1

RESEARCH ARTICLE 1 2

Zipcode RNA-binding Proteins and Membrane Trafficking Proteins 3

Cooperate to Transport Glutelin mRNAs in Rice Endosperm 4

5 6

Li Tian1*, Kelly A. Doroshenk1, Laining Zhang1, Masako Fukuda1,2, Haruhiko Washida1,3, 7 Toshihiro Kumamaru2, Thomas Okita1* 8

9 1 Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, 10 USA 11 2 Faculty of Agriculture, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395, Japan 12 3 Current address: Organic Nico Co., Ltd, Kyodai Katsura Venture Plaza, 1–36, Goryo-Ohara, 13 Nishikyo-ku, Kyoto 615–8245, Japan 14 * Correspondence should be addressed to li.tian@wsu.edu or okita@wsu.edu.15

16 Short Title: Glutelin mRNAs are transported on endosomes 17

18 One-sentence summary: Glutelin mRNAs are transported on endosomes through the direct 19 interactions of two RNA-binding proteins with two membrane trafficking factors. 20

21 The author responsible for distribution of materials integral to the findings presented in this 22 article in accordance with the policy described in the Instructions for Authors 23 (www.plantcell.org) is Thomas Okita (okita@wsu.edu) 24

25 26

ABSTRACT 27 28

In rice (Oryza sativa) endosperm cells, mRNAs encoding glutelin and prolamine are 29

translated on distinct cortical-endoplasmic reticulum (ER) subdomains (the cisternal-ER 30

and protein body (PB)-ER), a process that facilitates targeting of their proteins to 31

different endomembrane compartments. Although the cis- and trans-factors responsible 32

for mRNA localization have been defined over the years, how these mRNAs are 33

transported to the cortical ER has yet to be resolved. Here, we show that the two 34

interacting glutelin zipcode RNA-binding proteins (RBPs), RBP-P and RBP-L, form a 35

quaternary complex with the membrane fusion factors N-ethylmaleimide-sensitive factor 36

(NSF) and the small GTPase Rab5a, enabling mRNA transport on endosomes. Direct 37

interaction of RBP-L with Rab5a, between NSF and RBP-P, and between NSF and 38

Rab5a were established. Biochemical and microscopic analyses confirmed the co-39

localization of these RBPs with NSF on Rab5a-positive endosomes that carry glutelin 40

mRNAs. Analysis of a loss-of-function rab5a mutant showed that glutelin mRNA and 41

Plant Cell Advance Publication. Published on May 29, 2020, doi:10.1105/tpc.20.00111

©2020 American Society of Plant Biologists. All Rights Reserved

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the quaternary complex were mis-targeted to the extracellular paramural body structure 42

formed by aborted endosomal trafficking, further confirming the involvement of 43

endosomal trafficking in glutelin mRNA transport. Overall, these findings demonstrate 44

that mRNA localization in plants co-opts membrane trafficking via the acquisition of new 45

functional binding properties between RBPs and two essential membrane trafficking 46

factors, thus defining an endosomal anchoring mechanism in mRNA localization. 47

48

INTRODUCTION 49 50

Localization of mRNAs is a universal mechanism to efficiently drive protein 51

targeting in eukaryotes and prokaryotes. The targeting of mRNAs facilitates the 52

accumulation of the locally translated proteins to specific cellular compartments and, 53

hence, is an essential mechanism in establishing cell polarity, patterning, and fate 54

determination as well as protein sorting (Herbert and Costa, 2019; Hughes and Simmonds, 55

2019; Tian et al., 2019b, 2020). 56

mRNA localization occurs as a multi-step process. After transcription, cis-acting 57

elements (RNA zipcodes) are recognized and bound by trans-acting factors, mainly 58

RNA-binding proteins (RBPs) to form a primary mRNA–nucleoprotein (mRNP) 59

complex. After export to the cytoplasm, the mRNP complex undergoes extensive 60

remodeling with recruitment of new factors and detachment of others enabling 61

cytoskeletal-based transport to the destination site (Blower, 2013; Weis et al., 2013; Tian 62

and Okita, 2014). 63

Although extensive knowledge on mRNA localization has been acquired by studies in 64

Drosophila, yeast and mammalian cells, only a few examples have emerged from higher 65

plants. The best defined model in plants is storage protein mRNA localization in 66

developing rice (Oryza sativa) endosperm cells, where mRNAs encoding glutelin and 67

prolamine are recognized by zipcode RBPs and transported to two distinct cortical 68

endoplasmic reticulum (ER) subdomains, the cisternal-ER, and protein body-ER (PB-69

ER), respectively (Chou et al., 2019; Tian et al., 2019b). Translation of prolamine 70

mRNAs on the PB-ER results in the assembly of prolamine intracisternal granules that 71

form an ER-derived protein body I (PB-I), while glutelin precursors are exported to the 72

