Zipcode RNA-Binding Proteins and Membrane TraffickingProteins Cooperate to Transport Glutelin mRNAs inRice Endosperm[OPEN]

Li Tian,a,1 Kelly A. Doroshenk,a Laining Zhang,a Masako Fukuda,a,b Haruhiko Washida,a,2 Toshihiro Kumamaru,b

and Thomas Okitaa,1

a Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164–6340b Faculty of Agriculture, Kyushu University, Fukuoka 819–0395, Japan

ORCID IDs: 0000-0003-1497-7923 (L.T.); 0000-0002-6841-1733 (K.A.D.); 0000-0001-6806-1641 (L.Z.); 0000-0002-3225-4828 (M.F.);0000-0001-7372-9503 (H.W.); 0000-0003-4870-1247 (T.K.); 0000-0002-2246-0599 (T.O.)

In rice (Oryza sativa) endosperm cells, mRNAs encoding glutelin and prolamine are translated on distinct cortical-endoplasmic reticulum (ER) subdomains (the cisternal-ER and protein body-ER), a process that facilitates targeting oftheir proteins to different endomembrane compartments. Although the cis- and trans-factors responsible for mRNAlocalization have been defined over the years, how these mRNAs are transported to the cortical ER has yet to be resolved.Here, we show that the two interacting glutelin zipcode RNA binding proteins (RBPs), RBP-P and RBP-L, form a quaternarycomplex with the membrane fusion factors N-ethylmaleimide-sensitive factor (NSF) and the small GTPase Rab5a, enablingmRNA transport on endosomes. Direct interaction of RBP-L with Rab5a, between NSF and RBP-P, and between NSF andRab5a, were established. Biochemical and microscopic analyses confirmed the co-localization of these RBPs with NSF onRab5a-positive endosomes that carry glutelin mRNAs. Analysis of a loss-of-function rab5a mutant showed that glutelinmRNA and the quaternary complex were mis-targeted to the extracellular paramural body structure formed by abortedendosomal trafficking, further confirming the involvement of endosomal trafficking in glutelin mRNA transport. Overall,these findings demonstrate that mRNA localization in plants co-opts membrane trafficking via the acquisition of newfunctional binding properties between RBPs and two essential membrane trafficking factors, thus defining an endosomalanchoring mechanism in mRNA localization.

INTRODUCTION

Localization ofmRNAs is a universal mechanism to efficiently driveprotein targeting in eukaryotes and prokaryotes. The targeting ofmRNAs facilitates the accumulation of the locally translated pro-teins to specific cellular compartments and, hence, is an essentialmechanism in establishing cell polarity, patterning, and fate de-termination as well as protein sorting (Herbert and Costa, 2019;Hughes and Simmonds, 2019; Tian et al., 2019b, 2020.

mRNA localization occurs as a multistep process. After tran-scription, cis-acting elements (RNA zipcodes) are recognized andbound by trans-acting factors, mainly RNA binding proteins(RBPs) to form a primary mRNA–nucleoprotein (mRNP) complex.After export to the cytoplasm, the mRNP complex undergoesextensive remodeling with recruitment of new factors and de-tachment of others enabling cytoskeletal-based transport to thedestination site (Blower, 2013; Weis et al., 2013; Tian and Okita,2014).

Although extensive knowledge on mRNA localization has beenacquired by studies in Drosophila melanogaster, yeast (Saccha-romyces cerevisiae), and mammalian cells, only a few exampleshave emerged fromhigher plants. Thebest-definedmodel in plantsisstorageproteinmRNAlocalization indevelopingrice (Oryzasativa)endosperm cells, where mRNAs encoding glutelin and prolamineare recognized by zipcode RBPs and transported to two distinctcortical endoplasmic reticulum (ER) subdomains, the cisternal-ER,and protein body-ER (PB-ER), respectively (Chou et al., 2019; Tianetal., 2019b).TranslationofprolaminemRNAsonthePB-ERresultsin theassemblyofprolamine intracisternalgranules that formanER-derived protein body I (PB-I), while glutelin precursors are exportedtotheGolgiandthentransported toproteinstoragevacuoles (PSVs)for processing and storage (Chou et al., 2019; Tian et al., 2019b).Although several cytoskeleton-associated RBPs required formRNA localization have been identified (Doroshenk et al., 2009,2012), information on how thesemRNAs are transported to distinctER subdomains remains elusive.Emerging evidence from fungal model systems reveals the

intimate link of mRNA transport with membrane trafficking(Schmid et al., 2006; Jansen et al., 2014; Haag et al., 2015;Niessing et al., 2018). Several mRNAs from yeast, Candida albi-cans, and Ustilago maydis are co-transported with mobile ER orshuttling endosomes (Schmid et al., 2006; Jansen et al., 2014;Haagetal.,2015;Pohlmannetal.,2015;Niessingetal.,2018).ASH1as well as other mRNAs are co-transported on tubular ER thatmoves to theemergingbudordaughter cell in yeast.Thisprocess is

1 Address correspondence to li.tian@wsu.edu or okita@wsu.edu.2 Current address: Organic Nico Co., Ltd, Goryo-Ohara, Nishikyo-ku,Kyoto 615–8245, Japan.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is Thomas Okita (okita@wsu.edu).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.20.00111

The Plant Cell, Vol. 32: 2566–2581, August 2020, www.plantcell.org ã 2020 ASPB.

mediated by the RBPs She2p and She3p, with She2p havingmembrane binding properties and She3p serving as an adaptorprotein linking the mRNP-cER to Myo4P protein (Schmid et al.,2006; Niessing et al., 2018). The cdc3 mRNA is transported onshuttling endosomes in the smut fungus, U. maydis, a processrequiring localization of the RBP Rrm4 on the endosomes and theinteractionof amembrane-associated linker proteinUpa1withRrm4 (Pohlmann et al., 2015; Niessing et al., 2018). Specific adaptorproteins appear to be needed to hitch mRNPs on endosomes foractive transport over long distance. More recently, neuronal RNAgranuleshavebeenshown tohitchhikeonmoving lysosomesusingannexin11 as a tether (Liao et al., 2019). Although co-transport ofmRNAs with membranous compartments was proposed to bea common mechanism in higher eukaryotes (Jansen et al., 2014),whether the mechanism is utilized by higher plants remains to bedetermined.

Previous studies suggested that endocytosis and membranetrafficking likely play a role in mRNA localization in plants. Forexample, the loss of function of the small GTPase Rab5a and itscognate guanine nucleotide exchange factor (GEF) resulted indefects in endocytosis and membrane trafficking and the mis-targeting of glutelin proteins to the prolamine containing PB-I aswell as to the extracellular paramural body (PMB) in rice endo-sperm cells (Fukuda et al., 2011; Wen et al., 2015). As storageprotein targeting is regulated by their mRNA localization in riceendosperm cells, the mis-targeting of glutelin proteins in themutant suggests a relationship betweenendosomal transport andglutelin mRNA localization in rice. The extracellular distribution ofglutelinmRNAswithinPMBs fromamutant expressingadefective

GEF (Yang et al., 2018) further supports the possible involvementof endosomal trafficking in glutelin mRNA transport. However,direct evidence depicting the co-transport of glutelinmRNAswithshuttling endosomes and howendosomal trafficking are engagedinglutelinmRNA localizationhaveyet tobeestablished.Suchmis-targeting of glutelinmRNAs in rice lines expressingmutant Rab5aand GEF may simply be a consequence of pleiotropy.Recent studies (Tian et al., 2018, 2019a) identified two RBPs,

RBP-P and RBP-L, which contain two and three RNA recognitionmotif (RRM) domains, respectively. These RBPs specifically bindto the glutelin zipcode mRNA sequences and regulate glutelinmRNA localization. In this study, using these two glutelin zipcodeRBPs as entry points, we identified their interacting partners,N-ethylmaleimide-sensitive factor (NSF) and the small GTPaseRab5a,whichparticipate in endosomalmembrane trafficking. Thefour proteins may form a quaternary complex carrying glutelinmRNAs for active transport on endosomes to the cortical ERmembrane. The identification of these key linker proteins thatenable endosome-mediated mRNA transport in rice endospermcells provides new insights on how mRNAs can be distributed tospecific locations in eukaryotes.

