an arabidopsis prenylated rab acceptor 1 isoform, atpra1

An Arabidopsis Prenylated Rab Acceptor 1 Isoform,AtPRA1.B6, Displays Differential Inhibitory Effects onAnterograde Trafficking of Proteins at theEndoplasmic Reticulum1[W][OA]

Myoung Hui Lee, Chanjin Jung, Junho Lee, Soo Youn Kim, Yongjik Lee, and Inhwan Hwang*

Division of Molecular and Life Sciences (M.H.L., C.J., J.L., S.Y.K., I.H.) and Division of Integrative Biosciencesand Biotechnology (Y.L., I.H.), Pohang University of Science and Technology, Pohang 790–784, Korea

Prenylated Rab acceptors (PRAs), members of the Ypt-interacting protein family of small membrane proteins, are thought to aidthe targeting of prenylated Rabs to their respective endomembrane compartments. In plants, the Arabidopsis (Arabidopsis thaliana)PRA1 family contains 19 members that display varying degrees of sequence homology to animal PRA1 and localize to theendoplasmic reticulum (ER) and/or endosomes. However, the exact role of these proteins remains to be fully characterized. In thisstudy, the effect of AtPRA1.B6, a member of the AtPRA1 family, on the anterograde trafficking of proteins targeted to variousendomembrane compartments was investigated. High levels of AtPRA1.B6 resulted in differential inhibition of coat proteincomplex II vesicle-mediated anterograde trafficking. The trafficking of the vacuolar proteins sporamin:GFP (for green fluorescentprotein) and AALP:GFP, the secretory protein invertase:GFP, and the plasma membrane proteins PMP:GFP and H+-ATPase:GFPwas inhibited in a dose-dependent manner, while the trafficking of the Golgi-localized proteins ST:GFP and KAM1(DC):mRFPwasnot affected. Conversely, in RNA interference plants displaying lower levels of AtPRA1.B6 transcripts, the trafficking efficiency ofsporamin:GFP and AALP:GFP to the vacuole was increased. Localization and N-glycan pattern analyses of cargo proteinsrevealed that AtPRA1.B6-mediated inhibition of anterograde trafficking occurs at the ER. In addition, AtPRA1.B6 levels werecontrolled by cellular processes, including 26S proteasome-mediated proteolysis. Based on these results, we propose that AtPRA1.B6 is a negative regulator of coat protein complex II vesicle-mediated anterograde trafficking for a subset of proteins at the ER.

Many small integral membrane proteins are knownto play roles in the various steps of protein trafficking(Emery et al., 2000; Compton and Behrend, 2006). TheYpt-interacting protein (YIP) family displays an abilityfor binding Rabs/Ypts (Shisheva et al., 1994; Wadaet al., 1997; Yang et al., 1998; Liang and Li, 2000;Figueroa et al., 2001; Calero et al., 2002; Calero andCollins, 2002; Pfeffer and Aivazian, 2004; Spang, 2004).The YIP family members, containing multiple trans-membrane domains ranging from two to five, are di-vided into seven subfamilies (Yip1–Yip6 and Yif1) basedon sequence homology (Pfeffer and Aivazian, 2004).

Among the YIP family members, the biological roleof Yip3p/PRA1 (for prenylated Rab acceptor 1) hasbeen studied most extensively. In animal cells, PRA1localizes to the Golgi apparatus and endosomes, and itinteracts with multiple Rab proteins as well as theGDP dissociation inhibitor (GDI). Additionally, PRA1activates the dissociation of prenylated Rabs from theRab/GDI complex, facilitating the association of pre-nylated Rabs with target membranes. This indicatesthat PRA1/Yip3 plays a role in targeting prenylatedRabs to their specific compartments by acting as a GDIdisplacement factor (Abdul-Ghani et al., 2001; Sivarset al., 2003; Seabra and Wasmeier, 2004). PRA1 alsointeracts with the SNARE protein VAMP2 and mayplay a role in regulating vesicle fusion to target mem-branes. In contrast to PRA1 in animal cells, Yip3p, theortholog of animal PRA1, appears to be involved in adifferent physiological process. In yeast, Yip3p local-izes primarily to the Golgi, with a minor portionlocalizing to the endoplasmic reticulum (ER). Thelocalization of Rab proteins was not perturbed inyip3D. Furthermore, overexpression of Yip3p causesER membrane proliferation, a well-known phenotyperesulting from the inhibition of anterograde traffickingat the ER (Kaiser and Schekman, 1990). These resultsraise the possibility that Yip3p plays a role in thebiogenesis of coat protein complex II (COP II) vesicles

1 This work was supported by grants from the World ClassUniversity Project (no. R31–10105) and National Research Founda-tion (no. 20110000025) of the Ministry of Education, Science, andTechnology, and a grant from the Institute of Planning and Evalu-ation for Technology, the Ministry of Food, Agriculture, Forestryand Fisheries (Korea).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Inhwan Hwang ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.111.180810

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at the ER in yeast (Geng et al., 2005). Thus, the biologicalrole of PRA1 has not been fully characterized.

The biological role of PRA2 is even less known.PRA2 belongs to the Yip6 subfamily and localizes tothe ER (Abdul-Ghani et al., 2001; Pfeffer and Aivazian,2004). PRA2/PRA2F expression levels are elevated invarious cancer cells, and the encoded proteins are en-riched in synaptic vesicles in neuronal cells (Koomoaet al., 2008; Borsics et al., 2010). In neuronal cells, Yip6band JM4, also members of the Yip6 subfamily, areinvolved in regulating the ER exit of the neuron-specificNa+/K+ Glu transporter EAAC1 as well as selectedneurotransmitter transporters (Ruggiero et al., 2008).Yip6b interacts specifically with EAAC1, resulting in itsretention in the ER.

Similar to animal and yeast cells, plant cells alsocontain a large number of small membrane proteinsthat display varying degrees of sequence homology toanimal and yeast YIP proteins. In Arabidopsis (Arabi-dopsis thaliana), members of the AtPRA1 family havebeen studied for their expression and subcellularlocalization (Alvim Kamei et al., 2008). Upon expres-sion in transgenic plants, specific GFP-tagged AtPRA1family members localized to the ER and/or endo-somes. Additionally, AtPRA1.B6, a member of theAtPRA1 family, was recently demonstrated to localizeprimarily to the Golgi apparatus. However, at highexpression levels, AtPRA1.B6 localizes to the ER andthe Golgi apparatus (Jung et al., 2011). A PRA1 homo-log in rice (Oryza sativa), OsPRA1, interacts withOsRab7 and localizes to the prevacuolar compartment.Thus, OsPRA1 is believed to play a role in proteintrafficking to the vacuole (Heo et al., 2010).

In this study, the effect of AtPRA1.B6 on the traffick-ing of cargo proteins targeted to various endomem-brane compartments was investigated. As AtPRA1.B6levels increased due to ectopic expression, the antero-grade trafficking of a subset of proteins was inhibitedat the ER in a protein level-dependent manner. Con-versely, when AtPRA1.B6 levels were reduced using anRNA interference (RNAi) approach, the anterogradetrafficking of vacuolar proteins was enhanced.

