

of 0.01 and the test strain at OD

600

0.03.As shown in Figure 4A, when secGFP was coexpressed

with wild-type

At

Rab1b, the fluorescence signal was unal-tered in comparison with that of tissues inoculated with thesecGFP strain alone or with the secGFP strain plus a controlstrain expressing

b

-glucuronidase (GUS) rather than

At

Rab1b

layer of cortical cytoplasm, only the GFP-HDEL signal originating from the ER in the cortical cytoplasm adjacent to the anticlinal cell walls isclearly visible at this magnification.) The images are a projection of a 30

3

1-

m

m Z-series collected by confocal laser scanning microscopy.

(B)

High-resolution image of epidermal cells inoculated with secGFP and GFP-HDEL at OD

600

0.01, collected by confocal laser scanning micros-copy. The inset shows secGFP fluorescence in the apoplast (green) imaged in the presence of 5

m

g/mL plasma membrane–labeling dye FM4-64(red). To show the typical reticulate organization of the ER, the GFP-HDEL image is taken from an optical section passing through the corticalcytoplasm that lies immediately adjacent to the outer periclinal cell walls; the secGFP image is taken from a section passing through the centerof the cells and shows the anticlinal walls in cross-section with their associated secGFP fluorescence.Bar in

(A)

5

100

m

m for all images; bar in

(B)

5

10

m

m for both images.

Figure 2.

(continued).

2206 The Plant Cell

(pVKH-En6-GUS) (data not shown). In contrast, coexpres-sion of secGFP with

At

Rab1b(N121I) resulted in strong ac-cumulation of intracellular secGFP in many cells of theinfected region (Figure 4A). At higher resolution, GFP fluo-rescence was observed in a dynamic reticulate pattern thatwas very reminiscent of the ER visualized with GFP-HDEL(Figure 4B). This suggested that, like BFA,

At

Rab1b(N121I)may inhibit export of secGFP from the ER and thus lead toaccumulation of a stable fluorescent form of the protein inthis compartment. In contrast to some BFA experiments(Boevink et al., 1999), the morphology of the ER was not ap-preciably altered by expression of

At

Rab1b(N121I). Wenever observed secGFP accumulating in genuinely punctatestructures such as those generated by BFA or by Golgi-tar-geted GFP fusions (Boevink et al., 1998, 1999; Nebenführ etal., 1999). Occasionally, both GFP-HDEL and the secGFPthat accumulated in the presence of

At

Rab1b(N121I) ap-peared to reside in diffuse punctate structures of various di-mensions, but whenever these were viewed at greaterresolution, they always resolved into junctions of the ER net-work and were thus most probably imaging artifacts. Theaccumulation of secGFP fluorescence in the presence of

At

Rab1b(N121I) reached its maximum

z

2 days after infiltra-tion, as with secGFP at high inoculum densities, but the flu-orescence decreased much more slowly than for secGFP,being still visible 5 or 6 days later; this suggested that in thepresence of the Rab mutant, the stability of fluorescentsecGFP had been increased. Coexpression of GFP-HDELwith either wild-type or mutant

At

Rab1b resulted in no changein its apparent distribution or fluorescence intensity (data notshown), making it unlikely that the effect of

At

Rab1b(N121I)on secGFP fluorescence resulted from a general enhance-ment of GFP expression or detection.

These data suggest that the dominant inhibitory mutant

At

Rab1b(N121I) inhibits membrane traffic to the plasmamembrane and may act to prevent export of secGFP fromthe ER.

Inhibitory Effect of

At

Rab1b(N121I) Is Specifically Rescued by Coexpression of

At

Rab1b

Interpretation of the phenotypes of mutants such as

At

Rab1b(N121I) is complicated by the likelihood that theyact by sequestering an essential cellular cofactor in a non-productive complex and thus indirectly inhibit the capacityof the wild-type protein to perform its usual function. TheRas-like GTPases carrying the asparagine-to-isoleucinesubstitution that we introduced into

At

Rab1b(N121I) arepredicted to act by inhibiting the activity of a guanine nucle-otide exchange factor (GEF) that ordinarily promotes theconversion of the GTPase from its GDP-bound form to itsactivated GTP-bound form (Barbacid, 1987; Wilson et al.,1994; Jones et al., 1995). This poses a potential problemwhen the phenotype of such mutants is used to infer thenormal cellular activity of the wild-type GTPase. If the GEF

Figure 3. secGFP Reports Reversible Inhibition of Membrane Trafficby BFA.

(A) Confocal microscopy of GFP fluorescence in epidermal cells ofsamples of leaves infiltrated with Agrobacterium strains carrying ei-ther secGFP (left) or GFP-HDEL (right) after either 15 hr of incubationin water (Water) or 8 hr of incubation in 10 mg/mL BFA followed bywashing and a further 7-hr incubation in either 10 mg/mL BFA (BFA)or water (Washout). Bars 5 25 mm.(B) Protein extracts from samples incubated for either 15 hr in water(lanes 1), 15 hr in BFA (lanes 2), or 8 hr in BFA followed by washingand incubation for 7 hr in water (lanes 3) were subjected to SDS-PAGE and immunoblotted with antibodies raised against GFP. Threetimes as much extract was loaded in the samples from the unin-fected control (lane C) and the secGFP-treated tissues because thisprotein accumulated consistently less than did GFP-HDEL. The arrowindicates the position of the 30-kD molecular marker, and the arrow-heads denote the differences in mobility of the detected bands.

Rab1 GTPase Function in Plants 2207

(or other cellular factor) sequestered by the mutant GTPaseis required not only for the activity of the wild-type GTPasebut also for the activity of one or more functionally distinctproteins in the cell, then the mutant phenotype that is ob-served could arise in part or in full from inhibition of this sec-ond activity. To infer the cellular function of the wild-typeGTPase from this phenotype would thus be misleading.