RESULTS

RBP-P Interacts with Membrane Fusion Factor NSF

Previous studies (Doroshenk et al., 2014; Tian et al., 2018, 2019a)established that the glutelin zipcode RBPs RBP-P and RBP-L,

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which interact with each other, are essential for localization ofglutelin mRNAs to the cisternal-ER, as mutations in these RBPsled to themis-localization of glutelin mRNAs. To obtain additionalinsight into how glutelin mRNAs are transported to the ER, weperformed immunoprecipitation–mass spectrometry (IP-MS)studies using affinity-purified anti-RBP-P antibody (Figure 1A).Tandem mass spectrometry of a major polypeptide band ob-served in the IP generated with anti-RBP-P but not with controlanti-GFP identified a major interacting protein as NSF.

To determine whether RBP-P interacts directly with NSF, yeasttwo-hybrid (Y2H) studies were performed. In such two-hybridscreening, the two proteins of interest were fused to the acti-vating domain and the DNA binding domain of yeast GAL4transcription factor, respectively. Interaction of these proteinsrestoreGAL4 that, in turn, activate the transcription ofHis andAdereporter genes. While no background interaction of RBP-P andNSF with a complementary empty vector was observed, yeastcells carrying both RBP-P and NSF genes activated the reportergenes and survived in selection medium lacking His and Ade(Figure 1B). These results indicate that RBP-P and NSF interactunder stringent binding conditions.

To further substantiate the interaction between RBP-P andNSF, bimolecular fluorescence complementation (BiFC) analysesusing tobacco (Nicotiana tabacum) BY-2 cells was performed. Inaddition to supporting an interaction between protein pairs, BiFCalso provides intracellular information on where this interactionoccurs. RBP-P and NSF were fused to two complementary non-fluorescent fragments (n- and c-terminal halves) of enhanced

yellow fluorescent protein (EYFP), nEYFP and cEYFP, re-spectively, and co-transformed into live BY-2 cells. If the twoproteins interact, the two EYFP fragments are brought in closeproximity to reform the native protein structure enabling emissionof yellow fluorescence and, in turn, direct visualization of theprotein complex in live cells (Kerppola, 2006; Miller et al., 2015).EYFP fragments lacking one of the putative protein partners wereused as a negative control. As shown in Figures 2A to 2C,while nointeraction was detected between RBP-P or NSF with the emptyEYFP fragment controls, cells expressing both RBP-P and NSFfused to EYFP fragments emitted bright yellow fluorescence,indicating that RBP-P interacted with NSF. Closer examinationreveals that RBP-P/NSF complexes were distributed to the cy-toplasm as intensely bright clusters together with more looselydiffuse structures (Figure 2C).NSF is a soluble hexameric ATPase commonly found in the

cytoplasm of eukaryote cells (Mastick and Falick, 1997; Zhaoet al., 2007, 2010; Guo et al., 2017), which predominantly playsa major role as a chaperone in intracellular membrane fusionevents. Through its interaction with the adaptor protein, solubleNSF attachment protein (SNAP), NSF binds to soluble NSF ac-tivating protein receptor (SNARE) complexes and utilizes theenergy of Mg21-dependent ATP hydrolysis to disassemble theSNARE protein complex, and thus facilitating the recycling ofSNAREproteins for further cyclesofmembrane fusion (Zhaoet al.,2007; Chang et al., 2012; Ryu et al., 2015). During this process,SNAP serves as chaperone by stimulating the ATPase activity ofNSF and dissociating from NSF after ATP hydrolysis (Zhao et al.,

Figure 1. Identification of NSF as an Interacting Partner of RBP-P.

(A) Precipitation ofNSFbyRBP-P antibody as revealed by IP-MS. Left representation, immunoblot (IB) analysis to test the IP reliability; right representation,silver-stained SDS-polyacrylamide gel of eluted samples from anti(a)-GFP andRBP-P IPs. Input, startingmaterial of rice lysate; Ub, unbound fraction fromIPs; B, bound fraction (eluted samples) from IPs. Blue asterisk (*) indicates a modified form of RBP-P. The bands indicated by red and black arrows wereexcised for MS analysis, and NSF was identified as a specific protein precipitated by anti-RBP-P but not by anti-GFP.(B) Interaction between RBP-P and NSF revealed by Y2H assay. Yeast colonies co-transfected with pGBK and pGAD constructs were labeled 1 through 4(asdescribed in the top tabletop). –, empty vector. Yeast cells carrying thecorrespondinggenesweregrownonSD/-Leu/-Trpmediumasgrowthcontrol andSD/-Leu/-Trp/-His/-Ade/13-AT selection medium to detect their interaction. Note that only the yeast cells carrying both NSF and RBP-P survived on thestrict selection medium (bottom), suggesting that RBP-P interacts with NSF.

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Figure 2. RBP-P Interacts Indirectly with Rab5a through NSF, Forming a Complex Attached to Endosomes.

(A) to (C) RBP-P interacts with NSF as revealed by BiFC studies. BF, bright field; Merge, the merged images of BF and BiFC (middle); –, empty vector ascontrol.

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2007). This transient interaction between NSF and SNAP is onlydetected under conditions where a nonhydrolyzable ATP is usedas a substrate (Hanson et al., 1995; Barnard et al., 1997; Changet al., 2012). Alternatively, the addition of EDTA to chelate Mg21

and thus inhibit ATPase activity has been used to detect thetransient interaction betweenNSFandSNAP (Hansonet al., 1995;Barnard et al., 1997; Chang et al., 2012; Li et al., 2018).

Based on the established properties of NSF, we performed co-immunoprecipitation (Co-IP) experiments to assess the formationof RBP-P/NSF complexes in rice endosperm cells. Rice seed ly-sates supplementedwith 1mMof ATPand8mMofMgCl2 orEDTAwere incubated with affinity-purified anti-RBP-P and NSF anti-bodies (Figure 2D) or anti-GFP antibody, the latter used as a neg-ative control. No proteins were captured by the control GFPantibody, revealingthe reliabilityof the IPexperiments. Interestingly,RBP-PandNSFwere co-precipitated in thepresenceofMg21-ATPbut not when EDTAwas added to the seed lysates (Figure 2D). Thisresult indicates that formation of the RBP-P/NSF complex in riceendosperm cells requires Mg21-ATP, a condition distinct fromthose complexes involving the stable interaction betweenNSF andSNAP (Hanson et al., 1995; Barnard et al., 1997;Chang et al., 2012;Li et al., 2018). The differences in binding properties, as well as theabsence of SNAP inRBP-P IPs (Figure 2D), indicate that the eventsof membrane fusion requiring NSF-SNAP are not required forformation of the RBP-P/NSF complex. On the other hand, the in-teraction betweenNSFwith RBPs suggests that NSFmay functionin mRNA metabolism by its interaction with RBP-P.

RBP-P Indirectly Interacts with Rab5a through NSF

The interaction of the glutelin zipcode trans-factor RBP-P withNSF supports a close relationship between glutelin mRNAtransport with membrane trafficking. Previous studies demon-strated that Rab5a, an evolutionarily conserved key GTPase in-volved in the biogenesis of early endosomes and membranetrafficking in the cytoplasm (Woodman, 2000; Saito and Ueda,2009; Ito et al., 2018), is also required for glutelin mRNA locali-zation (Doroshenk et al., 2010) and vesicular membrane transportbetween the Golgi and PSV in rice endosperm cells (Wang et al.,2010; Fukuda et al., 2011). Therefore, we investigatedwhether the

abovementioned RBP-P/NSF complex is associated with Rab5ato regulate the endosomal transport of mRNAs.We first performed a transient expression study to confirm the

association of rice Rab5a (Figures 2E and 2F; SupplementalFigure 1) with endosomes. FM4-64 is a lipophilic dye that initiallylabels the plasmamembrane and, subsequently, internalizes withmembrane vesicles transported along the endocytic pathway(Vida and Emr, 1995; Ueda et al., 2001). In this experiment, BY-2protoplasts expressing GFP-Rab5a were treated with FM4-64 for15 min before observation. As shown in Figure 2F, red fluores-cencederived fromFM4-64wasevidenton theplasmamembraneand in internalized endosomes. GFP-Rab5a displayed a similardistribution pattern and co-localized with the internal FM4-64–labeled vesicles (Figure 2F), an observation confirming thatRab5a is associated with endosomes.WethenstudiedtherelationshipofRab5awiththeabovementioned