RESULTS

HA:AtPRA1.B6 Inhibits the Trafficking of Vacuolar

Proteins, Sporamin:GFP, and AALP:GFP

Previously, AtPRA1.B6 expressedwith anN-terminalhemagglutinin (HA) tag was demonstrated to localizeprimarily to the Golgi apparatus (Jung et al., 2011). Inaddition, when HA:AtPRA1.B6 was expressed at highlevels, it localized to the ER and the Golgi apparatus.Yeast Yip3, the likely ortholog of AtPRA1, exhibitssimilar localization patterns, with a primary localiza-tion to the Golgi apparatus and a minor portion to theER (Geng et al., 2005). To gain insight into the physi-ological role of AtPRA1.B6, the effect of high expressionlevels of AtPRA1.B6 on anterograde trafficking of var-

ious cargo proteins was examined. In a previous study,overexpression of Yip3 in yeast resulted in an inhibitionof anterograde trafficking (Geng et al., 2005). A similarobservation was made when Yip6b/GTRAP3-18 wasexpressed in animal cells (Ruggiero et al., 2008). Ininitial investigations, the utilized proteins were des-tined for the central vacuole. For example, sporamin:GFP, a chimera of the sweet potato (Ipomoea batatas)protein sporamin and GFP, is targeted to the vacuole,where it is processed to a 30-kD protein (Kim et al.,2001). Increasing amounts ofHA:AtPRA1.B6 along withsporamin:GFPwere cotransformed into protoplasts, andprotein extracts were used in western-blot analysisusing anti-GFP antibody to determine the traffickingefficiency. The ratio of processed proteins to the totalamount of expressed proteins (processed proteins plusfull-length proteins) was used to assess the traffickingefficiency. The trafficking efficiency of sporamin:GFPdecreased gradually with increasing amounts of HA:AtPRA1.B6 (Fig. 1A), indicating that vacuolar traffick-ing was inhibited by HA:AtPRA1.B6 in a dose-depen-dent manner. The expression of HA:AtPRA1.B6, asdetermined by anti-HA antibody, was proportional tothe amount of introduced plasmid DNA.

To further confirm that AtPRA1.B6 inhibits vacuolartrafficking, a second vacuolar protein, AALP:GFP, wasanalyzed. As a chimeric protein consisting of Arabi-dopsis aleurain-like protein (AALP) and GFP, thisprotein is targeted to the central vacuole, where it isprocessed proteolytically to produce a 30-kD protein(Sohn et al., 2003). AALP:GFP and HA:AtPRA1.B6 orempty vector (R6) were introduced into protoplasts,and these proteins were detected by western-blotanalysis using anti-GFP and anti-HA antibodies. Inthe presence of cotransformed HA:AtPRA1.B6, theamount of processed AALP:GFP protein was reducedsignificantly compared with the control transformedwith empty vector R6 (Fig. 1B). These data confirmthat HA:AtPRA1.B6 inhibits vacuolar trafficking.

To obtain independent evidence for inhibition, thelocalization of vacuolar cargo proteins was examinedin the presence of HA:AtPRA1.B6. HA:AtPRA1.B6 orR6 was cotransformed into protoplasts together withsporamin:GFP or AALP:GFP, and the localization ofthese cargo proteins was examined using a fluorescencemicroscope. In control protoplasts transformedwithR6,both sporamin:GFP and AALP:GFP produced the vac-uolar pattern in a majority of the protoplasts (Fig. 1C,panels a and e), and a minor population of protoplastsdisplayed a network pattern alone or both the vacuolarand network patterns. The vacuolar pattern indicatesthat sporamin:GFP and AALP:GFP were trafficked tothe central vacuole. However, in the presence of HA:AtPRA1.B6, both sporamin:GFP and AALP:GFP pri-marily produced the network pattern with a minorportion of the vacuolar pattern (Fig. 1C, panels c and g).This confirms that AtPRA1.B6 inhibits the vacuolartrafficking of sporamin:GFP and AALP:GFP.

To eliminate the possibility that the inhibition ofvacuolar trafficking by HA:AtPRA1.B6 was caused by

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high levels of HA:AtPRA1.B6 produced from the trans-formed HA:AtPRA1.B6 plasmid DNA in protoplasts,transgenic plants harboring HAx3:AtPRA1.B6 were ex-amined. Transgenic plants with HAx3:AtPRA1.B6 weregenerated previously (Jung et al., 2011). AtPRA1.B6 wastagged with a trimeric HA tag (HAx3) and placed underthe control of the dexamethasone-inducible promoter(Aoyama and Chua, 1997; Jung et al., 2011). Transgenicplants containing only a single copy ofHAx3:AtPRA1.B6(HAx3:AtPRA1.B6 plants) were grown on dexametha-sone-containing plates, and the HAx3:AtPRA1.B6 levelswere analyzed by western blotting using anti-HA anti-

body. The protein levels increased in a dose-dependentmanner when plants were incubated on plates supple-mented with varying concentrations of dexamethasone(Jung et al., 2011). The HAx3:AtPRA1.B6 levels in trans-genic plants were compared with the HA:AtPRA1.B6levels in protoplasts by western-blot analysis using anti-HA antibody. The HAx3:AtPRA1.B6 level in transgenicplants was significantly lower than the HA:AtPRA1.B6level in protoplasts transformed with 2 mg of HA:AtPRA1.B6 plasmid DNA (Supplemental Fig. S1).Since AtPRA1.B6 in transgenic plants and protoplastshas trimeric and monomeric HA tags, respectively, the

Figure 1. HA:AtPRA1.B6 expressed transiently in protoplasts inhibits the trafficking of sporamin:GFP and AALP:GFP in a dose-dependent manner. A, Inhibition of vacuolar trafficking of sporamin:GFP to the vacuole by HA:AtPRA1.B6 in a dose-dependentmanner. Sporamin:GFP was cotransformed into protoplasts with the indicated amounts of HA:AtPRA1.B6 or R6, and proteinextracts from protoplasts were analyzed by western-blot analysis using anti-GFPand anti-HA antibodies. Trafficking efficiency ofsporamin:GFP was determined by the signal intensity of the processed form over the total amount of expressed proteins. Threeindependent trafficking experiments were performed. Error bars indicate SD (n = 3). PRA1, HA:AtPRA1.B6. B, Inhibition ofvacuolar trafficking of AALP:GFP by HA:AtPRA1.B6. AALP:GFP was cotransformed with HA:AtPRA1.B6 or R6 into protoplasts,and protein extracts from protoplasts were analyzed by western-blot analysis using anti-GFPand anti-HA antibodies. In order tomeasure the trafficking efficiency of AALP:GFP, the amount of the processed form was quantified using software equipped toLAS3000 and expressed as a relative value to the total amount of protein. Error bars indicate SD (n = 3). C, Image analysis ofprotein trafficking inhibition by HA:AtPRA1.B6. Sporamin:GFP or AALP:GFP was introduced into protoplasts together with HA:AtPRA1.B6 or R6, and the localization of cargo proteins was examined. Three independent transformation experiments wereperformed for each sample. More than 100 protoplasts were examined to determine their localization patterns. Red signals showthe autofluorescence of chlorophyll (CH). Bars = 20 mm.

Inhibition of Anterograde Trafficking by AtPRA1.B6

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difference in the protein levels between HAx3:At-PRA1.B6 in transgenic plants and HA:AtPRA1.B6 inprotoplasts would be greater than the difference in theimmunoblot band intensity. Subsequently, whetherHAx3:AtPRA1.B6 produced in transgenic plants isable to inhibit the trafficking of proteins was investi-gated. HAx3:AtPRA1.B6 expression was induced withdexamethasone using two approaches, before andafter protoplast preparation. Protoplasts from dexa-methasone-treated and untreated HAx3:AtPRA1.B6plants were incubated in the absence and presence ofdexamethasone after transformation with cargo con-structs, respectively. The trafficking of proteins to thevacuole was examined by western-blot analysis usinganti-GFP. The trafficking efficiency of sporamin:GFPand AALP:GFP to the vacuole was significantly re-duced in the presence of HAx3:AtPRA1.B6 that hadbeen induced before protoplasting (Fig. 2). Similarly,the vacuolar trafficking of AALP:GFPwas inhibited byHAx3:AtPRA1.B6 that had been produced in proto-plasts at the same time with AALP:GFP (SupplementalFig. S2). The HAx3:AtPRA1.B6 protein level in thesesamples was confirmed by western-blot analysis usinganti-HA antibody. The results indicate that the stableexpression of HAx3:AtPRA1.B6 in transgenic plantsalso inhibits the anterograde trafficking of vacuolarcargoes.