In practice, that theoretical objection has not been a prac-tical impediment to the use of dominant inhibitory Rab GTP-ases of this type, which have proved to be specific inhibitorsof their respective Rab GTPase subclass when tested ei-ther in vivo or in vitro in other eukaryotic systems (Tisdaleet al., 1992; Wilson et al., 1994; Jones et al., 1995). How-ever, no information is available about the specificity ofRab GEFs in plants, nor do we know the biochemical basisof the antimorphic behavior of

At

Rab1b(N121I); therefore,we tested empirically for the specificity of

At

Rab1b(N121I)action in vivo.

Our starting assumption was that the interaction between

At

Rab1b(N121I) and a GEF or other essential cofactor is inequilibrium with the interaction between the same factorand the wild-type GTPase. If so, coexpressing the wild typein large amounts should alleviate the inhibitory effect of

At

Rab1b(N121I) on the activity of wild-type

At

Rab1b. If thecofactor sequestered by

At

Rab1b(N121I) is required only forthe activity of either

At

Rab1b or proteins with the samefunction as

At

Rab1b, this coexpression of wild-type GTPaseshould completely restore the trafficking function that is dis-rupted by

At

Rab1b(N121I) in the secGFP transport assay.Alternately, perhaps

At

Rab1b(N121I) sequesters a GEF orother cofactor that is required for the activity of both

At

Rab1b and one or more proteins with a trafficking func-tion distinct from that of

At

Rab1b; if so, the accumulation ofsecGFP could result in part or in full from the loss of thissecond activity. In this case, the disrupted trafficking func-tion will not be restored by coexpression of

At

Rab1b andmay even be further inhibited because of the increased com-petition for a common cofactor. Thus, by asking whethercoexpression of wild-type

At

Rab1b could restore normalmembrane traffic to an assay inhibited by

At

Rab1b(N121I),

Figure 4.

Inhibition of secGFP Transport by an

At

Rab1b Mutant.

(A)

Low-magnification images of GFP fluorescence in epidermal

cells of leaves infiltrated with strains carrying secGFP, GFP-HDEL,or secGFP plus either wild-type or mutant

At

Rab1b as indicated.

At

Rab1b(N121I) causes accumulation of secGFP in the ER. This isvisible as a reticulate network when the optical section passesthrough the cytoplasm underlying the periclinal cell walls; in mostcells, however, the section passes through the anticlinal walls, andthe network in the underlying cortical cytoplasm is not resolved.

(B)

High-resolution images of the intracellular accumulation ofsecGFP and GFP-HDEL. The distribution of secGFP accumulating inthe presence of

At

Rab1b(N121I) resembles that of GFP-HDEL. Ineach case, the optical sections pass through a portion of the corticalcytoplasm beneath the periclinal cell walls.Bar in

(A)

5

25

m

m for all images; bar in

(B)

5

10

m

m for both images.

2208 The Plant Cell

we were able to test empirically whether secGFP accumu-lates through loss of

At

Rab1b function.Coexpression of AtRab1b(N121I) and the wild type at

equal inoculum densities completely rescued the secGFPtrafficking defect associated with AtRab1b(N121I), as shown inFigure 5A. When viewed at high resolution, secGFP fluores-cence in these samples was comparable to that obtainedwith secGFP alone (Figure 2B; data not shown). Inoculationof AtRab1b(N121I) at twice its usual inoculum density, orcoinoculation with control strains expressing GUS ratherthan AtRab1b, resulted in no decrease in secGFP accumu-lation (data not shown). Furthermore, when GFP-HDEL wascoexpressed with AtRab1 and AtRab1(N121I), neither theintensity nor the distribution of the fluorescent signal was al-tered (Figure 5B), indicating that the absence of secGFP flu-orescence in the rescue experiment is unlikely to haveresulted from a nonspecific influence of the two AtRab1bconstructs on GFP expression, detection, or localization. Onthe basis of these observations, we suggest that the traffick-ing defect associated with AtRab1b(N121I) is specific to atrafficking function in which AtRab1b can act. Consistentwith this interpretation, the inhibition of secGFP transport byAtRab1b(N121I) was not rescued by coexpression withAtRab8c, a member of a different but closely related Rabsubclass, Rab8 (Moore et al., 1995; Bischoff et al., 1999),which is predicted to perform a different trafficking functionfrom that of AtRAB1b (Figure 5C).

The rescue test thus demonstrates that AtRab1b is suf-ficient to restore the trafficking function inhibited byAtRab1b(N121I). On morphological grounds, we proposethat AtRab1b(N121I) causes secGFP to accumulate in theER, which suggests that AtRab1b promotes continued ex-port of secGFP from the ER.

An N-Glycosylated GFP Variant Is Processed to an Endoglycosidase-H–Resistant Form in the Golgi

To investigate the hypothesis that the secGFP accumulatingin the presence of AtRab1b(N121I) has not been transportedthrough the Golgi stack, we developed an N-glycosylatedGFP derivative. Before the action of mannosidase II in theGolgi, N-glycans are sensitive to removal by endoglycosi-dase H (endoH) (Staehelin and Moore, 1995; Dupree andSherrier, 1998). Consequently, if membrane traffic is inhib-ited upstream of the Golgi compartment in which manno-sidase II acts, then a secreted N-glycosylated GFP isexpected to accumulate an endoH-sensitive form. Where inthe Golgi stack the mannosidase II acts is not known, but amedial compartment is thought likely (Staehelin and Moore,1995; Dupree and Sherrier, 1998).

We initially modified the coding sequence of GFP-HDELto introduce a potential N-glycosylation site in a 10-residuesynthetic peptide incorporated between the cleavage site ofthe ER targeting signal and the GFP coding region. The re-sulting protein, N-GFP-HDEL, retained bright fluorescence

Figure 5. sec-GFP Accumulation Phenotype of AtRab1b(N121I) IsRescued by Coexpression of Wild-Type AtRab1b but Not AtRab8c.