RBP-P/NSF complex. Co-IP studies were performed using affinity-purifiedantibodies toRab5a,RBP-P, andNSF.All threeproteinswerefound in IPs generated by anti-Rab5a, anti-RBP-P, and anti-NSF(Figure 2D), suggesting that they form a multiprotein complex inrice endosperm cells. Protein complex formation apparently requiredactiveATPasehydrolysisasall threeprotein interactionscouldonlybesimultaneously detected in the presence of Mg21 and ATP and notwhen EDTA and ATP were included (Figure 2D).To further characterize this protein interactome and the for-

mation of their complexes, BiFC (Figures 2G to 2J) studies wereperformed. Interaction between NSF and Rab5a was observed inthe tested cells, and their complexes existed in the cytoplasm. Bycontrast, Rab5a does not interact with RBP-P (Figure 2J) and,therefore, the association of RBP-P with Rab5a in the co-immunoprecipitates (Figure 2D) is mediated through the in-teraction between NSF and Rab5a.Rab GTPases are highly conserved small proteins and share

two conserved regions that are required for conformationalswitching between active and inactive states of GTP hydrolysis(Figure 2E; Supplemental Figure 1). The switch regions are dis-ordered in an inactive conformation and adopt a well-definedconformation in the active stage (Stenmark and Olkkonen,2001).Aminoacidsubstitutionswithin theswitchregionsaffects theirconformational switching. While the GDP-fixed Rab5a (G45D)

Figure 2. (continued).

(D)SNAP-independent formation of a protein complex consisting of RBP-P, NSF, and Rab5a as revealed byCo-IP. IP studieswere conductedwith anti(a)-GFP,RBP-P,NSF, orRab5aantibodies in thepresenceofATPandMgCl2 orwithEDTA.GFPantibodywasusedasanegative control. Input, the starting ricelysate. Note that the association ofRBP-PwithNSFandRab5a requiresMgCl2 andATP, unlike the interaction of SNAPwithNSFandRab5a,which requiresEDTA.(E)Schematic structureofRab5a in rice. The twoswitch regionsare indicated asSwitch1andSwitch2. The twomutation sites (G45DandQ70L) togeneratethe GDP- and GTP-fixed forms of Rab5a, respectively, are indicated in red.(F)GFP-Rab5a (green) co-localized with FM4-64-labeled (magenta) plasmamembrane and endosomal compartment. Co-localization of GFP-Rab5a withFM4-64 endosomes are denoted by arrows.(G) to (J) Rab5a interacts with NSF (I), but not to RBP-P (J), as shown by BiFC.(K) and (L) NSF interacts with both GDP-bound ([J], Rab5aG45D) and GTP-bound ([K], Rab5aQ70L) Rab5a.(M) and (N) RBP-P/NSF complexes labeled by BiFC (yellow) co-localize with RFP-Rab5a (magenta) in the cytoplasm. In (M) and (N), co-localized RBP-P/NSF complexes with endosomal Rab5a are indicated by white arrows. Non-colocalization of RBP-P/NSF complexes and Rab5a-postive endosome areindicated by empty arrowheads. (M) The punctate RBP-P/NSF structures align with the particle-like, endosome structures of RFP-Rab5a. (N)RBP-P/NSFcomplexes co-localize with the membrane-associated, GTP-fixed Rab5aQ70L, indicating RBP-P/NSF complexes are associated with active endosomes.(O) Co-localization of RBP-P/NSF complexes with FM4-64 labeled endosomes (magenta) are denoted by arrows. Scale bars, 20 mm.

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mutation in switch 1 inhibits the conformation switching to an activeGTPase stage and restricts Rab5a in a GDP-bound form, a Q70Lmutation inswitch2 locks it inaconstitutivelyactiveGTP-boundform(Lee et al., 2009). Only theGTP-bound form of Rab5 aremembrane-bound during endosomal trafficking (Yuan and Song, 2020).

To investigate whether the NSF-Rab5a complex is associatedwith active endosomes, the interaction of NSF with the twostructural forms of Rab5a containingG45D or Q70Lwas analyzedby BiFC (Figures 2K and 2L). Although NSF directly interacts withboth GDP- (Rab5aG45D) and GTP- (Rab5aQ70L) forms of Rab5a,their complexes are observed as two distinct populations. Whenbound to the GTP-fixed Rab5a (Q70L) form (Figure 2L), the NSFcomplex was distributed as endosomal punctate structures. Bycontrast, the NSF complex constituted by GDP-fixed Rab5a(G45D) showed a diffuse distribution throughout the cytoplasm(Figure 2K). These results are consistent with the view that theGTP-fixedRab5a (Q70L) form ismembrane-boundwhile theGDP-fixed Rab5a (G45D) is mainly soluble.

The direct interaction between NSF and Rab5a provides an in-direct link to RBP-P and, in turn, RNA localization. To confirm theassociation of the RBP–P/NSF complex to Rab5a-positive endo-somes,weperformeda three-way co-localization test consistingofRBP-P and NSF as BiFC interacting partners in BY-2 cells ex-pressing Rab5a tagged with the red fluorescence protein (RFP;Figures 2M and 2N). Florescence analysis of BY-2 cells showedthat RFP-Rab5aWT was distributed as diffuse signals throughoutthe cytoplasm and as punctate structures (endosomes) near theplasma membrane (Figure 2M, middle). By contrast, RFP-Rab5aQ70L was present only as punctate structures (Figure 2N,middle) indicating that the activated GTPase form of Rab5a isassociated with endosomes. The fluorescence distribution patternseen for RFP-Rab5aWT is consistent with membrane-free Rab5abeing distributed in the cytoplasm and active Rab5a associatedwith endosomes (Yuan and Song, 2020).

As earlier seen inFigure 2C,BiFC-linkedRBP–P/NSFcomplexesare distributed as fluorescent punctate structures in BY-2 cells(Figures 2M and 2N). These RBP-P/NSF complexes co-localizedwith RFP-Rab5a–associated endosomes in the cytoplasm, espe-ciallywithmembrane-associatedGTP-fixed (Q70L) Rab5a (Figures2Mand 2N). To further confirm that the RBP-P/NSF complexes areassociated with endosomes, we used FM4-64, which specificallylabels these small membrane compartments (Figure 2O). Thefluorescent signals from the RBP-P/NSF complexes co-localizedwithendosomalcompartments labeledbyFM4-64,providingdirectevidencethatRBP–P/NSFcomplexesco-localizewithendosomes.Consistent with the abovementioned Co-IP analysis (Figure 2D)where RBP-P, NSF, and Rab5a may co-assemble into a multi-protein complex in rice endosperm cells, the BiFC results suggestthat RBP-P is associated with endosomes through an NSF-mediated interaction with Rab5a.

It should be noted that not all RBP–P/NSF complexes co-localized with the Rab5a-linked endosomes (Figures 2M and2N, open arrowheads). Similarly, the location of several Rab5a-active endosomes (Figures 2M and 2N, open arrowheads) do notcoincide with RBP–P/NSF complexes. The lack of overlap in thedistribution of a population of RBP–P/NSF complexes and Rab5endosomes suggests the multiple roles of these proteins in RNAlocalization and membrane trafficking.