HA:AtPRA1.B6 Inhibits the Trafficking of Secretory andPlasma Membrane Proteins

Next, we examined whether AtPRA1.B6 inhibits thetrafficking of secretory and plasma membrane pro-teins. Invertase:GFP was selected as a secretory pro-

tein. Invertase:GFP, a fusion of secretory invertase andGFP, is efficiently secreted into the medium (Kim et al.,2005). Invertase:GFP was introduced into protoplaststogether with HA:AtPRA1.B6 or R6, and the secretionof invertase:GFP was examined by fluorescence mi-croscopy. In control protoplasts transformed withR6, a GFP signal was not detected in protoplasts(Fig. 3A, panel a). In a previous study, invertase:GFPwas shown to be secreted efficiently into the incuba-tion medium (Kim et al., 2005). Thus, the lack of a GFP

Figure 2. HAx3:AtPRA1.B6 expressed stably in transgenic plants in-hibits the trafficking of sporamin:GFP and AALP:GFP. Protoplasts wereprepared from HAx3:AtPRA1.B6 or pTA transgenic plants treated withdexamethasone for 1 d and transformed with sporamin:GFP or AALP:GFP. Trafficking of sporamin:GFP and AALP:GFP was examined 24 hafter transformation by western-blot analysis using anti-GFP. Expressionof HAx3:AtPRA1.B6 was examined by western blotting using anti-HAantibody. pTA, pTA plants; PRA1, HAx3:AtPRA1.B6 plants.

Figure 3. HA:AtPRA1.B6 inhibits trafficking of the secretory proteininvertase:GFP. A, Inhibition of invertase:GFP trafficking by HA:AtPRA1.B6. Invertase:GFP was contransformed with AtPRA1.B6 or R6 intoprotoplasts, and the localization of invertase:GFP was examined.Chloroplasts (CH) were detected by red autofluorescence of chloro-phyll. Bars = 20 mm. B, Western-blot analysis. Invertase:GFP wascotransformedwithHA:AtPRA1.B6 or R6, and the protein extracts fromprotoplasts and incubation medium were analyzed by western-blotanalysis using anti-GFP and anti-HA. To quantify trafficking efficiency,the band intensity was determined using software equipped toLAS3000. Trafficking efficiency was determined by the relative amountof secreted invertase:GFP over the total expressed proteins (proteins inboth the medium and protoplasts). In order to measure the traffickingefficiency of invertase:GFP, the amount of secreted protein was quan-tified using software equipped to LAS3000 and expressed as a relativevalue to the total amount of protein. Error bars indicate SD (n = 3). R6,Empty vector; PRA1, HA:AtPRA1.B6; M, protein extracts from theincubation medium; P, protoplast extracts. C, Inhibition of invertase:GFP trafficking by HAx3:AtPRA1.B6 expressed in transgenic plants.Protoplasts from pTA or HAx3:AtPRA1.B6 transgenic plants treatedwith dexamethasone (30 mM) for 1 d were transformed with invertase:GFP, and trafficking efficiency was examined by western-blot analysisusing anti-GFP antibody. Expression of HAx3:AtPRA1.B6 was detectedwith anti-HA antibody. Proteins from the incubation medium wereincluded in the analysis. Actin detected with anti-actin antibody wasused as a loading control. The asterisk indicates a 30-kD nonspecificband detected by anti-HA antibody.

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signal in the protoplasts was likely a result of theefficient secretion of invertase:GFP into the medium.However, when coexpressed with HA:AtPRA1.B6, in-vertase:GFP produced the network pattern in themajority of protoplasts (Fig. 3A, panel c), indicatingthat HA:AtPRA1.B6 inhibits the secretion of invertase:GFP to the medium. To confirm this finding at thebiochemical level, protein extracts were prepared fromprotoplasts and analyzed by western blotting usinganti-GFP antibody. Proteins from the incubation me-dium were included in the analysis. In control proto-plasts transformed with R6, 89% of the invertase:GFPwas secreted into the medium (Fig. 3B). However,when coexpressed with HA:AtPRA1.B6, 57% of theinvertase:GFP was secreted into the medium. Theseresults confirm that HA:AtPRA1.B6 inhibits the anter-ograde trafficking of secretory proteins. To eliminatethe possibility that an excess of transiently expressedAtPRA1.B6 in protoplasts caused the inhibition ofinvertase:GFP secretion, protoplasts were preparedfrom HAx3:AtPRA1.B6 plants that had been treatedwith dexamethasone for 1 d and transformed withinvertase:GFP. Subsequently, the secretion of inver-tase:GFP was examined by western-blot analysis us-ing anti-GFP antibody. As a control, protoplasts fromplants harboring the empty expression vector pTA(pTA plants) were included. The amount of invertase:GFP in the medium was decreased to lower levels incomparison with the control protoplasts from pTA

plants (Fig. 3C). This confirms that AtPRA1.B6 inhibitsthe trafficking of invertase:GFP.

Next, the trafficking of proteins to the plasma mem-brane was examined using H+-ATPase:GFP and PMP:GFP. H+-ATPase:GFP, a chimeric protein betweenplasma membrane H+-ATPase and GFP, is localizedto the plasma membrane (Kim et al., 2001). Recently,plasma membrane protein (PMP):GFP, a chimeric pro-tein generated using the N-terminal 40-amino acidsegment containing the transmembrane domain ofAt1g53610, a protein predicted to localize to theplasma membrane, and GFP was shown to localizeto the plasma membrane (Lee et al., 2011). Thesereporter protein constructs were introduced into pro-toplasts together with HA:AtPRA1.B6 or R6, and theirlocalization was examined using a fluorescence mi-croscope. In control protoplasts transformed with R6,both proteins produced a ring pattern, indicating thatthey were targeted to the plasma membrane (Fig. 4). Incontrast, when coexpressed with HA:AtPRA1.B6, theprotoplasts that had been transformed with H+-ATPase:GFP or PMP:GFP produced various localization pat-terns, such as aggregation, network, and plasma mem-brane patterns (Fig. 4). In the presence of HA:AtPRA1.B6, the plasma membrane pattern of H+-ATPase:GFPand PMP:GFP was reduced to 67% and 50%, respec-tively, from 97%. This suggests that HA:AtPRA1.B6inhibits the trafficking of these proteins to the plasmamembrane. We examined whether H+-ATPase:GFP and

Figure 4. HA:AtPRA1.B6 inhibits trafficking ofplasma membrane proteins H+-ATPase:GFP andPMP:GFP. H+-ATPase:GFP (A) or PMP:GFP (B)was introduced into protoplasts together withempty expression vector R6, HA:AtPRA1.B6, orSar-1[H74L], and localization of the plasmamembrane proteins was examined To quantifythe localization pattern, transformation was per-formed three times for each sample, and eachtime more than 200 transformed protoplasts wereanalyzed to determine the localization. Theplasma membrane pattern was expressed as arelative value to the total number of transformedprotoplasts. Error bars indicate SD (n = 3). PRA1,HA:AtPRA1.B6; CH, chloroplast. Bars = 20 mm.