(A) Low-magnification images of epidermal cells expressing secGFP,either alone or in combination with the AtRab1b derivative, as indi-cated at bottom left. secGFP accumulates in the presence ofAtRab1b(N121I) (bottom left), but when wild-type and mutant AtRab1bare coexpressed (bottom right), secGFP accumulation is suppressedto amounts similar to those observed in controls that express onlysecGFP (top left) or secGFP and wild-type AtRab1b alone (top right).(B) Low-magnification (at top) and high-magnification (at bottom)confocal images of epidermal cells expressing GFP-HDEL alone or incombination with AtRab1b and AtRab1b(N121I), as specified at bot-tom left of each image. Coexpression with the AtRab1b constructsaffected neither the intensity nor the pattern of GFP-HDEL accumula-tion, as indicated by the upper and lower images, respectively.(C) Low-magnification images of epidermal cells expressingsecGFP. The wild-type AtRab8c does not induce intracellular accu-mulation of secGFP (left) and cannot suppress the effects ofAtRab1b(N121I) (right).Bar in (A) 5 25 mm for all images; bars in (B) 5 25 mm for the upperimages and 10 mm for the lower images; bar in (C) 5 25 mm for bothimages.

Rab1 GTPase Function in Plants 2209

in the ER (Figure 6) and acquired an N-glycan that remainedin an endoH-sensitive form, as shown in Figure 7A. When theequivalent synthetic peptide was included in secGFP, theresulting protein, N-secGFP, was glycosylated and showedfluorescence and stability properties similar to those de-scribed for secGFP. In the presence of AtRab1b(N121I),N-secGFP accumulated in a typical reticulate pattern, anaccumulation inhibited by coexpression with AtRab1b butnot AtRab8c, as previously observed with secGFP (data notshown). In the presence of AtRab1b(N121I), N-secGFP ac-cumulated in a form that was sensitive to digestion by en-doH, consistent with an ER location (data not shown).Unfortunately, however, trafficking of N-secGFP to the apo-plast in control treatments appeared to result in loss of thesynthetic glycosylated peptide from the N terminus of thefusion protein (data not shown), so we were unable to deter-mine whether the glycan on N-secGFP ordinarily is pro-cessed to an endoH-resistant form during transport throughthe Golgi stack. In an attempt to solve this problem, we in-troduced several other synthetic and natural N-glycosylationsequences into the N terminus of secGFP; however, wewere unable to identify a variant that exhibited fluorescenceand retained the N-terminal glycosylated extension whentransported to the apoplast.

Because GFP is more stable when targeted to the plantGolgi (Boevink et al. 1998, 1999), we suspect that proteoly-sis of secGFP and N-secGFP may occur in a post-Golgicompartment. To generate a stable glycosylated GFP forwhich the glycan was demonstrably modified in the Golgi,we replaced the N-terminal signal peptide of N-secGFP withthe N-terminal 53 residues of the rat trans-Golgi enzymesialyltransferase (ST) to generate N-ST-GFP (Figure 1). Thisenzyme is targeted to the trans-Golgi cisternae when ex-pressed in Arabidopsis (Wee et al., 1998). These N-terminal53 residues, which include the cytoplasmic and transmem-brane domains, are sufficient to target GFP to the Golgi intobacco (Boevink et al., 1998) and were expected to targetN-ST-GFP to this organelle. As shown in Figure 6, confocalimaging of epidermal cells transiently expressing N-ST-GFPrevealed that fluorescence was confined to numerous mobilepunctate structures typical of Golgi stacks labeled with GFP(Boevink et al., 1998; Nebenführ et al., 1999). Furthermore,immunoblot analysis showed that N-ST-GFP accumulatedin an endoH-resistant form, which migrated as expectedwith an apparent molecular mass of 35 kD (Figure 7A). Theseobservations suggest that N-ST-GFP resides in or passesthrough the mannosidase II–containing compartment of theGolgi.

Cells consistently accumulated less N-ST-GFP than GFP-HDEL (Figure 7A), even when the expression of N-ST-GFPwas increased until its accumulation in the Golgi was maxi-mized and the Golgi were brighter than the ER of cells ex-pressing N-GFP-HDEL, as in Figure 6. This suggests eitherthat GFP is more fluorescent in the Golgi than in the ER or,more probably, that the total volume of the Golgi apparatusis less than that of the ER, such that GFP is at a relatively

Figure 6. Effects of AtRab1b(N121I) on Intracellular Distribution ofN-ST-GFP.

Projections of 24 3 1-mm confocal optical sections through epider-mal cells expressing N-ST-GFP, N-GFP-HDEL, or N-ST-GFP plusAtRab1b(N121I), as indicated. In the cells expressing N-ST-GFPalone, fluorescence is observed in a series of discrete mobile punc-tate structures typical of the plant Golgi apparatus. In the presenceof AtRab1b(N121I), the Golgi are still labeled, but fluorescence isalso seen in a reticulate network similar to that observed withN-GFP-HDEL. The arrow points to a nuclear membrane present inthe section of one of these cells. Autofluorescence is visible in thechloroplasts of guard cells (arrowheads). Bars 5 10 mm.

2210 The Plant Cell

high concentration in each Golgi stack, accounting for therelative brightness of the stacks. Our inability to increase thetotal quantity of N-ST-GFP in the Golgi to amounts similar tothose in the ER suggests a limit on the capacity of the Golgito accumulate this protein. We next asked whether coex-pression with AtRab1b(N121I) alters the transport and pro-cessing of N-ST-GFP.