RBP-L Is Involved in the RBP–P/NSF/Rab5a Complex

We had previously demonstrated that RBP-P co-assembles withRBP-L to formaproteincomplex that isessential for storageproteinmRNA localization (Doroshenk et al., 2014; Tian et al., 2018; Tianet al., 2019a). To determine whether RBP-L is also involved in in-teractingwithNSForRab5a,BiFCstudieswereconducted (Figures3A to 3F). Such analysis showed that RBP-L interacts directly withRab5a but not with NSF (Figures 3A to 3C). The RBP–L/Rab5aWT

complex is observed as twodistinct populations.Onepopulation isdistributed as endosomal punctate structureswith the bulk locatedclose to the plasma membrane. The co-localization of RBP–L/Rab5aWT with endosomes is supported by their close associationwith FM4-64–labeled endosomal compartments (Figure 3D). Asecond population of RBP–L/Rab5aWT is viewed as a diffuse cloudaround thenucleus.Similar to theRBP–P/NSF/Rab5acomplex, theinteraction of RBP-L with Rab5a was not dependent on the func-tional state of Rab5a as it interacts with both the GTP-bound Ra-b5aQ70L and GDP-bound Rab5aG45D (Figures 3E and 3F), althoughthe distribution patterns are distinct. RBP–L/Rab5aG45D is distrib-uted mainly as a diffuse cloud around the nucleus and near theplasma membrane, whereas RBP–L/Rab5aQ70L is observed pre-dominantly as some discrete endosomal punctate structures.To further determine whether RBP-L is associated with the

RBP–P/NSF/Rab5a complex, we performed Co-IP with para-formaldehyde (PFA)-fixed seed extracts to optimize the capture ofany potential dynamic endosome-associated complexes formedin vivo. Such results showed that, irrespective of the antibody usedin the initial IP, theRBP-P,RBP-L,NSF,andRab5aweredetected inIPs generated by all four antibodies (Figure 3G). By contrast, SNAPis only present in IPs obtainedwith anti-NSF and anti-Rab5 but notin IPs generated by antibodies to RBP-P and RBP-L (Figure 3G).These results are consistent with the Co-IP results depicted inFigure2DwhereSNAPispresent in IPsgeneratedwithantibodies toNSF and Rab5a in the presence of EDTA but not in the presenceof MgCl2. Overall, these results are consistent with the view thatNSF-Rab5a complexes exist as two separate populations. OneNSF–Rab5a population together with SNAP participates in Rab5-mediated endosomal fusion (Woodman, 2000; Zhao et al., 2007),whereNSFandSNAPdisassemble theSNAREcomplex formedbyRab5-mediatedmembrane fusion. A second NSF–Rab5a complexcontains RBP-P and RBP-L and is independent of SNAP.To further address whether RBP-P and RBP-L are attached to

endosomes together, sequential IPs were performed (Figure 3H).In this analysis, the RBP–P/NSF/Rab5a complexes precipitatedby anti-RBP-P antibodies were next subjected to an additional IPusing anti-RBP-L. All four proteins were detected in the second IPgenerated by anti-RBP-L, suggesting that RBP-P, RBP-L, NSF,and Rab5 co-assemble to form a quaternary protein complex.

The Quaternary Complex Attaches To Active EndosomesCarries Glutelin mRNAs

To investigate whether the quaternary complex binds glutelinmRNA, we performed RNA-IP analysis to detect the in vivo as-sociation of the complex with glutelin mRNAs (Figure 3I). In thisanalysis, antibodies to RBP-P, RBP-L, NSF, and Rab5a wereutilized to capture the associated RNA–protein complexes, and

Glutelin mRNAs Are Transported on Endosomes 2571

the RNA was subsequently isolated from the IP fractions andsubjected to RT-PCR using specific primers to amplify glutelintranscripts. Compared with the negative empty-resin control andanti-GFP antibody, glutelin mRNAs were highly enriched in IPsgenerated by all four antibodies (Figure 3I). The mRNA amountassociatedwithNSFandRab5awas lower than that of RBP-PandRBP-L, a result consistent with the roles of these proteins inmembrane fusion events. Overall, these results support the viewthat the quaternary complex contains glutelin mRNAs.

Based on these results, a working model of cytosolic glutelinmRNA transport is proposed in Figure 4. The mRNP complexcontaining glutelin mRNA, RBP-P, andRBP-L is bound to Rab5a-associated endosomes through a four-way interactome, i.e., thedirect interaction of RBP-P with RBP-L, of RBP-L with Rab5a, ofNSF with RBP-P, and of Rab5a with NSF. The GTP-bound Rab5ais associated with endosomes (Yuan and Song, 2020), as sug-gested by distribution of this activated Rab5a form as endosomalpunctate structures (Figures 2N and 3F).

Figure 3. RBP-L Interacts Directly with Rab5a, Which Collectively Forms Multiprotein Complexes with RBP-P and NSF to Transport Glutelin mRNAs onEndosomes.

(A) to (C) RBP-L interacts directly with Rab5a (C) but not to NSF (B). Note that the RBP-L/Rab5a complex is detected in both the nucleus and cytoplasm.(D)RBP-L/Rab5acomplexes (yellow) co-localizedwithFM4-64 labeledendosomes (magenta) that are locatedclose to theplasmamembrane.Co-localizedsignals are indicated by arrows.(E) and (F) in vivo interactions of RBP-L with GDP- ([E], Rab5aG45D) or GTP-bound ([F], Rab5aQ70L) Rab5a.(G)Association of RBP-P andRBP-LwithNSF andRab5a as revealed by IP. The IP experimentwas conductedwith developing seeds treatedwith 1%PFAto preserve labile protein complexes. Input, the starting rice lysate; Control, GFP antibody; IB, immunoblot.(H)RBP-P, RBP-L, NSF, andRab5a form a ternary complex, as revealed by sequential IPs. The first IPwas conductedwith anti(a)-GFP (negative control) orRBP-P antibodies. Proteins captured by RBP-P antibody were subjected to a second IP using RBP-L antibody. Subsequent immunoblot analysis showedthe presence of NSF and Rab5a together with RBP-P and RBP-L. Input, the starting rice lysate; FT, flow-through (unbound fraction); E, bound fraction; W1and W2, washing fractions.(I)TheRBP-P/RBP-L/NSF/Rab5acomplex containsglutelinmRNAsbasedonRNA-IP.RNAsextracted fromeach IPgeneratedbyanti-RBP-P, anti-RBP-L,anti-GFP, or empty resin (– CT) were subjected to RT-PCR using glutelin-specific primers and resolved by agarose gel electrophoresis. GFP antibody(a-GFP) andempty resin (–CT)were usedasnegative controls. Input, PCRproducts usingcDNAsynthesized from total RNAs.Actinwasusedas thecontrolgene to verify the specific binding of the complex to glutelin RNAs. Scale bars, 20 mm.

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Loss of Function of Rab5a Results in Mis-targeting ofGlutelin mRNAs

To provide evidence in support of thismodel, we analyzed a rab5amutant EM960 (Fukuda et al., 2011) expressing a GDP-fixed(G45D) Rab5a (Figure 5A). Similar to the phenotype shown inthe EM956mutant lackingRab5a (Fukuda et al., 2011) or amutantline expressing a defective Rab5a effector GEF (Wen et al., 2015),normal endosomal trafficking is disrupted in the endosperm cellsof GDP-fixed rab5a mutant and leads to the formation of PMBs(Figures 5B and 5C), an aborted endosome complex containingmis-sorted endomembrane proteins. These extracellular PMBs,which contain numerous electron-dense vesicles, are located inthe space between the invaginating plasma membrane and thecell wall in the mutant endosperm cells (Figures 5B and 5C).

To investigate the co-localization of RBP-P, RBP-L, and NSFwith Rab5a and the subcellular localization of their complex in riceendosperm cells, we performed double immuno-fluorescencelabeling on thin sections of rice developing seeds using anti-bodies raised against each of the four proteins. Although the bulkof these proteins were evidently independent of Rab5, there wasample evidence for co-localization of RBP-P, RBP-L, and NSFwith Rab5a. The co-localization of these proteins with Rab5a wasapparent as punctate structures in the cytoplasm, particularly inthe cortical region underneath the plasmamembrane (Figures 6A,6C, and 6E), an intracellular location enriched in Rab5a-mediatedendosomeactivity (Chavrier et al., 1990; Fischer vonMollard et al.,1994). To directly assess the co-localization of these proteins, thefluorescence intensity profiles of these proteins were quantifiedalong a specific linear distance (Figure 6, right panels). Thefluorescence signals for the proteins examined overlapped sub-stantially, indicating that RBP-P, RBP-L, and NSF co-localized toRab5a-labeled endosomal compartments in rice endospermcells. The independent distribution of RBP-P, RBP-L, and NSFwith Rab5a was also evident in the BiFC/RFP double labeling(Figures 2M and 2N), which is indicative of their roles in othercellularprocesses.Thisview isalsosupportedby theCo-IP results