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PMP:GFP are transported by COPII vesicles. H+-ATPase:GFP or PMP:GFP was cotransformed into protoplaststogether with Sar-1[H74L], and localization of theseproteins was examined. Sar-1[H74L], a dominant nega-tive mutant of Sar-1, inhibits COPII-dependent antero-grade trafficking from the ER to the Golgi apparatus(Takeuchi et al., 2000). In the presence of Sar-1[H74L],both proteins were not targeted to the plasma mem-brane (Fig. 4), indicating that they are transported to theplasma membrane in a COPII-dependent manner.

HA:AtPRA1.B6 Does Not Inhibit the Trafficking ofProteins Localized to the Golgi Apparatus

The effect of AtPRA1.B6 on the trafficking of Golgi-localized proteins was also investigated. ST:GFP, achimeric protein containing rat sialyltransferase andGFP, has been shown to localize to the Golgi appara-tus (Wee et al., 1998; Kim et al., 2001). In this study, ST:GFP was introduced into protoplasts together withHA:AtPRA1.B6 or R6, and the localization of ST:GFPwas examined. Surprisingly, ST:GFP produced a typ-ical punctate staining pattern in protoplasts trans-formed with HA:AtPRA1.B6, similar to the controlprotoplasts transformed with R6 (Fig. 5A, panels aand c). To examine whether the ST:GFP-positivepunctate stains represented the Golgi apparatus, thelocalization of ST:GFP was compared with that ofendogenous g-COP, a COPI component localized tothe Golgi apparatus (Pimpl et al., 2000). The localiza-tion of ST:GFP and g-COP was determined directly byGFP fluorescence and immunohistochemistry usinganti-g-COP antibody (Pimpl et al., 2000), respectively.In both the presence and absence of HA:AtPRA1.B6,the endogenous g-COP and ST:GFP produced punc-tate staining patterns (Fig. 5A, panels e, f, i, and j).Moreover, the punctate patterns closely overlappedone another (Fig. 5A, panels g and k), indicating thatHA:AtPRA1.B6 does not inhibit the trafficking of ST:GFP to the Golgi apparatus. The production of HA:AtPRA1.B6 in transformed protoplasts was confirmedby western-blot analysis using anti-HA antibody (Fig.5B).

To confirm this result at the biochemical level, endo-glycosidase H (Endo H) sensitivity of the N-glycansof ST:GFP was utilized. The N-glycans of ER-localizedproteins are sensitive to Endo H, whereas the N-glycansof Golgi-localized proteins are resistant to Endo H(Frigerio et al., 1998). Protein extracts from protoplaststransformed with ST:GFP were treated with or withoutEndo H and analyzed by western blotting using anti-GFP antibody. Both in the presence and absence of HA:AtPRA1.B6, themajority of ST:GFPwas resistant to EndoH (Fig. 5C), thus confirming that ST:GFP localizes to theGolgi apparatus. Furthermore, HAx3:AtPRA1.B6 ex-pressed in transgenic plants also did not inhibit thetrafficking of ST:GFP to the Golgi apparatus (Supple-mental Fig. S3).

To determine whether this result is specific to ST:GFP,another Golgi reporter protein was utilized. KAM1

Figure 5. HA:AtPRA1.B6 does not inhibit the trafficking of ST:GFP tothe Golgi apparatus. A, Localization of ST:GFP to the Golgi apparatus inthe presence of HA:AtPRA1.B6. Wild-type protoplasts were trans-formed with the indicated constructs, and localization of ST:GFP andg-COP was examined directly by GFP fluorescence and by immuno-staining with anti-g-COP antibody, respectively. R6, Empty vector;PRA1, HA:AtPRA1.B6; CH, chloroplast. Bars = 20 mm. Three inde-pendent transformation experiments were performed with each sam-ple. In the case of ST:GFP +R6 and ST:GFP+HA:PRA1, more than 100protoplasts were examined to determine the localization. In the case ofST:GFP+R6+r-COP and ST:GFP+HA:PRA1+r-COP, more than 60 pro-toplasts were examined for the localization. B, Western-blot analysis ofHA:AtPRA1.B6 expression. Protein extracts from transformed proto-plasts (A) were analyzed by western blotting using anti-HA. C, EndoH-resistant form of ST:GFP N-glycans in the presence of HA:AtPRA1.B6. ST:GFP was cotransformed with the indicated amounts of AtPRA1.B6 or R6, and protein extracts from the transformed protoplasts weretreated with (+) or without (2) Endo H. Subsequently, the proteinextracts were analyzed by western blotting using anti-GFP antibody. Inorder to measure the trafficking efficiency of ST:GFP to the Golgiapparatus in the presence of AtPRA1.B6, the amount of the EndoH-resistant form was quantified using software equipped to LAS3000and expressed as a relative value to the total amount of protein. Errorbars indicate SD (n = 3).

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(DC):mRFP, a chimera between the Golgi-localized C-terminal deletion construct of Katamari1 (KAM1) andmonomeric red fluorescent protein (mRFP; Tamuraet al., 2005), is targeted to the cis-Golgi. KAM1(DC):

mRFP was introduced into protoplasts together withHA:AtPRA1.B6 or R6, and the localization of KAM1(DC):mRFP was examined. In both the presence andabsence of HA:AtPRA1.B6, KAM1(DC):mRFP pro-duced the punctate staining pattern (Fig. 6A, panelsa and c), suggesting localization to the Golgi appara-tus. To confirm that KAM1(DC):mRFP localizes to theGolgi apparatus even in the presence of HA:AtPRA1.B6, KAM1(DC):mRFP and ST:GFP were introducedinto protoplasts together with HA:AtPRA1.B6 or R6and the localization of the proteins was examined.Again, both KAM1(DC):mRFP and ST:GFP colocalizedto the punctate stains regardless of HA:AtPRA1.B6expression (Fig. 6A, panels e–g and i–k). These resultsindicate that HA:AtPRA1.B6 does not inhibit the traf-ficking of KAM1(DC):mRFP to the Golgi apparatus. Toeliminate the possibility that HA:AtPRA1.B6 inhibitsboth KAM1(DC):mRFP and ST:GFP and causes themto accumulate in the ER as punctate stains, as observedwith Sar-1:RFP at the ER exit site, protoplasts wereprepared from transgenic plants expressing ST:GFP(Lee et al., 2002). These protoplasts were cotrans-formed with KAM1(DC):mRFP and HA:AtPRA1.B6 orR6, and the localization of these proteins was exam-ined. In both the presence and absence of HA:AtPRA1.B6, KAM1(DC):mRFP colocalized with stably express-ed ST:GFP at punctate stains (Fig. 6B, panels a–c and e–g), indicating that HA:AtPRA1.B6 does not inhibit the

Figure 6. HA:AtPRA1.B6 does not inhibit the trafficking of KAM1(DC):mRFP to the Golgi apparatus. A and B, Localization of KAM1(DC):mRFP to the Golgi apparatus upon coexpressing HA:AtPRA1.B6.Protoplasts from wild-type plants (A) or ST:GFP transgenic plants (B)were transformed with the indicated constructs, and the localization ofthese proteins was examined directly by RFP or GFP fluorescence. Ineach transformation, 10 mg of HA:AtPRA1.B6 was introduced. Bars =20 mm. Three independent transformation experiments were performedfor each sample, and more than 60 protoplasts were analyzed for thelocalization each time. C, Differential inhibition of anterograde traf-ficking by coexpressing HA:AtPRA1.B6. Protoplasts were cotrans-formed with three plasmids, HA:AtPRA1.B6 (10 mg), invertase:GFP (5mg), and KAM1(DC):mRFP (5 mg), and the localization of these proteinswas examined. Bars = 20 mm. Three independent transformationexperiments were performed, and more than 30 protoplasts wereexamined to determine the localization.