N-ST-GFP Accumulates an EndoH-Sensitive Form in the Presence of AtRab1b(N121I)

Coexpression of N-ST-GFP with AtRab1b(N121I) changedits intracellular distribution, causing it to accumulate addi-tionally in a typically ER-like reticulate network associatedwith the Golgi stacks (Figure 6). This reticulate network wasnot observed when N-ST-GFP was coexpressed with wild-type AtRab1b or when the wild-type and mutant Rab pro-teins were coexpressed together with N-ST-GFP; therefore,

this network most probably represents the ER. This interpre-tation was supported by immunoblot analysis of N-ST-GFP,which revealed that coexpression with AtRab1b(N121I) in-duced the accumulation of an additional endoH-sensitiveform of N-ST-GFP that apparently had not encounteredGolgi mannosidase II (Figure 7B). The residual endoH-resis-tant form of N-ST-GFP presumably represents the Golgi-localized population identified in confocal images (Figure 6).We observed that the labeling of the reticulate compartmentin the presence of AtRab1b(N121I) was far more transientwith N-ST-GFP than with secGFP or N-secGFP, and thatthe loss of N-ST-GFP fluorescence in this compartment wasaccompanied by the loss of the endoH-sensitive form. Thisis consistent with the view that the sensitive form resides inthe network, the resistant form in the Golgi.

These observations suggest that AtRab1b(N121I) inhibitsthe transport of GFP fusions before they reach the mannosi-dase II compartment of the Golgi. They strongly suggest thatthe reticulate compartment in which the GFP fusions accu-mulate is pre-Golgi and therefore is most probably the ER.They also show that AtRab1b(N121I) can inhibit the normaltrafficking of both a membrane protein such as N-ST-GFPand soluble proteins such as secGFP and N-secGFP.

Expression of AtRab1b(N121I) Disrupts Normal Movement of Golgi Stacks on the ER Network

Plant Golgi stacks are characterized by periods of rapidvectorial movement punctuated by periods of relative sta-sis in which individual stacks cease directed movementand oscillate in a fashion resembling Brownian motionwithin a small area little larger than the Golgi stack itself(Boevink et al., 1998; Nebenführ et al., 1999). However, thesignificance of this Golgi behavior for membrane traffic be-tween the ER and Golgi in higher plants is unclear. Unex-pectedly, confocal imaging of cells coexpressing N-ST-GFPand AtRab1b(N121I) revealed that vectorial Golgi movementwas markedly reduced and in most cells was essentiallyabolished. In these cells, the Golgi stacks simply underwentminor oscillatory movements reminiscent of the movementsthat characterize periods of stasis in control cells. Apartfrom the altered Golgi behavior, the cells appeared to beotherwise healthy; they continued to exclude propidium io-dide and appeared to maintain an unaltered ER network thatexhibited its characteristic movement. Moving images ofthese cells are viewable at http://www.plants.ox.ac.uk/moore/index.html. To convey the extent of Golgi movement in astatic image, we took 60 successive images of a single opti-cal section every 1.5 sec and projected them on top of eachother (shown in Figure 8). In this projected image, Golgistacks that exhibited rapid vectorial movement over the pe-riod of observation appeared at many different positions,tracing out their path across the composite image as de-picted in Figure 8A. In control cells expressing only N-ST-GFP,the movement of Golgi stacks was so extensive that entire

Figure 7. Effect of AtRab1b(N121I) on Processing of N-Glycosy-lated GFP Derivatives.

(A) Extracts of leaf tissues expressing N-GFP-HDEL or N-ST-GFPwere incubated either with (1) or without (2) endoH and analyzed bySDS-PAGE and immunoblotting with a GFP C-terminal antibody(N15; ABCAM). The N-glycans on GFP-HDEL, but not those onN-ST-GFP, are sensitive to removal by endoH, as illustrated by ashift of z2 kD in the apparent molecular mass of the major band.(B) Cells expressing N-ST-GFP alone or N-ST-GFP plus AtRab1b(N121I)were analyzed as described for (A). Coexpression of N-ST-GFP withAtRab1b(N121I) resulted in the accumulation of an endoH-sensitivepopulation of N-ST-GFP. This example shows that slightly less thanhalf of the N-ST-GFP had accumulated in an endoH-sensitive form.Although in other experiments the endoH-sensitive population wassometimes slightly more abundant than the resistant form, it wasnever observed to be the predominant species.

Rab1 GTPase Function in Plants 2211

Figure 8. AtRab1b(N116I) Inhibits Normal Golgi Movement.

Confocal laser scanning microscopy of cells coexpressing N-ST-GFP with wild-type AtRab1b (A), AtRab1b(N121I) (B) and (C), or wild-typeAtRab1b plus AtRab1b(N121I) (D). Cells were imaged every 1.5 sec over a 90-sec period. At left in (A) to (D) is the first image from each series,and at right is the final image. At center is the projection of the entire series of 60 images in which moving Golgi trace out their tracks. Cells suchas those in (B) and at the bottom of (C) represent z20% of the epidermal cell population and exhibited some Golgi movement, although this wasless extensive than in controls (cf. rows [A] or [D] with [B] and [C]), whereas the other two cells shown in (C) exhibited essentially no Golgimovement and such cells represented z60% of the epidermal cell population.

2212 The Plant Cell

regions of cytoplasm were covered by these tracks (Figure8A). In contrast, stacks that did not exhibit vectorial move-ment throughout the 90-sec period of observation appear inthe projection as single dots, the diameter of which reflectsthe amplitude of oscillation of the Golgi stack. We estimatethat in control cells, <2% of the total population of Golgi arestatic. Strikingly, in the projections of cells coexpressingN-ST-GFP and AtRab1b(N121I), between 50 and 90% ofthe Golgi are static over the 90-sec observation period (Fig-ures 8B and 8C). In cells that exhibit vectorial Golgi move-ment, the extent of movement is much less than for thecontrols (cf. Figures 8A and 8B).

As with the secGFP transport assay, wild-type AtRab1bdid not alter Golgi movement (data not shown), but it couldrestore normal Golgi movement when coexpressed withAtRab1b(N121I) (Figure 8D). This suggests that Golgi move-ment can be inhibited by loss of AtRab1b activity alone,which implies that AtRab1b activity is required for the nor-mal regulation of plant Golgi movement. Thus, in addition tothe acto-myosin system, AtRab1b is now also implicated inthe control of plant Golgi movements, providing experimen-tal evidence of an intriguing link between Golgi mobility andthe control of membrane traffic from the ER.