(Figures2Fand3H)where IPsbyantibodies toRBP-P,RBP-L, andNSFcontainedonlyasmall proportionof the totalRab5aamounts.The RBPs retain their co-localization with Rab5a in the rab5a

mutant (Figures 6Band6D), an expectedobservation asRBP-Laswell as NSF interact with both the GDP- and GTP-fixed Rab5aforms (Figures 2K, 2L, 3E, and 3F). These protein complexes existas punctate structures within the PMBs in the rab5a mutant,(Figures 6B, 6D, and 6F). Hence, disruption of membrane traf-ficking in the rab5amutant displaces not only endosomal proteinsto the extracellular PMBs but also RBP–mRNA complexes.Transmission electron microscopy (TEM) analysis further con-

firmed the co-localization of RBP-P and RBP-L with Rab5a onendosomes in wild-type and aborted endosome vesicles withinPMBs in rab5a mutant (Figure 7). In wild type, Rab5a-mediatedendosomes were observed as electron-dense vesicles with an ir-regular shape likely due toendosomal fusion (Figures 7A to7E).Co-localization of RBP-P and RBP-L with Rab5a was observed onthose endosomes, further suggesting that mRNP complexes,carrying glutelin mRNAs bound by RBP-P and RBP-L, are trans-ported on endosomes. In the rab5amutant line, normal endosomaltrafficking is disrupted, resulting in the displacement of mRNA-associatedendosomes to theextracellular PMBs (Figures7F to7J).To further address whether glutelin mRNAs are associated with

the Rab5a-mediated endosomes, in situ RT-PCR on developingendosperm sections was performed to locate glutelin mRNAs. Inthis experiment, Rhodamine B dye was used to specifically stainthe PB-ER (Muench et al., 2000) while glutelin mRNAs were la-beled with Alexa488-dUTP by in situ RT-PCR using glutelin-specific primers (Figure 8). In wild type, glutelin mRNAs are lo-calized on the cisternal-ER separate from PB-ER (Figure 8). Inrab5a mutant cells, however, they were mainly distributed to thePMBs and with smaller amounts associated with the PB-ER.These results indicate that normal glutelin mRNA localization isdisrupted in the rab5a mutant. This result resonates with the re-tention of the complexes formed by RBP-P, RBP-L, NSF, andRab5a in PMBs (Figures 6 and 7), further supporting a Rab5a-dependent endosome transport of glutelin mRNAs.

Figure 4. A Working Model of Glutelin mRNA Transport via Trafficking Endosomes to the Cortical ER.

In rice endospermcells, glutelinmRNA isboundby the scaffold complex ofRBP-PandRBP-L, forminganmRNPcomplex. Throughdirect orNSF-mediatedindirect interaction with membrane-associated Rab5a, RBP-L and RBP-P link the mRNP complex to endosomes for active transport via the cytoskeleton.Rab5aeffectors (e, light orange)maybe involved tostabilize thequaternary complex andmodulateGTP-boundactiveRab5a-derivedendosomal traffickingon actin filaments in rice endosperm cells. Other unknown RBPs or factors, showed in light gray shapes, may also be involved to constitute the mRNPcomplex and define the linkage onto endosomes.

Glutelin mRNAs Are Transported on Endosomes 2573

Taken all together, the results described here support the viewthatglutelinmRNAsare transported to thecisternal-ERmembranevia their hitchhiking on endosomes. In the rab5amutant, however,glutelin mRNA-containing mRNP complexes are not transportedto their normal location but are mis-targeted to the PB-ER anddisplaced to the extracellular PMBs because of the disruption innormal endosomal trafficking.

DISCUSSION

Thestudyof ricestorageproteinmRNA localizationasamodelhasprovided considerable information on why and how plant mRNAsare localized to specific ER compartments. Although zipcode cis-elements of glutelin and prolamine mRNAs and several key RBPshave been identified (Hamada et al., 2003; Washida et al., 2009;Doroshenk et al., 2012; Washida et al., 2012; Tian et al., 2018,2019a), the mechanism by which glutelin and prolamine mRNAsare transported to distinct subdomains of the ER membrane hasyet to be established. Here, we report that rice endosperm cellsemploy Rab5a and NSF—proteins traditionally known for theiressential roles in endosomal trafficking and membrane fusionevents—as adaptor proteins linking mRNA-protein complexes toendosomes for active transport of glutelin mRNAs.

Active transport of mRNAs along cytoskeleton networks byhitchhiking on motile, membrane-bound organelles has beenreported in mammalian and fungi cells. In human axons, the RasGAP SH3 domain binding protein1-labeled RNA granules werefound to co-localize and co-traffic with moving lysosomes along

microtubules (Liao et al., 2019). The membranous amyotrophiclateral sclerosis-associated phosphoinositide binding protein,annexin A11, functions as a molecular tether between RNAgranules and lysosomes (Liao et al., 2019). In addition to mRNAs,neuronal precursor microRNAs are transported to the tip ofgrowing axons by hitchhiking on late endosomes/lysosomes(Corradi et al., 2020).Endosomal transport of mRNAs has been defined in the fila-

mentous fungus U. maydis, where mRNAs are transportedthroughout the growing hyphae via endosome trafficking on mi-crotubules (Baumann et al., 2012; Göhre et al., 2012; Vollmeisteret al., 2012; Pohlmann et al., 2015; Niessing et al., 2018). mRNPcomplexes carrying cdc3mRNAs are attached to endosomes viathe adaptor protein Upa1, which associates with the endosomalmembrane through its FYVE domain and two RBPs, Pab1 andRrm4, through its PABP-associated motif2 and PAM2-like do-mains, respectively (Pohlmann et al., 2015).Unlike the abovementioned examples in mammalian and fungi

cells, higher plants apparently adapted the two membrane traf-ficking factors, NSF and Rab5a, to mediate transport of storageprotein mRNAs. The linking of glutelin mRNP complexes toendosomes is mediated by the four-way interactions of NSF andRab5a with the two glutelin zipcode RBPs, RBP-P and RBP-L.These interacting proteins likely form a quaternary complex en-abling endosomal transport of glutelin mRNAs. Such protein–protein interactions are accomplished through a gain in bindingproperties by Rab5a and NSF, and the glutelin zipcodemRNA binding proteins, RBP-P and RBP-L, which allow for the

Figure 5. Rab5a Mutation Leads to Abnormal Trafficking of Endosomes and Formation of Extracellular PMBs.

(A)Schematic representationof theRab5amutationsite in the rab5amutant.AG134Abasesubstitutionwithin theRab5agene resulted inaG45Daminoacidreplacement.(B) Formation of PMBs (white asterisks) was observed in endosperm cells of rab5amutant through lightmicroscopy observations on seed sections stainedwith 1% Toluidine blue. Scale bar, 25 mm.(C) Ultrastructure of PMBs formed in rab5a mutant due to aborted endosomal trafficking in comparison to wild-type (WT) endosperm cells.Cell wall and PMB boundaries are indicated bymagenta and green dashed lines, respectively. SG, starch granules; orange *, PB-I; blue *, PSVs. Scale bar,1 mm.

2574 The Plant Cell

heterotypic interaction of an RBP with a membrane fusion factor(RBP-P/NSF and RBP-L/Rab5a). The interaction of an RBP witha membrane trafficking factor, i.e., among RBP-P with NSF andRBP-L and Rab5a, as well as between NSF and Rab5a, are un-expected findings as such interactions have not been reported inother organisms. It remains unclear whether the interactions ofthese membrane trafficking factors to RBPs and even betweenthemselves is an inherent property or whether they are uniqueto higher plants. While Upa1-mediated endosomal mRNAtransport is proposed as evolutionarily conserved in fungi (Mülleret al., 2019), further investigation is needed to assess whether

endosomalmRNA transport by theNSF-Rab5a-RBPmachinery isa widespread phenomenon in transporting mRNAs among higherplants and other eukaryotic organisms.It appears that the recognition of NSF and Rab5a with RBPs is

highly selective. These non-random interactions highlight thediverse binding capabilities of NSF, Rab5a, as well as for RBP-Pand RBP-L in rice endosperm cells. Both RBP-P and RBP-Lcontain RRMs, with the former having two RRM motifs and thelatter having three RRM motifs. Due to the conservation of theRRM motifs, the recognition of NSF and Rab5a to RBP-P andRBP-L, respectively, is likely via the unique N- and C-terminal

Figure 6. Rab5a Mutation Leads to the Transport of the RBP-P/RBP-L/NSF/Rab5a Ternary Complex to PMBs.