Figure 7. HA:AtPRA1.B6 inhibits ER-to-Golgi trafficking of PMP:GFP-glyNC. Protoplasts were cotransformed with PMP:GFP-glyNC, incu-bated with (+) or without (2) tunicamycin, and analyzed by westernblotting using anti-GFP. Protoplasts were cotransformed with PMP:GFP-glyNC (5 mg) and HA:AtPRA1.B6 (10 mg) or R6 (10 mg). Proteinextracts from the transformed protoplasts were treated with (+) orwithout (2) Endo H and analyzed by western blotting using anti-HAand anti-GFP antibodies. In order to measure the trafficking efficiencyof PMP:GFP-glyNC to the Golgi apparatus, the amount of the EndoH-resistant form was quantified using software equipped to LAS3000and expressed as a relative value to the total amount of protein. Errorbars indicate SD (n = 3). gly, Glycosylated form; un-gly; unglycosylatedform; R6, empty vector; PRA1, HA:AtPRA1.B6.



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trafficking of KAM1(DC):mRFP to the Golgi appara-tus. These results clearly demonstrate that AtPRA1.B6does not inhibit the trafficking of Golgi-residentproteins.

To further confirm the differential effect of AtPRA1.B6 on anterograde trafficking and to eliminate thepossibility that the lack of inhibitory effect on Golgi-localized proteins is caused by low levels of HA:AtPRA1.B6 in a subpopulation of transformed proto-plasts, the trafficking of these proteins was examinedin protoplasts cotransformed with three constructs,KAM1(DC):mRFP, invertase:GFP, and HA:AtPRA1.B6.GFP and RFP signals of invertase:GFP and KAM1(DC):mRFP produced the network pattern and the punctatestaining pattern, respectively, in a single protoplast(Fig. 6C). This clearly demonstrates that AtPRA1.B6inhibits the trafficking of invertase:GFP but not ofGolgi-localized KAM1(DC):mRFP.

HA:AtPRA1.B6 Inhibits Anterograde Trafficking at

the ER

The results shown in Figure 6 prompted an exami-nation of where the AtPRA1.B6-mediated inhibitionof anterograde trafficking occurs. If the AtPRA1.B6-mediated inhibition occurs at the post-Golgi compart-ment, AtPRA1.B6 would not inhibit the Golgi-localizedproteins. To define which step is inhibited by AtPRA1.B6 during anterograde trafficking, the Endo H sensi-tivity of the N-glycans of cargo proteins was used(Frigerio et al., 1998; Park et al., 2004). PMP:GFP wasmodified by addingN-glycosylation sites to both the Nand C termini to yield PMP:GFP-glyNC (Park et al.,2004). Initially, the N-glycosylation of PMP:GFP-glyNC was examined. Protoplasts transformed withPMP:GFP-glyNCwere incubated with or without tuni-camycin, an inhibitor of N-glycosylation, and proteinextracts from the transformed protoplasts were ana-lyzed by western blotting using anti-GFP antibody.

PMP:GFP-glyNC from tunicamycin-treated proto-plasts migrated faster than that from tunicamycin-untreated protoplasts (Fig. 7, left panel), confirmingthat PMP:GFP-glyNC is N-glycosylated. Next, PMP:GFP-glyNC was introduced into protoplasts togetherwith HA:AtPRA1.B6 or R6. Subsequently, proteinsfrom transformed protoplasts were treated with orwithout Endo H and analyzed by western blottingusing anti-GFP antibody. In the absence of HA:At-PRA1.B6, the majority of PMP:GFP-glyNC was resis-tant to Endo H (Fig. 7, lane 2 in right panel), consistentwith the image analysis (Fig. 4, panel e). In contrast, inthe presence of HA:AtPRA1.B6, the majority of PMP:GFP-glyNC was sensitive to Endo H (Fig. 7, lane 4 inright panel), indicating that PMP:GFP-glycNC local-ized to the ER. These results suggest that AtPRA1.B6inhibits anterograde trafficking of plasma membraneproteins at the ER. In addition, the results weresupported by the accumulation of vacuolar proteinsin the ER in the presence of HA:AtPRA1.B6 (Fig. 1C,panels c and g).

The ER-Localized C-Terminal Deletion Mutant DC1, But

Not the Prevacuolar Compartment-Localized N-TerminalDeletion Mutant DN2, Inhibits Anterograde Traffickingof Sporamin:GFP and Invertase:GFP

Two AtPRA1.B6 deletion mutants, DC1 and DN2,which have a deletion of the C-terminal 37 andN-terminal 36 amino acid residues of AtPRA1.B6,localize to the ER and prevacuolar compartment,respectively (Jung et al., 2011). The trafficking of pro-teins to the vacuole or apoplasts was examined in thepresence of these mutants. Sporamin:GFP or invertase:GFPwas cotransformed into protoplasts together withHA:AtPRA1.B6, HA:DC1, HA:DN2, or R6, and theirtrafficking efficiency was determined by western-blotanalysis using anti-GFP antibody. In the case of inver-tase:GFP, proteins from the incubation medium were

Figure 8. The C-terminal but not the N-terminaldeletion mutants inhibit anterograde trafficking ofsporamin:GFP and invertase:GFP. Sporamin:GFPor invertase:GFPwas transformed into protoplaststogether withHA:AtPRA1.B6,HA:DC1,HA:DN2,or R6, and their trafficking was examined bywestern-blot analysis using anti-GFP antibody.The trafficking efficiencies of AALP:GFP andsporamin:GFP were quantified using softwareequipped to LAS3000. The amounts of secretedinvertase:GFP and processed sporamin:GFP wereexpressed as relative values to the total amountsof invertase:GFP and sporamin:GFP, respectively.Error bars indicate SD (n = 3). R6, Empty vector;PRA1, HA:AtPRA1.B6; DN2, HA:DN2; DC1, HA:DC1; M, proteins from the medium, P, proteinextracts from protoplasts.

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included in the analysis. HA:DC1, but not HA:DN2,inhibited the trafficking of sporamin:GFP and inver-tase:GFP (Fig. 8), as observed with wild-type HA:AtPRA1.B6. These results indicate that the localizationof HA:PRA1.B6 to the ER is important for the inhibi-tion. The expression of HA:AtPRA1.B6, HA:DC1, andHA:DN2 was confirmed by western-blot analysis us-ing anti-HA antibody.

COPII Vesicles Are Involved in Anterograde TraffickingRegardless of Whether the ER-to-Golgi Trafficking IsInhibited by AtPRA1.B6

In anterograde trafficking from the ER to the Golgiapparatus, one group of proteins was subjected toAtPRA1.B6-mediated inhibition and the other groupof proteins was not. To investigate the differentialinhibition of anterograde trafficking by AtPRA1.B6,any differences in the trafficking pathways due to thegroups of proteins were examined. COPII vesicles areinvolved in the ER-to-Golgi trafficking in plant cells.Accordingly, cargo proteins (AALP:GFP, sporamin:

GFP, invertase:GFP, and PMP:GFP-glyNC) subject toAtPRA1.B6-mediated trafficking inhibition at the ERwere examined to determine whether they were trans-ported from the ER to the Golgi apparatus by COPIIvesicles. Protoplasts were transformed with AALP:GFP, sporamin:GFP, invertase:GFP, or PMP:GFP-glyNC,together with Sar-1[H74L], a dominant negative Sar-1mutant that inhibits anterograde trafficking from theER (Takeuchi et al., 2000; Phillipson et al., 2001; Sohnet al., 2003), and the localization of these proteins wasexamined by fluorescence microscopy or western-blotanalysis using anti-GFP antibody. All of these proteins,AALP:GFP, sporamin:GFP, invertase:GFP, and PMP:GFP-glyNC, produced a network pattern (Fig. 9A,panels a–d). Additionally, in the presence of Sar-1[H74L], the majority of AALP:GFP and sporamin:GFPwas detected as precursors and invertase:GFP wasdetected primarily in the protoplasts, with almost nosecretion into the medium (Fig. 9B). In the case ofPMP:GFP-glyNC, the majority of the protein wasdetected as Endo H-sensitive forms. These resultsconfirmed that all these cargo proteins are transportedby COPII vesicles.