DISCUSSION

Investigation of membrane traffic on the biosynthetic secre-tory pathway in plants has been hindered by the absence ofsimple in vitro or in vivo transport assays. Here, we showthat secreted and N-glycosylated variants of GFP can beused effectively to report on membrane traffic between theER and the Golgi apparatus in tobacco epidermal cells. Thebasis of the assay is the observation that secreted GFP ex-hibits less fluorescence than do the forms retained withinthe ER or Golgi apparatus. This differential fluorescenceprovides a convenient assay of the ability of drugs or trans-genes to interfere with normal trafficking of GFP to the apo-plast. The use of transient expression allows transgenes tobe tested rapidly and is applicable for use with transgeneswhose expression would preclude the regeneration of trans-genic plants. The weaker fluorescence observed with apo-plastic GFP may arise at least in part from the lesser stabilityof the protein in this compartment compared with that in theER. secGFP consistently accumulated in lesser amountsthan did the ER-localized GFP-HDEL, and its predominantform was a proteolytic product estimated to be z2 kDsmaller than expected. This product was not detected by anantiserum raised against a C-terminal peptide of GFP (H.Batoko and I. Moore, unpublished results), so this unex-pected form is probably truncated at the C terminus. Thetruncation may contribute to the low stability or weak fluo-rescence of apoplastic GFP, although some GFP variantsthat have lost z10 residues from their C terminus are knownto retain fluorescence in the ER and cytoplasm (Gadella et

al., 1999). Thus, perhaps the apoplast provides a poor envi-ronment for the formation or maintenance of fluorescentGFP molecules.

Fusions to ST show that GFP can be stable in the lumenof the plant Golgi apparatus (Boevink et al., 1998), probablyuntil reaching the trans-Golgi compartments (Wee et al.,1998); therefore, the secGFP assay potentially can detectperturbation in membrane trafficking before this compart-ment is reached. Still to be determined is whether the assayis able to report on subsequent steps in the secretory path-way, given uncertainty as to whether secGFP would emit agreater fluorescent signal in post-Golgi structures than inthe apoplast.

We have used the GFP assay to identify a function foran Arabidopsis Rab GTPase in membrane traffic to theGolgi apparatus. In the presence of the antimorphic alleleAtRAB1b(N121I), secGFP accumulated in a reticulate net-work that was morphologically indistinguishable from theER. Consistent with this interpretation, in the presence ofAtRab1b(N121I), an N-glycosylated derivative of GFP accu-mulated in an endoH-sensitive form that had not been mod-ified by Golgi mannosidase II. Where mannosidase II actswithin the plant Golgi stack is not known, although a mediallocation is considered likely (Staehelin and Moore, 1995;Dupree and Sherrier, 1998). The accumulation of an endoH-resistant form would also be observed if AtRab1b(N121I)were to inhibit transport between cis- and medial Golgicompartments rather than inhibit the transport from ER toGolgi, as we propose. We think inhibition of the formertransport is unlikely, given recent demonstrations that aGFP fusion to mannosidase I accumulates in cis-Golgi com-partments and results in a typically punctate distribution, ashas been described for the ST-GFP fusion (Boevink et al.,1998; Nebenführ et al., 1999). However, in the presence ofAtRab1b(N121I), we observed no such punctate distributionof secGFP, only a reticulate network typical of ER-targetedGFP fusions.

Notably, the AtRab1(N121I) did not appear to result in thevesiculation of the Golgi apparatus, which is seen with theequivalent Hs-Rab1a mutant in mammalian cells (Wilson etal., 1994) or in the accumulation in the ER, as happens withthe equivalent YPT1 mutant in Saccharomyces cerevisiae(Schmitt et al., 1988; Jedd et al., 1995). Confirming theseobservations by electron microscopy of tobacco epidermalcells will be of interest. However, the apparently different ef-fects of dominant inhibitory Rab1 mutants in the various or-ganisms may reflect specific differences in the organizationof ER–Golgi membrane traffic in each, and the molecularbasis of these differences may become clearer as more fac-tors involved in ER–Golgi traffic in plants are characterized.

The mechanism of action of AtRab1b(N121I) remains tobe determined, but antimorphic GTPase mutants such asthis are believed to act by inhibiting the activity of a GEF(Jones et al., 1995; Rybin et al., 1996; Cool et al., 1999).GEFs promote the conversion of the GTPase to its activeGTP-bound form by catalyzing the release of GDP from the

Rab1 GTPase Function in Plants 2213

inactive GDP-bound form, facilitating the binding of GTP.The asparagine-to-isoleucine substitution in the GNKxDmotif of the GTPase appears to reduce the affinity of theprotein for guanine nucleotides (Cherfils and Chardin, 1999)and stabilizes the nucleotide-free form of the protein thatarises, usually only transiently, during the nucleotide ex-change reaction. This nucleotide-free state forms a high-affinity complex with the GEF; accordingly, the mutant isbelieved to competitively sequester the GEF in a nonpro-ductive complex that precludes normal rates of nucleotideexchange on the wild-type GTPase.

Although no functional data are yet available on regulatoryproteins of plant Rab GTPases, the inhibitory activity ofAtRab1b(N121I) probably results from the titration of someessential cofactor (such as a GEF) with which AtRab1b in-teracts, although the identity of this factor is not known.When interpreting the phenotypes of such antimorphic al-leles, one must consider the possibility that the mutant alleleelicits a pleiotropic phenotype through titration of a cofactorthat is required by more than one Rab GTPase or titration ofsome other cellular factor. Whatever the mechanism of ac-tion of AtRab1b(N121I), the ability of AtRab1b, but notAtRab8c, to rescue the secGFP accumulation phenotype in-dicates that the AtRab1b(N121I) phenotype probably arisesspecifically from the loss of an activity that can be suppliedby the wild-type AtRab1b protein; this would reveal a rolefor AtRab1b in transport between the ER and Golgi. Thefailure of AtRab8c to rescue the phenotype is importantbecause it is a member of the plant Rab subclass that ismost closely related to the Rab1 subclass, which includesAtRab1b (Bischoff et al., 1999). This supports the view thatthe AtRab1b(N121I) phenotype is specific for the Rab1 sub-class. Inhibition of a GEF specific for the Rab1 subclasswould be consistent with the current understanding of RabGTPase action in other eukaryotes. Once considered pro-miscuous, Rab GEFs in yeasts and mammals are nowthought probably to be specific for individual Rab sub-classes. This specificity is inferred from the specificity of ac-tion of antimorphic alleles, such as AtRab1b(N121I), thatinhibit Rab GEF activity (Jones et al., 1995; Walch-Solimenaet al., 1997; Day et al., 1998) and from the direct analysisof the few Rab GEFs that have been identified (Walch-Solimena et al., 1997; Wang et al., 2000).