(A) to (F) Intracellular locationofRBP-P ([A]and [B],magenta),RBP-L ([C]and [D],magenta),NSF ([E]and [F],magenta), andRab5a ([A] to [F], green) inwild-type ([A], [C], and [E]) and rab5a mutant ([B], [D], and [F]) rice endosperm cells as revealed by immunofluorescence labeling.Co-localization patterns of RBP-P, RBP-L, and NSF with Rab5a are shown in the Merge representation, with rectangular areas enlarged in the adjacentfourth representations. Fluorescence intensity graphs on the very right show the relative strength of the magenta and green fluorescence signals asmeasured by scanning the region indicated by the white line in the fourth representation. X- and y axes represent the fluorescence intensity and position ofthe signals (pixels), respectively, as evaluatedby thesoftwareFUJI (ImageJ). PMBsare indicatedbyasterisk (*) andco-localizationsignals arehighlightedbywhite arrowheads. Scale bar, 20 mm (the left three representations); 10 mm (the rightmost representations).

Glutelin mRNAs Are Transported on Endosomes 2575

regions that flank the RRMmotifs. Indeed, our preliminary results(Supplemental Figure 2) show that the N-terminal end of RBP-P isessential for its interaction with NSF. Through the selective rec-ognition of NSF and Rab5a to other specific RBPs, these mem-brane trafficking factors could serve as the core componentsenabling endosome-coupled mRNA transport with the RBPsspecifying the mRNA species.

NSF is a homo-hexamer with three domains: the N-terminaldomain (NSF-N), required for SNAP-SNARE binding; the ATP

binding domain 1, responsible for ATPase activity; and the ATPbinding domain 2, responsible for hexamerization (Tagaya et al.,1993; Zhao et al., 2007). The NSF-N domain is the mainprotein–protein interaction site for NSF binding to other proteins.NSF-N domain is likely responsible for its interaction with the Nterminus of RBP-P in rice as well (Supplemental Figure 2). Se-quence alignment of NSF homologues from rice, Arabidopsis(Arabidopsis thaliana), Drosophila, yeast, and human show thatthe N-terminal region displays considerable sequence diversityamong these species (Supplemental Figure 3). Further structuralstudies are required to investigate their bindingmechanismand todeterminewhether this kind of interaction occurs in other species.Rab5a interacts with a large number of proteins, including its

regulators (activating effectors and inhibitors), linkers tomolecularmotors, membranous factors, components of membrane fusioncomplexes, protein kinases, and phosphatases (Hervé andBourmeyster, 2018; Pylypenko et al., 2018). Many of these in-teracting proteins function as Rab5a effectors to activate endo-somal transport and tightly control the specific functions of Rab5ain membrane composition and modification, cytoskeleton regu-lation, and intracellular trafficking (Hervé and Bourmeyster, 2018;Pylypenko et al., 2018). Although NSF has been reported to in-teract with several Rab5a effectors to drive membrane fusionduring endosomal docking (McBride et al., 1999;Grosshans et al.,2006), no direct relationship between Rab5a with NSF or witha RBP had been established until now. These findings will stim-ulate future research efforts on identifying new alternative roles ofRab5a and NSF beyond membrane fusion.Although the GTPase activity of Rab5a is not required for the

apparent formation of the quaternary protein complex as the fourproteins still co-localized in theG45DRab5a line (Figures 6 and 7),Rab5 GTPase activity is required for proper mRNA transport.Endosome formation andmembrane trafficking are dependent onRab5GTPase activity (Woodman, 2000; Zeigerer et al., 2012).NullRab5a activity results in aborted endosomal transport in themutant, which, in turn, disrupts glutelin mRNA transport on en-dosomes and causes its mis-localization to the PB-ER as well asdisplacement to the extracellular PMBs (Figure 8). Therefore, theactive GTPase activity of Rab5a is essential for the transport ofglutelin mRNAs on endosomes. This also raises the question ofwhether NSF and RBP-L act as Rab5a effectors to modulate therole of Rab5a in mRNA transport and localization. While this hy-pothesis needs further examination, other effectors, functionallyequivalent toRabaptin-5andEEA1 that interactwithbothNSFandRab5a (McBride et al., 1999; Grosshans et al., 2006), may also beinvolved. These effectors, including the abovementioned GEF,may allow further stabilization of the linkage of mRNP complex toactive endosomes and regulate endosomal mRNA transport viathe cytoskeleton (Figure 4).The identification andstudyof rice linesexpressingmutations in

Rab5a were instrumental in establishing its involvement in bothRNA and membrane trafficking in developing rice endosperm.While Rab5 is an essential growth factor, loss of Rab5a only hada slight effect on rice growth and development as its activity iscomplemented by other Rab5 activities. Rice expresses threeother Rab5 genes, Rab5b, Rab5c, and Rab5d (SupplementalFigure 4). Like Rab5a, Rab5c is a conventional type, while Rab5bandRab5dareplant-specific type homologous to theArabidopsis

Figure 7. Retention of RBP-P and RBP-L on the Aborted Endosomes inthePMBsof rab5aMutantasRevealedby Immunocytochemistry andTEM.

(A) to (J) Localization of RBP-P ([A] to [C], [F], and [G]) or RBP-L ([D], [E],and [H] to [J]) on Rab5a-labeled endosomes in wild-type ([A] to [E]) andrab5a mutant ([F] to [J]) endosperm cells. RBPs (RBP-P and RBP-L) andRab5a were labeled with 15-nm (blue arrowheads) and 10-nm (red arrows)gold particles, respectively.(B), (C), (E), (G), (I), and (J) Enlarged areas (rectangle) shown in (A), (D), (F),and (H). Cell wall and PMB boundaries are indicated by red and yellowdashed lines, respectively.(A), (D), (F), and (H) Scale bar, 2 mm.(B), (C), (E), (G), (I), and (J) Scale bar, 200 nm.Note that Rab5a-mediated endosomes are observed as electron-densevesicleswith an irregular shape that is likely due to endosomal fusion ([A] to[E]) and because the aborted endosome vesicles are trapped within thePMBs in the rab5a mutant ([F] to [J]).

2576 The Plant Cell

plant-specific Rab5F1/ARA6. During rice growth and develop-ment, the loss of Rab5a is offset by Rab5c and possibly byRab5b and Rab5d. Although the other Rab5 isoforms can com-pensate for Rab5a, they are only able to partially fill this role asrab5a rice linesgrowslowerandflower later thannormal, likelydueto their significant lower expression compared with Rab5a(Supplemental Figure 5). At the grain filling stage where there ismassive protein transport from the ER via the Golgi to the storagevacuole, the reduced expression of these other Rab5 isoformsfails to maintain normal membrane trafficking—resulting in thesecretion of storage proteins, the formation of PMBs, and mis-localization of glutelin mRNAs.

Efforts to identify rice lines expressing defective NSF have notbeen productive, likely because of the importance of its ATPaseactivity in membrane fusion. Mutations in NSF were reported toinduce severe defect and cell lethality in several organisms(Boulianne and Trimble, 1995; Golby et al., 2001; Mohtashamiet al., 2001; Horsnell et al., 2002; Zhao et al., 2007). For example,Drosophila expresses two NSF isoforms, dNSF1 and dNSF2.dNSF1 is dominant in the adult central nervous system whiledNSF2 is broadly distributed at the larval/adult stages of de-velopment, respectively (Boulianne and Trimble, 1995). WhiledNSF1 null mutants perish as pharate adults, dNSF2 deletionmediates a recessive lethal phenotype, which is not even rescuedby the addition of a dNSF2 transgene (Golby et al., 2001; Moh-tashamietal., 2001). Inyeast, loss-of-functionmutationof theNSFgene, Sec18, resulted in a dominant lethal phenotype (Horsnellet al., 2002). These studies suggest that NSF mutations arepleiotropic and cause severe growth problems. In Arabidopsis,even a subtle mutation of NSF caused severe abnormal Golgimorphology (Tanabashi et al., 2018). Given that NSF is coded bya single gene copy in the rice genome, mutations that affect NSFactivity will likely confer a strong lethal phenotype in rice.