Figure 9. AALP:GFP, sporamin:GFP, invertase:GFP,PMP:GFP-glyNC, and ST:GFPare transported to theGolgi apparatus by COPII vesicles in protoplasts.Sar-1[H74L] or empty vector R6 was cotrans-formed into protoplasts together with AALP:GFP,sporamin:GFP, invertase:GFP, PMP:GFP-glyNC,or ST:GFP, and trafficking of these proteins wasexamined by fluorescence microscopy (A) orwestern-blot analysis using anti-GFP antibody(B). In the case of invertase:GFP, proteins fromthe incubation medium were included in theanalysis. Protein extracts containing PMP:GFP-glyNC were treated with (+) or without (2) EndoH before western-blot analysis. The traffickingefficiency of PMP:GFP-glyNC was quantified us-ing software equipped to LAS3000. The amountof the Endo H-resistant form was expressed as arelative value to the total amount of protein. Errorbars indicate SD (n = 3). Spo:GFP, Sporamin:GFP;P, protein extracts from protoplasts; M, proteinsfrom the medium. Bars = 20 mm.



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Previously, ST:GFP was reported to be transportedto the Golgi apparatus in protoplasts of leaf tissues(Batoko et al., 2000). To confirm this, protoplasts weretransformed with ST:GFP together with Sar-1[H74L],and the localization of ST:GFP was examined. In thepresence of Sar-1[H74L], ST:GFP produced a networkpattern (Fig. 9A, panel e), an indication of ER locali-zation. This confirms that ST:GFP is transported in aCOPII-dependent manner.

RNAi Plants Expressing Low Levels of AtPRA1.B6Exhibit Enhanced Vacuolar Trafficking

As high levels of AtPRA1.B6 inhibit anterogradetrafficking of a subset of cargo proteins, the effect of

low levels of AtPRA1.B6 on anterograde traffickingwas investigated. RNAi plants were generated using a317-bp fragment of AtPRA1.B6 (Wesley et al., 2001), asatpra1.b6 mutant plants were not available. Two inde-pendent RNAi lines (lines 10 and 14) were selected,and the AtPRA1.B6 transcript levels were examined byquantitative reverse transcription (qRT)-PCR usinggene-specific primers. Figure 10A reveals that thetranscript levels of AtPRA1.B6 were specifically de-creased in RNAi plants. The transcript level ofAtPRA1.D, a homolog and member of the AtPRA1family used as a negative control, was not affected. Next,the protein trafficking efficiency of vacuolar proteinswasexamined in protoplasts prepared from the RNAi plants.The vacuolar trafficking efficiency of AALP:GFP wasincreased in protoplasts prepared from two independentlines of AtPRA1.B6 RNAi plants compared with that inwild-type plants (Fig. 10B). Similarly, the vacuolar traf-ficking efficiency of sporamin:GFP was also enhanced inAtPRA1.B6 RNAi plants, indicating that lower levelsof AtPRA1.B6 result in an increase in anterogradetrafficking efficiency.

AtPRA1.B6 Levels Are Decreased by 26S

Proteasome-Dependent Degradation

As anterograde trafficking of a subset of proteinswas shown to be sensitive to the AtPRA1.B6 level, theintriguing possibility that the AtPRA1.B6 levels aresubject to regulation in plant cells was investigated. Incells, protein levels can be lowered by 26S proteasome-mediated proteolysis (Smalle and Vierstra, 2004). Toexamine whether AtPRA1.B6 proteins are degradedby the 26S proteasome, the effect of MG132, an inhib-itor of the 26S proteasome (Lee and Goldberg, 1998),on the level of AtPRA1.B6 in protoplasts was deter-mined. Protoplasts transformed with HA:AtPRA1.B6were treated with MG132 or dimethyl sulfoxide(DMSO) 12 h after transformation, followed by anadditional incubation of 12 or 24 h. Protein levels wereexamined by western-blot analysis using anti-HAantibody and compared at three time points (0, 12,and 24 h) after MG132 or DMSO treatment. In theDMSO-treated protoplasts, the HA:AtPRA1.B6 levelsrose to a peak at 12 h and then declined to a lower levelat 24 h (Fig. 11). In contrast, in MG132-treated proto-plasts, the HA:AtPRA1.B6 levels continuously in-creased until the 24-h time point. Furthermore, theHA:AtPRA1.B6 levels were much higher in MG132-treated protoplasts than in DMSO-treated protoplastsat both 12 and 24 h. As a control, the level of ER-localized GKX, a chimeric fusion protein containingthe KKXX motif for ER retrieval from the Golgicomplex, was examined as a cargo protein marker(Benghezal et al., 2000; Lee et al., 2009). The GKX levelwas not affected by MG132 treatment, indicating that26S proteasome-dependent proteolysis is specific toHA:AtPRA1.B6. The loading control Lhcb4, detectedwith anti-Lhcb4 antibody, was at an equal level in allsamples. In addition, public microarray data revealed

Figure 10. Trafficking efficiency of vacuolar proteins is increased inAtPRA1.B6 RNAi plants. A, qRT-PCR analysis of AtPRA1.B6 transcriptlevels in RNAi plants. Total RNA was obtained from RNAi plants andused for qRT-PCR analysis with gene-specific primers. As a negativecontrol, AtPRA1.D, a homolog of AtPRA1.B6, was included. Actin levelswere used as an internal control. WT, Wild-type plants; RNAi, AtPRA1.B6 RNAi plants. Error bars indicate SD (n = 3). B, Trafficking efficiency inRNAi plants. Protoplasts from two independent RNAi plants (lines 10 and14) were transformed with sporamin:GFP or AALP:GFP, and theirtrafficking to the vacuole was examined by western-blot analysis usinganti-GFP antibody. The trafficking efficiencies of AALP:GFP and spora-min:GFP were quantified in wild-type and RNAi plants. The amounts ofthe processed forms were expressed as relative values to the total amountof proteins. Error bars indicate SD (n = 3). Spo:GFP, Sporamin:GFP.

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that expression of AtPRA1.B6 is induced by multipleenvironmental stresses (http://www.genevestigator.com). Taken together, these results strongly suggestthat AtPRA1.B6 levels are regulated by cellular pro-cesses including 26S proteasome-mediated proteolysisin plant cells.