Why transport of N-ST-GFP to the Golgi was not com-pletely inhibited by AtRab1b(N121I) or why it accumulated inthe ER more transiently than secGFP did is not clear. Per-haps before the membrane traffic was inhibited by mutantAtRab1b, some N-ST-GFP was transported to the Golgi,where it accumulated in a stable and fluorescent form; incontrast, secGFP transported to the apoplast was unstableand therefore was not detected. Although we cannot ex-clude this trivial explanation, we were not able to preventsome residual accumulation in the Golgi either by varyingthe inoculum of the AtRab1b(N121I) strain or by infiltratingleaves with the AtRab1b(N121I) strain at 1 or 2 days beforeexposure to the N-ST-GFP strain. Consequently, perhaps

AtRab1b(N121I) never completely inhibits export from theER. Possibly, tobacco epidermal cells possess a second ERexport mechanism that is not dependent on AtRAB1b, andN-ST-GFP and maybe secGFP can be transported by thismechanism. Interestingly, the Rab1 subclass is unusuallylarge and diverse in plants (e.g., Arabidopsis has at least sixgenes sharing as little as 74% identity compared with twoloci that are 91% identical in mammals and a single locus inSaccharomyces and Schizosaccharomyces); perhaps theseadditional Rab1 proteins are involved in mechanisms of ER–Golgi traffic that are insensitive to AtRab1b(N121I).

When AtRab1b(N121I) was coexpressed with the glyco-sylated Golgi marker N-ST-GFP, we consistently observed aresidual endoH-resistant form in addition to the new endoH-sensitive form. Perhaps some of the endoH-resistant mole-cules were generated in the ER, through either redistributionof Golgi processing enzymes to this compartment or the ac-cumulation in this compartment of newly synthesized pro-cessing enzymes for which transport to the Golgi had beeninhibited. However, coexpression of AtRab1b(N121I) withN-GFP-HDEL did not result in the appearance of endoH-resis-tant forms of this marker, although whether N-GFP-HDEL isa substrate for the glycan-processing activities normallyfound in the Golgi remains to be established. However,given the clear presence of residual Golgi-localized N-ST-GFP in the presence of AtRab1b(N121I), the simplest inter-pretation is that the endoH-resistant molecules representthis Golgi-localized population.

Our analysis of N-ST-GFP transport unexpectedly re-vealed that expression of AtRab1b(N121I) resulted in eitherpartial or complete inhibition of Golgi movement on the ERnetwork. The movement of plant Golgi stacks on an actinnetwork coextensive with the ER has been demonstratedrecently by the expression of ST-GFP in tobacco epidermalcells. Individual Golgi stacks alternate between periods ofrapid vectorial movement and periods of nondirected oscil-lation reminiscent of Brownian motion (Boevink et al., 1998).A recent analysis of Golgi movement in cultured tobaccocells also identified these two modes of Golgi movementand demonstrated that the probability of individual stacksentering periods of vectorial movement or stasis variedmarkedly in different regions of the cytoplasm of a singlecell (Nebenführ et al., 1999). Certain cytoplasmic positionscould be identified in which a succession of Golgi stacksindividually entered periods of stasis and then resumedvectorial movement. Furthermore, an individual Golgistack was able to remain in a period of stasis while an adja-cent stack engaged in rapid vectorial movement, suggestingthat the control of Golgi movement is devolved to the indi-vidual stack. These unexpected observations have led tothe formulation of at least two distinct hypotheses to ac-count for membrane exchange between the ER and thehighly dynamic Golgi stacks of plant cells. The vacuumcleaner model postulates that rapidly moving Golgi stacksencounter and fuse with ER-derived vesicles as they travelon the actin/ER network (Boevink et al., 1998). The alternate

2214 The Plant Cell

“filling station” model postulates that membrane traffic be-tween these two organelles occurs during the periods ofstasis, when the Golgi may be positioned near putative ex-port sites on the ER (Nebenführ et al., 1999). However, an-other explanation of these periods of stasis is that theysimply represent discontinuities in the actin network, in whichGolgi stacks are immobilized before reengaging the network(Nebenführ et al., 1999). So although the distribution of theER and the movement of the Golgi stacks clearly are closelyassociated with the same actin network, no evidence links anyof these properties to membrane traffic between the two or-ganelles. Our observation that expression of AtRab1b(N121I)inhibits the trafficking of secGFP from ER to Golgi and alsoresults in the inhibition or cessation of Golgi movement pro-vides evidence of a possible mechanistic link between Golgitraffic and movement. To investigate these possibilitiesmore fully, it is important to quantify the rate of ER–Golgitraffic in cells in which their Golgi are immobilized by treat-ing the cells with inhibitors of the acto-myosin system or byexpression of AtRab1b(N121I). Identification of sites of vesi-cle production on the ER will also be informative, as will es-tablishing whether these coincide with the positions at whichGolgi stacks cease their bouts of rapid vectorial movement.