Although beyond the scope of this study, NSF mutations thataffectbinding toRBP-Pwouldbeaviableapproach toobtain furtherinsighton its role inmRNAtrafficking.Asdiscussedearlier, theNSF-N domain is likely responsible for its interactionwith the N terminusofRBP-P.Selectedresidues intheNSF-Nregioncanbereplacedbyaminoacids that alter chargeorconformationand then tested for itsprotein-interactive properties by Y2H analysis. Mutations in NSFthat abolish its interactionwith RBP-P but notwithRab5awould bepotential sites for genetic alteration by clustered regularly inter-spaced short palindromic repeats (CRISPR) technology and,thereby, disrupting glutelinmRNA transport butmaintaining normalfunction in membrane vesicle transport.Overall, this study provides evidence on how glutelin mRNPs are

able tohitchhikeon traffickingendosomes in riceendospermcellsbyexploiting the binding properties of RBPs and membrane traffickingfactors, NSF and Rab5a. These findings will provide the basis forfuture research on membrane trafficking-mediated mRNA transportand the unique functions of NSF and Rab5a in this cellular process.

METHODS

Plant Materials and Growth Conditions

The rice wild type (Oryza sativa ‘Japonica’ variety TC65) and rab5amutantline EM960 (Fukuda et al., 2011) were potted in Sunshine Brand #1 soil(Sungro Horticulture) and grown in walk-in growth chambers with a diurnalcycle of 12-h light/12-h dark at 27°C and a lighting intensity of 400 to700 mmol m22 s21 using a combination of metal halide and high-pressuresodium lamps.

Antibodies

Full-length cDNAs encoding RBP-P, RBP-L, NSF, SNAP, BiP, and GFPwerecloned intopET30a forHis-tagged fusionproteinexpression.TheHis-

Figure 8. Rab5a Mutation Leads to Mis-targeting of Glutelin mRNAs to PB-ER and the PMBs as Assessed by In Situ RT-PCR.

The PB-ER was stained by Rhodamine B dye (magenta), and glutelin mRNAs were labeled by in situ RT-PCR in the presence of Alexa-488-UTP (green). Inwild-type (WT) endospermcells (top), glutelinmRNAs are localized on the cisternal-ERadjacent to thePB-ER. In rab5amutant (bottom), glutelinmRNAs aremis-targeted to the PMBs (asterisk) and PB-ER (arrows). Fluorescence intensity graphs on the very right show the relative position of magenta and greenfluorescencesignals in the regions indicatedby rectangle in theMerge representations.X-andyaxes represent thefluorescence intensity andpositionof thesignals (pixels), respectively, evaluatedby the software FUJI (ImageJ).Note the extensive overlap in thedistributionsbetweenPB-ERandglutelinmRNAs inrab5a but not in wild type. Scale bar, 20 mm.

Glutelin mRNAs Are Transported on Endosomes 2577

tagged proteins were then purified and used to immunize New ZealandWhite rabbits for antibody production. Anti-Rab5a rabbit and mouse an-tibodies were generated from an earlier study by Fukuda et al. (2011). Forimmunofluorescence labeling, Alexa Fluor-488–labeled goat anti-mouseIgG antibody (cat. no. A32723; Invitrogen) and Alexa Fluor-594–labeledgoat anti-rabbit IgG antibody (cat. no. A32740; Invitrogen) were used assecondary antibodies. For the immunogold labeling, Electron Microscopy(EM)-grade, 10-nm–gold-conjugatedgoat anti-mouse IgG (cat. no.25,128;Electron Microscopy Science) and EM-grade 15-nm–gold-conjugatedgoat anti-rabbit IgG (cat. no. 25,112; Electron Microscopy Science) wereused as secondary antibodies.

IP Analysis

Most IP experiments were conducted as described by Doroshenk et al.(2014) except that incubations with antibody were conducted in thepresence or absence of 1 mM of ATP/GTP, 8 mM of MgCl2, or EDTA. Tomaximize capture of transient mRNP complexes on endosomes, de-velopingseedswere treatedwith1%(w/v)PFA.Detailedprocedures for IP-MS, the identification of NSF protein, and PFA fixation for the enhanced IPare described below.

IP-MS Analyses

Affinity-purified RBP-P or GFP antibodies were prepared using immobi-lized metal affinity chromatography based on irreversibly oxidized Co(III)-IDA resin as previously described byCrofts et al. (2010) and cross linked toProtein A/G agarose resin using the Pierce Crosslink ImmunoprecipitationKit according to themanufacturer’s instructions (ThermoFisher Scientific).Protein extraction and Co-IP experiments were performed at 4°C unlessindicated. Twogof dehulledmid-developingwild-type rice seedharvested12 to 14 d after flowering were frozen in liquid nitrogen and ground toapowder. Proteinswere extracted in 6mLof IPbuffer (20mMof Tris-HCl atpH 7.5, 0.15 M of NaCl, 1 mM of EDTA, and 0.5% v/v Nonidet P-40)containing 13 protease inhibitor cocktail and 0.53 phosphatase inhibitor(Sigma-Aldrich). The crude extract was clarified by twice centrifuging at12,000g for 10min. The resulting supernatant was gently rotated overnightwith agarose resin to eliminate nonspecific interactions. Seven-hundredmicroliters of the unbound protein fraction were added to columns con-tainingRBP-P orGFPantibodies cross linked toProtein A/G resin or nakedresin and incubated ;7 h with rotation. The columns were washed fivetimes with IP buffer and once with 13 conditioning buffer, and boundproteins were eluted with 50 mL of IgG elution buffer (Thermo FisherScientific). The antibody-conjugated Protein A/G resin columns were re-generated by washing with 13 Coupling Buffer (Thermo Fisher Scientific)followed by IP buffer. Fresh, precleared rice-seed extract was added andincubated overnight. The columns were washed and proteins eluted,as above.

Eluted proteins froma total of fiveCo-IP experiments using anti-RBP-P,anti-GFP antibodies, or naked resin were pooled, then precipitated withaddition of 100% (w/v) trichloroacetic acid (TCA) to a 20% (w/v) finalconcentration. The TCA precipitate was washed with acetone and thenresuspended in 30mL of SDS sample buffer containing 4Mof urea and 5%(v/v) b-mercaptoethanol. Protein samples were resolved on 10% SDS-PAGE gels and stained using a silver-nitrate–staining protocol (Chevalletet al., 2006). Because the protein profile of the minus antibody control IPlooked very similar to the GFP co-immunoprecipitate, proteins from onlythe GFP and RBP-P co-immunoprecipitates were analyzed by massspectrometry. Gel slices corresponding to similar positions within eachlane were excised, in-gel–trypsin digested, and subjected to liquidchromatography–tandemMS as previously described by Doroshenk et al.(2009). Proteins were identified by searching the O. sativa NationalCenter for Biotechnology Information non-redundant database (134,548

sequences) using the software Mascot (www.matrixscience.com) aspreviously described by Doroshenk et al. (2009).

IP with 1% PFA Fixation

Antibodies raised against RBP-L, RBP-P, NSF, or Rab5a were affinity-purified asmentioned above. Twenty microliters of resuspended protein AMagbeads slurry (25% [w/v] slurry; GenScript) in a 1.5-mL tube waswashed twice with 13 PBS and then incubated overnight at 4°S underrotation with 30 mg of affinity-purified antibody (diluted to 500 mL by 13PBS).Antibodieswerecross linked toprotein-AMagbeadsusing thePierceCrosslink Immunoprecipitation Kit (Thermo Fisher Scientific).