DISCUSSION

This study provides evidence that AtPRA1.B6 func-tions as a negative regulator of the anterograde traf-ficking of a subset of proteins at the ER. Theanterograde trafficking efficiency was inversely corre-lated with the protein levels of AtPRA1.B6. For twovacuolar proteins (sporamin:GFP and AALP:GFP), anapoplast-secreted protein (invertase:GFP), and twoplasma membrane proteins (H+-ATPase:GFP andPMP:GFP), overexpression of AtPRA1.B6 inhibits theanterograde trafficking. In contrast, suppression ofAtPRA1.B6 in RNAi plants resulted in an increase intrafficking efficiency. The inhibition of anterogradetrafficking by high levels of AtPRA1.B6 homologs isnot unique to plants. Similarly, overexpression of yeastand animal homologs, Yip3p and Yip6b, respectively,inhibits ER-to-Golgi transport in yeast and animal cells(Geng et al., 2005; Ruggiero et al., 2008). In addition,the overexpression of JM4/Yip6a suppresses the traf-ficking of the CC chemokine receptor 5 to the cellsurface (Schweneker et al., 2005). To demonstrate theeffect of AtPRA1.B6 levels on protein trafficking, thisstudy employed two artificial approaches, ectopicoverexpression and RNAi-mediated suppression, toincrease and decrease AtPRA1.B6 levels, respectively.This hypothesis requires the AtPRA1.B6 levels to becontrolled by cellular processes. Indeed, AtPRA1.B6levels are down-regulated by the 26S proteasome.Moreover, under certain conditions, AtPRA1.B6 levelsmay be altered by biotic or abiotic stresses, accordingto microarray data in the public database (http://www.genevestigator.com).The AtPRA1.B6-mediated inhibition of anterograde

trafficking occurs at the ER. This conclusion is based onthe fact that the cargo proteins inhibited by AtPRA1.B6accumulated in the ER, as determined by the Endo H

sensitivity of N-glycans and the localization of cargoproteins. In support of this finding, the C-terminal dele-tion mutant DC1 that localized primarily to the ER, butnot the prevacuolar compartment-localized DN2 (Junget al., 2011), also inhibited the anterograde trafficking ofvacuolar proteins. This raises the intriguing question ofhow the Golgi-localized AtPRA1.B6 is able to function asa negative regulator of anterograde trafficking at the ER.One simple explanation is that the mislocalization ofAtPRA1.B6 to the ER, resulting from overexpression,causes anterograde trafficking at the ER. Another possi-bility is that although themajority ofAtPRA1.B6 localizesto the Golgi apparatus, a small amount also localizes tothe ER. The ER-localized AtPRA1.B6 may not be easilydetected at normal expression levels but may becomevisible at high levels, as observed in the case of over-expression. It is possible that the ER-localizedAtPRA1.B6functions as a negative regulator of anterograde traffick-ing of a subset of proteins. This second explanation isfavored. Indeed, a minor portion of Yip3p localizes to theER, in addition to themajor portion localized to the Golgiapparatus in yeast. Similarly, overexpression of Yip3pcauses inhibition of anterograde trafficking at the ER(Geng et al., 2005).

Despite the fact that AtPRA1.B6-mediated inhibi-tion of trafficking occurs at the ER, trafficking of ST:GFP and KAM1(DC):mRFP to the Golgi apparatus wasnot affected by AtPRA1.B6. These data indicate thatAtPRA1.B6-mediated inhibition is specific to a subsetof cargo proteins that are transported by the Golgi-dependent anterograde trafficking pathway. In addi-tion, these results argue strongly against the possibilitythat inhibition is caused by the nonspecific effects ofHA:AtPRA1.B6 overexpression. Furthermore, the in-hibitory effect of AtPRA1.B6 on anterograde traffick-ing appears to differ from that of SNARE SYP31 andSYP81 overexpression. SNARE overexpression in-hibits the trafficking of not only secreted a-amylasebut also the Golgi-localized proteins ST:GFP and Man:GFP (Bubeck et al., 2008). However, the mechanism bywhich Golgi-localized proteins escape the inhibition isnot clearly understood. As reported previously withother vacuolar and secretory proteins in plant cells(Phillipson et al., 2001; daSilva et al., 2004), all of the

Figure 11. AtPRA1.B6 protein levels are lowered by 26S proteasome-mediated degradation. Protoplasts transformed with HA:AtPRA1.B6 or GKX as a negative control were treated with DMSO or MG132 12 h after transformation, followed by additionalincubation of 12 or 24 h, and protein extracts were analyzed by western-blot analysis using anti-HA antibody. As a loadingcontrol, Lhcb4 was detected with anti-Lhcb4 antibody. PRA1, HA:AtPRA1.B6.



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cargo proteins were transported by COPII vesiclesregardless of whether their trafficking was inhibitedby AtPRA1.B6 or not. Thus, one possible explanationfor the differential inhibition is that plant cells maycontain multiple types of COPII vesicles that aredifferentially used depending on the cargo proteins.Indeed, in animal cells and yeast cells, multiple typesof COPII vesicles exist (Tang et al., 1999; Wendeleret al., 2007). Among COPII vesicle components, Sec24plays a critical role in selecting cargo proteins for theCOPII vesicle (Miller et al., 2003). In yeast cells, thedeletion of LST1, encoding an isoform of Sec24, in-hibits the trafficking of Pma1p from the ER but doesnot affect the trafficking of other proteins (Roberget al., 1999). In animal cells, Wendeler et al. (2007) re-ported the differential effects of Sec24 isoform-specificsilencing on the transport of membrane proteins. Sim-ilarly, in Arabidopsis, multiple isoforms of Sec24 exist,supporting the possibility that multiple types of COPIIvesicles also exist in plant cells. Consistent with thisinference, mutations in each of these Sec24 isoformsresulted in different phenotypes (Faso et al., 2009).Additionally, Arabidopsis contains two Sar-1 isoformsthat exhibit differences in their localizations and ef-fects on protein secretion. Again, the presence of twoSar-1 isoforms supports the inference that multipleCOPII vesicles exist in plants (Hanton et al., 2008).However, it is currently unknown whether these mul-tiple Sec24 and Sar-1 isoforms account for the differ-ential inhibition of anterograde trafficking by AtPRA1.B6. The differential inhibitory effect of AtPRA1.B6 canalso be observed if the COPII-dependent exit of cargoproteins from the ER is mediated by multiple path-ways. Indeed, certain cargoes, such as GONST1, CASP,and KAT, exit the ER in a sorting signal-dependentmanner, whereas a-amylase is secreted from the ER bythe default or bulk flow pathway (Denecke et al., 1990;Phillipson et al., 2001; Hanton et al., 2005; Sieben et al.,2008).

PRA1 is proposed to play a role in targeting prenyl-ated Rabs to the Golgi apparatus and endosomes bycatalyzing their dissociation from Rab/GDI complexesin the cytoplasm (Sivars et al., 2003). Similarly, Arabi-dopsis PRA1 homologs have been shown to interactwith Rabs (Alvim Kamei et al., 2008). Thus, AtPRA1.B6 may regulate the behavior of Rab proteins involvedin COPII vesicle formation in a cargo-specific manner.For this reason, high levels of AtPRA1.B6 may inhibitthe activity of Rabs. In animal cells, PRA1 inhibits theextraction of membrane-bound Rab GTPase by GDI1(Hutt et al., 2000), which in turn may affect the activityof Rabs. Additionally, Yip6b/GTRAP3-18, a member ofthe Yip6 subfamily that includes PRA2, has an inhibi-tory effect on Rab1, a protein involved in ER-to-Golgitrafficking. Interestingly, the inhibitory effect of Yip6b/GTRAB3-18 can be reversed by an excess of Rab1(Maier et al., 2009). In yeast cells, Yip3p also forms acomplex with Ypt1p, a protein involved in ER-to-Golgitrafficking (Geng et al., 2005). However, the overex-pression or deletion of Yip3p does not affect the be-

havior of Rab proteins (Geng et al., 2005), raising thepossibility that the inhibitory effect of Yip3p on proteintrafficking may occur through a mechanism that doesnot require Rab proteins. Originally, Yip3p was de-tected as a binding partner of Yip1p, an integral mem-brane protein and a member of the Yip1 subfamilylocalized to the Golgi apparatus (Otte et al., 2001; Caleroand Collins, 2002; Pfeffer and Aivazian, 2004). Yip1p, inconjunction with Yif1p, a member of the Yif1 subfamilyin yeast, plays an essential role in COPII vesicle bio-genesis. In the absence of Yip1p, COPII vesicle buddingdoes not occur and cargo accumulates in the ER(Heidtman et al., 2003). In fact, Yip3p has been recentlyidentified as a component of COPII vesicles (Chen et al.,2004). Furthermore, overexpression of Yip3p causes ERmembrane proliferation, likely due to the blockage ofCOPII vesicle budding (Geng et al., 2005). However, it isnot known whether AtPRA1.B6 is a component ofCOPII vesicles or is involved in COPII vesicle buddingin plant cells. Further study is necessary to understandthe detailed mechanism of AtPRA1.B6 action in proteintrafficking in plant cells.