METHODS

Molecular Cloning and Generation of Mutant AtRab1b(N121I)

Standard molecular techniques as described by Ausubel et al. (1999)were followed. The wild-type cDNA of AtRAB1b (Bischoff et al.,1999) was amplified by the polymerase chain reaction (PCR) from theexpressed sequence tag clone z26415 by using the upstream primerPAR1 (59-ACCGCTCGAGAACCATGAATCCTGAGTACG-39) and thedownstream primer PAR2 (59-TAGATGTCGACGGATCCTCAA-GTTGAGCAGCAGCCG-39). The PCR product was digested andsubcloned as a XhoI-SalI fragment into pBluescript SK1 (Strat-agene). A double-stranded oligonucleotide constructed with PARNI1(59-AACAAACTCCTTGTTGGAATCAAGTCAGATCTTACTGAAAAC-AGAGCCATTCCTTATGAA-39) and PARNI2 (59-CTTGGCAGTTTCATA-AGGAATGGCTCTGTTTTCAGTAAGATCTGACTTGATTGATTCCAA-CAAG-39) was used to replace a HincII-StyI fragment in the wild-typecoding region. The double-stranded oligonucleotide introduced botha single-point mutation, the asparagine-to-isoleucine substitution(letters in boldface), and an additional HinfI restriction site (under-lined letters) to generate AtRab1b(N121I). The wild-type and the mu-tant cDNAs were checked by sequencing with primers T3 and T7 andthen were cloned as an XhoI-BamHI fragment into SalI and BamHIsites of the expression cassette of the binary vector pVKH18-EN6-GUS, under control of an enhanced cauliflower mosaic virus (CaMV)35S promoter, a tobacco mosaic virus–V sequence as translationenhancer, and a nopaline synthase polyadenylation signal, all de-rived from pE6113GUS (Mitsuhara et al., 1996). Plasmid pVKH18-EN6-GUS had been generated by inserting the expression cassetteof pE6113GUS (Mitsuhara et al., 1996) as a PvuII-HindIII fragmentinto the Ecl136II-HindIII sites in the polylinker of pVKH18, a derivativeof pVK18 (Moore et al., 1998) that carries a hygromycin-selectable

marker in place of the methotrexate resistance marker of pVK18 (I.Moore, unpublished results).

To create pVKH-GFP-HDEL, the coding region of mGFP5 fused toan Arabidopsis chitinase signal peptide from plasmid pBIN-mGFP5-ER (gift of Dr. J. Haseloff, University of Cambridge, UK) was sub-cloned into the expression vector pVKH18-EN6 as an XbaI and SacIfragment, replacing the b-glucuronidase (GUS) coding region to yieldgreen fluorescent protein (GFP)–HDEL (Figure 1). The C-terminal tet-rapeptide HDEL of GFP-HDEL was replaced by a c-Myc epitope byPCR with primers 59-CCGGATCCAACAATGAAGACTAATCT-39 and59-TTGGATCCTTACAAATCCTCCTCAGAGATAAGCTTCTGCTC-39. ThePCR product was cloned into pVKH18-EN6-gus, replacing the GUScoding sequence to generate pVKH-secGFP.

To engineer the glycosylated form of GFP, we amplified the signalpeptide in mGFP5-ER by using Taq/Pwo (Hybaid Ltd., Ashford, UK)and primer SGFP1 (59-GCTCTAGAACCACCATGAAGACTAATCTTT-TTC-39) combined with SGFP29 (59-AACCTCGAGCCGGTAACGGTT-CCATTTGAAACAAGTTCTCCAGTCGCAGTGAATTCGGCCGAGGAT-AAT-39). The resulting PCR product, which encoded the signal pep-tide with a 10-residue linker downstream, was digested with XbaIand XhoI and then was used to replace the signal peptide of secGFPto yield pVKH-N-secGFP. The new GFP coding region is thus ex-tended at the N terminus by a 10-residue region containing an N-gly-cosylation site (see Figure 1). The synthetic peptide harboring theglycosylation site was designed according to Gavel and von Heijne(1990). Primers SGFP1 and HGFP39 (59-AAGGATCCTTAAAGCTCAT-CATGAAGATCTTCGGAAATAAGCTTCTGCTCTTTGTATAGTTCATC-39)were used to amplify the N-secGFP coding region, introducing a C-ter-minal HDEL tetrapeptide to generate N-GFP-HDEL. The PCR productwas digested by XbaI and BamHI and then was cloned into pVKH18-En6-gus, replacing the GUS coding region. N-ST-GFP was con-structed by replacing the signal peptide of N-secGFP with the sialyl-transferase (ST) transmembrane domain (Boevink et al., 1998) andleaving the 10-residue N-glycosylated linker between the transmem-brane domain and the GFP. Details on all of the constructs are avail-able on request.

Plant Material and Agrobacterium tumefaciens–Mediated Transient Expression

Nicotiana tabacum SR1 (cv Petit Havana) seeds were disinfectedwith 70% ethanol and plated on Murashige and Skoog basal salt me-dium (Sigma) containing 0.8% agar (Difco). After incubation for 2weeks at 20 to 228C, the seedlings were transferred to soil and theresulting plants were maintained at 218C. Each expression vectorwas introduced into A. tumefaciens strain GV3101 (pMP90) (Konczand Schell, 1986) by electroporation. A single colony from the trans-formants was used to inoculate 2 mL of YEB medium (per liter: 5 g ofbeef extract, 1 g of yeast extract, 5 g of sucrose, and 0.5 g ofMgSO4·7H2O), supplemented with 100 mg/mL kanamycin, 10 mg/mLgentamycin, and 1 mM isopropyl-b-D-thiogalactopyranoside. Thebacterial culture was incubated at 28 to 308C with agitation untilreaching the stationary growth phase. One milliliter of culture wastransferred into an Eppendorf tube, and the bacteria were pelleted bycentrifugation at 2200g for 5 min in a microcentrifuge at room tem-perature. The pellet was washed twice with 1 mL of the infiltrationbuffer (50 mM Mes, pH 5.6, 2 mM Na3PO4, 0.5% glucose [w/v], and100 mM acetosyringone [Aldrich]) and then resuspended in 1 mL ofthe same buffer. The bacterial suspension was diluted with infiltrationbuffer to adjust the inoculum concentration to the stated final OD600

Rab1 GTPase Function in Plants 2215

value. The inoculum was delivered to the lamina tissues of tobaccoleaves by gentle pressure infiltration through the stomata of the lowerepidermis, by using a 1-mL syringe without a needle. For experi-ments requiring coinfection of more than one construct, bacterialstrains containing the constructs were mixed before performing theleaf infection, with the inoculum of each construct adjusted to the re-quired final OD600. The infected area of the leaf was delimited and la-beled with an indelible pen, and the plant was incubated undernormal growing conditions as above.