Dehulled developing rice seeds were fixed with 1% (w/v) PFA in themodified phosphate-buffered saline buffer (1.76mMof KH2PO4, 10mM ofNa2HPO4, 136 mM of NaCl, 2.6 mM of KCl, 5 mM of MgCl2, and 10% [v/v]glycerol) under vacuum for 30 min. The reaction was stopped by vac-uuming in quenching buffer (0.333 M of Tris and 10% [v/v] glycerol) for10min. Ten to 20 aleurone-layer peels collected from the fixed seeds wereground to a fine powder using an ice-cold mortar and pestle in 800 mL oflysis buffer (50mMof Tris-HCl at pH 7.5, 150mMof NaCl, 10mMof EDTA,0.5% [v/v] NP-40, 0.1% [v/v] Triton X-100, 13proteinase inhibitor cocktail,100mg/mLof cycloheximide, and 20units/mLofRiboLockRNase Inhibitor[Thermo Fisher Scientific]), and then centrifuged at 1,000 g for 5 min toremove starch followed by centrifugation at 12,000g for 10 min. The clearsupernatantwasadded to thepreparedMagbeads for incubationovernightat 4°Swith gentle rotation. After washing twice with lysis buffer, the boundfractions were eluted with an IgG elution buffer (Thermo Fisher Scientific)and neutralized by addition of 1:10 volume of 1Mof Tris-HCl at pH 8.8. Theneutralized elution samples were analyzed by immunoblotting.

In Situ RT-PCR

In situ RT-PCR on developing rice seed sections was performed as pre-viously described byWashida et al. (2009). The specific primers Glutelin-F59-CCCTCAAGCATACAGGCGTG-39 andGlutelin-R 59-CGCTCTCTTGATTGCACTTGTCC-39 were used in the PCR to amplify glutelin RNAs.

Construction of Vectors

Gene sequences of RBP-P, NSF, and their truncated forms were clonedinto pGAD T7 and pGBK T7 vectors and used as preys and baits, re-spectively, in Y2H analyses for detection of protein–protein interaction.BiFC vectors of pSAT1-nEYFP-C1 and pSAT1-cEYFP-C1-B for N- andC-terminal EYFP fusion, respectively, were obtained from the ArabidopsisBiological Resource Center (https://www.arabidopsis.org). The cDNAsequences of RBP-P, RBP-L, NSF, Rab5a, and its mutant forms (Ra-b5aQ70L and Rab5aG45D) were cloned into pSAT1-nEYFP-C1 or pSAT1-cEYFP-C1-B vectors for BiFC analysis. GFP- or RFP-fusion vector drivenby double 35S promoters was constructed by replacing the N-terminalEYFP in pSAT1-nEYFP-C1 with GFP cDNA or RFP cDNA sequences. ThecDNA sequences of NSF and Rab5awere then cloned into a GFP- or RFP-fusion vector to obtain RFP protein fusions.

RNA-IP, Y2H, and BiFC Assays

Experimental procedures for these studies were performed as previouslydescribed by Doroshenk et al. (2014) and Tian et al. (2018, 2019a). Briefly,for RNA-IP analysis, developing rice seeds, collected 10 to 14 d afterflowering, were subjected to 1% (w/v) PFA fixation and extracts preparedand used for IP asmentioned above. The elution samples obtained from IPwere incubated at 70°C for 45 min to reverse the RNA–protein cross linksfollowedbyRNAextractionusingTRIzol (Invitrogen), cDNAsynthesisusingM-MLV reverse transcriptase (Promega), and PCR with 20 to 25 cycles of

2578 The Plant Cell

amplification using glutelin- and ACTIN-specific primers (Glutelin-F andGlutelin-Rasmentionedabove for in situRT-PCR,Actin-F59-TCCATCTTGGCATCTCTCAG-39, and Actin-R 59-GTACCCGCATCAGGCATCT-39).The antibodies used in the RNA-IP were affinity-purified as aforemen-tioned. In Y2H analysis, synthetic dropout (SD) growth media without Leuand Trp (SD/-Leu/-Trp) was used to screen positive transformants andselection media without Leu, Trp, His, and Ade (SD/-Leu/-Trp/-His/-Ademedium) supplementedwith 3mMof 3-amintriazole (3-AT; SD/-Leu/-Trp/-His/-Adenine/13-AT) or 40 mg/L of X-a-Gal to verify protein–proteininteraction.

BY-2 suspension cells were used for BiFC analyses. In brief, BY-2 cellswere treatedwithcellwall digestionbuffer (1%[w/v] cellulase [OnozukaRS;PhytoTechnology Laboratories], 0.05% [w/v] pectolyase [Seishin Phar-maceutical], 0.2% [w/v] Driselase [Sigma-Aldrich], 20mMof KCl, 10mMofCaCl2, 20mMof2-[N-morpholino]ethanesulfonic acidhydrate, and0.5MofSuc at pH 5.7) at room temperature for 3 h. After washing withW5 solution(154mMofNaCl, 125mMofCaCl2, 5mMofKCl, and5mMofGlc at pH5.8to 6.0), the BY-2 protoplasts were subjected to PEG-mediated trans-formation with the vectors of pSAT1-nEYFP-C1 and pSAT1-cEYFP-C1-Bas previously described by Tian et al. (2018). After culture at 26°C for 16 h,the BiFC fluorescence images were observed using a model no. SP-8confocal microscope (Leica). Negative controls using empty vectors werealso examined to check the reliability of the transformation procedure. Thelocalization pattern of target proteins or complexes was determined byexaminingat leastfivedifferentprotoplastcells. Toconfirmthe involvementof Rab5a and the corresponding complexes in endocytic pathway, pro-toplast incubationwas treatedwith FM4-64 endocytic tracer (Invitrogen) ata final concentration of 10 mM for 15 to 30 min before observation.

Microscopy

Light microscopy was performed on 10-mm–thick sections of developingrice seed samples embedded in LR-white resin. The sections were posi-tioned on X-tra slides (Leica), stained by 1% Toluidine blue and observedusing a model no. BH-2 Light microscope (Olympus). Co-localization testof RBP-P, RBP-L, and NSF with Rab5a in rice endosperm cells wasperformed through double-immunolabeling using the rabbit anti-RBP-P,RBP-L, and NSF antibodies and mouse anti-Rab5a antibodies (see An-tibodies) on 1-mm–thick LR-white sections as described in Fukuda et al.(2011), and observed under an Epi-Fluorescent Microscope (Leitz) witha model no. DFC425C camera (Leica). The fluorescence intensity of greenand red signalswas analyzed using the plot profile tool in the software FUJI(ImageJ). TEM analysis was performed as described in Tian et al. (2018).

Accession Numbers

Sequence data from this article can be found in the GenBank/EuropeanMolecular Biology Laboratory data libraries under National Center forBiotechnology Information accession numbers shown in the legends ofSupplemental Figures 3 and 4.

Supplemental Data

Supplemental Figure 1. Sequence information of Rab5a in rice.

Supplemental Figure 2. Possible binding domains of RBP-P with NSFrevealed by Y2H analysis.

Supplemental Figure 3. Protein sequence alignment of NSF.

Supplemental Figure 4. Protein sequence alignment of Rab5isoforms.

Supplemental Figure 5. Expression profile of Rab5 isoforms in riceplants.

ACKNOWLEDGMENTS

We thank Ai Nagamine for her help in the construction of BiFC plasmids,and the Franceschi Microscopy and Imaging Center at Washington StateUniversity for technical support. This work was supported by the NationalScience Foundation (grants MCB-1444610 and IOS-1701061), the USDANational Institute of Food and Agriculture (Hatch umbrella project899–1015621 and project WNP00119), and the Japan Society for thePromotion of Science (to M.F. and T.K.).

AUTHOR CONTRIBUTIONS

L.T. designed the study; K.A.D. identified NSF as interacting partner ofRBP-P through IP-MS; L.T. discovered interaction of RBPs and NSF withRab5a and conducted BiFC, RNA-IP, Y2H, light microscopy, and TEManalyses; L.T. and L.Z. conducted Co-IP analysis; M.F. conducted immu-nofluorescence microscopy; L.T. constructed vectors; L.T. and H.W.conducted in situ RT-PCR; T.K. provided rab5a mutant; T.O. supervisedthe project; L.T. and T.O. wrote the article.

Received February 27, 2020; revised May 12, 2020; accepted May 24,2020; published May 29, 2020.

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Glutelin mRNAs Are Transported on Endosomes 2581

DOI 10.1105/tpc.20.00111; originally published online May 29, 2020; 2020;32;2566-2581Plant Cell

and Thomas OkitaLi Tian, Kelly A. Doroshenk, Laining Zhang, Masako Fukuda, Haruhiko Washida, Toshihiro Kumamaru

Glutelin mRNAs in Rice EndospermZipcode RNA-Binding Proteins and Membrane Trafficking Proteins Cooperate to Transport

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