MATERIALS AND METHODS

Growth of Plants

Arabidopsis (Arabidopsis thaliana ecotype Columbia) plants were grown on

B5 plates in a culture room under 70% relative humidity and a 16-h-light/8-h-

dark cycle. A second set of plants were grown in soil in a greenhouse under

70% relative humidity and a 16-h-light/8-h-dark cycle. A third set of plants

were grown on B5 plates supplemented with dexamethasone (30 mM). Leaf

tissues obtained from 15- to 18-d-old plants were used immediately for

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

Generation of AtPRA1.B6 RNAi Plants and qRT-PCR

To generate AtPRA1.B6 RNAi transgenic plants, a 317-bp fragment (nu-

cleotide positions 1–317) of the AtPRA1.B6 coding region was amplified by

PCR using primers 5#-ATGGCTTCTCCTCTTCTCCC-3# and 5#-GCCAGC-

GATGCGAGGAGG-3# and ligated to the RNAi vector (Wesley et al., 2001).

The construct was introduced into plants by the floral dip method (Clough

and Bent, 1998).

To examine the transcript levels of AtPRA1.B6, total RNA was prepared

using an RNAqueous Kit (Ambion) with a TURBO DNA-Free Kit (Applied

Biosystems). cDNA was synthesized using a high-capacity cDNA Reverse

Transcription Kit (Applied Biosystems). qRT-PCR was performed on the Step

One Plus Real Time PCR System (Applied Biosystems). Plasmid DNA

containing the corresponding cDNAs was used as a template to generate a

calibration curve. The primers used for PCR were as follows: 5#-CCAACTTC-

GACTACTCCTGATCAA-3# and 5#-CGCGTAGAGAGCTCAGCAACT-3# for

AtPRA1.B6 and 5#-GGATGATCTTGTGAGTGACGATCT-3# and 5#-ACCAC-

CGCCAGAGGTAGAGA-3# for AtPRA1.D.

Construction of Plasmids

Various AtPRA1.B6 full-length and deletion constructs were reported

previously (Jung et al., 2011).

PMP cDNA (At1g53610) was amplified using the primers 5#-CCGC-

TCGAGGGAAACTATTCTCTCCCATC-3# and 5#-CGGGATCCACCCGTTT-

CCAAATCTCGTG-3# (Lee et al., 2011). To generate PMP:GFP-glyNC

containing N-glycosylation sites on both the N and C termini, PCR was

performed using the primers 5#-GCTCTAGAATGTCGCATAGAAATCAAAC-

GTCTTTGATTCAAAGCAATGCCTCGGAGCCTGTTGTTAAT-3# and 5#-ATA-

AGAATGCGGCCGCTCGTTTGATTTCTATGCTTGTACAGCTCGTC-3# with

PMP:GFP as a template. The PCR product was ligated to a pUC-based

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expression vector containing the 35S cauliflower mosaic virus promoter and nos

terminator. All PCR products were confirmed by sequencing.

Transient Expression, Immunofluorescence Staining,

and Microscopy

Plasmids were purified using Qiagen columns and introduced into

Arabidopsis leaf cell protoplasts by polyethylene glycol-mediated transfor-

mation (Jin et al., 2001; Kim et al., 2001). Protoplasts were prepared from leaf

tissues of wild-type, ST:GFP, or HAx3:AtPRA1.B6 plants (Lee et al., 2002; Jung

et al., 2011). Before preparing protoplasts, the HAx3:AtPRA1.B6 plants were

treated with dexamethasone (30 mM) for 1 d. In addition, protoplasts from

dexamethasone-untreated HAx3:AtPRA1.B6 plants were incubated with dexa-

methasone (30 mM) after transformation. The expression of these constructs was

monitored at various time points after transformation. Images of GFP fluo-

rescence were obtained from intact protoplasts placed on glass slides and

covered with a coverslip. For immunostaining, protoplasts on coverslips were

fixed with 4% (v/v) paraformaldehyde, as described previously (Frigerio

et al., 1998; Park et al., 2004). Fixed protoplasts were labeled with anti-g-COP

antibody (Pimpl et al., 2000). Cells were washed with buffer (10 mM Tris, pH

7.4, 0.9% [w/v] NaCl, 0.25% [w/v] gelatin, 0.02% [w/v] SDS, and 0.1% [v/v]

Triton X-100), and secondary immunostaining was performed for 3 h using

tetramethylrhodamine-5-(and-6)-isothiocyanate-labeled goat anti-rabbit anti-

bodies (Molecular Probes). Immunostained protoplasts were mounted in

medium (120 mM Tris, pH 8.4, and 30% glycerol) containing Mowiol 4-88

(Calbiochem).

Images were obtained using a fluorescence microscope (Axioplan 2; Carl

Zeiss) equipped with a 403/0.75 objective (Plan-NEOFLUAR) and a cooled

CCD camera (Senicam; PCO Imaging) at 20�C. Two filter sets were used:

XF116 (exciter, 474AF20; dichroic, 500DRLP; emitter, 510AF23) and XF117

(exciter, 540AF30; dichroic, 570DRLP; emitter, 585ALP [Omega Optical]).

Images were processed with Adobe Photoshop (version 7.0).

Western-Blot Analysis of HA-Tagged AtPRA1.B6Expressed in Protoplasts and Transgenic Plants

Transformed protoplasts were harvested at appropriate time points after

transformation and resuspended in lysis buffer (250 mM Suc, 25 mM HEPES-

NaOH [pH 7.4], 150 mM NaCl, 3 mM EDTA, 2 mM EGTA, 1 mM phenyl-

methylsulfonyl fluoride, and protease inhibitor cocktail [Roche Diagnostics]).

Protoplasts were lysed by repeated freeze-thaw cycles followed by brief

sonication on ice. The lysates were centrifuged at 5,000g for 5 min to remove

cellular debris. Protein extracts were analyzed by western blotting using the

appropriate antibodies. Protein blots were developed using western-blot

detection solution (Supex), and images were obtained using a LAS3000 image-

capture system (Fujifilm).

Endo H Digestion

For Endo H treatment, transformed protoplast extracts were denatured by

adding 100 mL of 23 denaturation buffer (1% SDS and 2% b-mercaptoeth-

anol) and, subsequently, boiling for 10min. After cooling to 22�C, 100 mL of 23reaction buffer (100 mM sodium citrate, pH 5.5) was added. The sample (100

mL) was mixed with 1 mL of Endo H (1 unit mL21). Digestions were conducted

for 1 h at 37�C and were terminated by the addition of 13 SDS-PAGE loading

buffer. Control samples were treated in the same way but without Endo H.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Protein levels of HA:AtPRA1.B6 in protoplasts

and HAx3:AtPRA1.B6 in transgenic plants.

Supplemental Figure S2. Trafficking of AALP:GFP to the vacuole is

inhibited by HAx3:AtPRA1.B6 induced in protoplasts.

Supplemental Figure S3. Trafficking of ST:GFP to the Golgi apparatus is

inhibited by HAx3:AtPRA1.B6 induced in protoplasts.

Received May 26, 2011; accepted August 4, 2011; published August 9, 2011.

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an arabidopsis prenylated rab acceptor 1 isoform, atpra1

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