Sampling, Brefeldin A Treatment, and Imaging

If not otherwise stated, GFP-dependent fluorescence was analyzed48 hr after infection in cells of the lower epidermis. Routinely, the ex-pression pattern and intracellular distribution of GFP fluorescencewere initially assessed by using an epifluorescence microscope be-fore the confocal microscope. Pieces of leaf were randomly cut fromthe infected area and mounted in water for microscopic observa-tions. For treatment with brefeldin A (BFA), leaf pieces were excised35 hr after infection and floated on 10 mg/mL BFA solution or wateras control. The BFA effect, as assessed by GFP fluorescence in thecell under an epifluorescence microscope, was evident after 6 hr. Tomonitor the reversibility of BFA effects, we thoroughly washed theleaf piece in water after 8 hr of incubation with the drug; half of thepiece was kept in the BFA solution and the other half was incubatedin water. The samples were examined 7 hr later. Samples that re-ceived 15 hr of BFA treatment gave the same result as those incu-bated with the drug for only 8 hr.

Confocal imaging (with a Zeiss [Wellwyn, UK] CLSM 410 or Bio-Rad MRC 1024) was performed on samples mounted in water. Opti-cal sectioning and time-lapse scanning were acquired by using theBio-Rad MRC 1024 imaging system. The software Confocal Assis-tant (Bio-Rad), LSMdummy (Zeiss), and Photoshop 5.0 were used forpostacquisition image processing.

Protein Extraction, Endoglycosidase Treatment, and Protein Gel Blot Analysis

Proteins were extracted from BFA-treated and control samples (z200mg fresh tissues) by grinding them in liquid N2 followed by homoge-nization in two volumes of the extraction buffer: 75 mM Tris, pH 6.8,1 mM EDTA, 2% SDS, 3% b-mercaptoethanol, and 1 mM phenyl-methylsulfonyl fluoride. The homogenate was cleared by centrifuga-tion for 5 min at 1100g in a microcentrifuge, and soluble proteins inthe supernatant were precipitated in 6% trichloroacetic acid (v/v) for20 min on ice. The mixture was centrifuged for 15 min at 12,000g,and the resulting pellet was washed twice with acetone, dissolvedin loading buffer for SDS-PAGE, and boiled for 5 min before loading.

Protein extracts for endoglycosidase H (endoH) digestion wereprepared from samples of infected leaf areas (300 to 400 mg freshweight) frozen in liquid N2 . The tissue was ground in a microcentri-fuge tube by using a plastic pestle in the presence of two volumes ofthe following extraction buffer: 50 mM sodium citrate, pH 5.5, 5%SDS (w/v), 0.01% BSA (w/v), 2% b-mercapthoethanol, 150 mMNaCl, and 10 mL (per 300 mg of plant material) of a protease inhibitorcocktail (Sigma). The mixture was boiled for 10 min and cleared bycentrifugation at 48C in a cooled microcentrifuge. The supernatantwas transferred to a microcentrifuge tube and stored on ice. Aliquotswere frozen in liquid N2 and stored at 2808C until used. For endogly-

cosidase digestion, buffer G5 (New England BioLabs, Hitchin, UK)was added to the protein extract thawed on ice to achieve a workingconcentration of buffer, which resulted in a twofold dilution of theprotein extract. Ten to 40 mL of the extract was incubated for 1 hr at378C in the presence of 1 mL (1000 units) of EndoHf (New EnglandBioLabs). Control samples were treated identically, but no endogly-cosidase was added. The reaction was stopped by adding an equalvolume of 2 3 SDS-PAGE loading buffer.

For SDS-PAGE, we used a 12% acrylamide gel and Protean III ap-paratus (Bio-Rad). After electrophoresis, proteins were transferred topolyvinylidene difluoride or nitrocellulose membrane for 2 hr in trans-fer buffer according to Burnette (1981). The filter was blocked for 1 hrin Tris-buffered saline containing 0.1% (v/v) Tween 20 (Sigma), 1%BSA ([w/v]; Fraction V; Roche Biochemicals, Lewes, UK), and 0.1%goat serum (v/v) (Sigma). Antibody to full-length GFP (Santa CruzBiotechnology, Santa Cruz, CA, or Molecular Probes, Leiden, TheNetherlands) or an antibody raised against the GFP C-terminal peptide(N15; ABCAM, Cambridge, UK) was used at 1/2000 dilution to probethe membrane for 1 hr. Alkaline phosphatase–conjugated secondaryantibody (Sigma) was used at 1/10,000 dilution (1-hr incubation), andthe results were determined with the Sigma Fast 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium method, used according tothe manufacturer’s recommendations.

ACKNOWLEDGMENTS

We thank Dr. Masashi Ugaki for the enhanced 35S cassette, Dr. JimHaseloff for pBIN-mGFP5ER, Claude Saint-Jore for the ST-GFP con-struct, Dr. Edmund Wee for cloning N-ST-GFP, and Dr. AndreasMeyer for assistance with confocal imaging. This work was sup-ported by a grant to I.M. from the Biotechnology and BiologicalSciences Research Council (UK).

Received July 17, 2000; accepted September 6, 2000.

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DOI 10.1105/tpc.12.11.2201 2000;12;2201-2217Plant Cell

Henri Batoko, Huan-Quan Zheng, Chris Hawes and Ian MooreApparatus and for Normal Golgi Movement in Plants

A Rab1 GTPase Is Required for Transport between the Endoplasmic Reticulum and Golgi

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