Golgi and then transported to protein storage vacuoles (PSVs) for processing and storage 73

3

(Chou et al., 2019; Tian et al., 2019b). Although several cytoskeleton-associated RBPs 74

required for mRNA localization have been identified (Doroshenk et al., 2009, 2012), 75

information on how these mRNAs are transported to distinct ER subdomains remains 76

elusive. 77

Emerging evidence from fungal model systems reveals the intimate link of mRNA 78

transport with membrane trafficking (Schmid et al., 2006; Jansen et al., 2014; Haag et al., 79

2015; Niessing et al., 2018). Several mRNAs from Saccharomyces cerevisiae, Candida 80

albicans, and Ustilago maydis are co-transported with mobile ER or shuttling endosomes 81

(Schmid et al., 2006; Jansen et al., 2014; Haag et al., 2015; Pohlmann et al., 2015; 82

Niessing et al., 2018). ASH1 as well as other mRNAs are co-transported on tubular ER 83

that moves to the emerging bud or daughter cell in Saccharomyces cerevisiae. This 84

process is mediated by the RBPs She2p and She3p, with She2p having membrane 85

binding properties and She3p serving as an adaptor protein linking the mRNP-cER to 86

Myo4P protein (Schmid et al., 2006; Niessing et al., 2018). The cdc3 mRNA is 87

transported on shuttling endosomes in the smut fungus, Ustilago maydis, a process 88

requiring localization of the RBP Rrm4 on the endosomes and the interaction of a 89

membrane-associated linker protein Upa1 with Rrm 4 (Pohlmann et al., 2015; Niessing et 90

al., 2018). Specific adaptor proteins appear to be needed to hitch mRNPs on endosomes 91

for active transport over long distance. More recently, neuronal RNA granules have been 92

shown to hitchhike on moving lysosomes using annexin11 as a tether (Liao et al., 2019). 93

Although co-transport of mRNAs with membranous compartments was proposed to be a 94

common mechanism in higher eukaryotes (Jansen et al., 2014), whether the mechanism is 95

utilized by higher plants remains to be determined. 96

Previous studies suggested that endocytosis and membrane trafficking likely play a 97

role in mRNA localization in plants. For example the loss-of-function of the small 98

GTPase Rab5a and its cognate guanine nucleotide exchange factor (GEF) resulted in 99

defects in endocytosis and membrane trafficking and the mis-targeting of glutelin 100

proteins to the prolamine containing PB-I as well as to the extracellular paramural body 101

(PMB) in rice endosperm cells (Fukuda et al., 2011; Wen et al., 2015). As storage protein 102

targeting is regulated by their mRNA localization in rice endosperm cells, the mis-103

targeting of glutelin proteins in the mutant suggests a relationship between endosomal 104

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transport and glutelin mRNA localization in rice. The extracellular distribution of glutelin 105

mRNAs within PMBs from a mutant expressing a defective GEF (Yang et al., 2018) 106

further supports the possible involvement of endosomal trafficking in glutelin mRNA 107

transport. However, direct evidence depicting the co-transport of glutelin mRNAs with 108

shuttling endosomes and how endosomal trafficking are engaged in glutelin mRNA 109

localization have yet to be established. Such mis-targeting of glutelin mRNAs in rice 110

lines expressing mutant Rab5a and GEF may simply be a consequence of pleiotropy. 111

Recent studies (Tian et al., 2018; Tian et al., 2019a) identified two RNA-binding 112

proteins, RBP-P and RBP-L, which contain two and three RNA recognition motif (RRM) 113

domains, respectively. These RBPs specifically bind to the glutelin zipcode mRNA 114

sequences and regulate glutelin mRNA localization. In this study, using these two 115

glutelin zipcode RBPs as entry points, we identified their interacting partners, N-116

ethylmaleimide-sensitive factor (NSF) and the small GTPase Rab5a, which participate in 117

endosomal membrane trafficking. The four proteins may form a quaternary complex 118

carrying glutelin mRNAs for active transport on endosomes to the cortical ER membrane. 119

The identification of these key linker proteins that enable endosome-mediated mRNA 120

transport in rice endosperm cells provides new insights on how mRNAs can be 121

distributed to specific locations in eukaryotes. 122

123

RESULTS 124

RBP-P interacts with membrane fusion factor NSF 125

Previous studies (Doroshenk et al., 2014; Tian et al., 2018; Tian et al., 2019a) 126

established that the glutelin zipcode RNA-binding proteins RBP-P and RBP-L, which 127

interact with each other, are essential for localization of glutelin mRNAs to the cisternal-128

ER, as mutations in these RBPs led to the mis-localization of glutelin mRNAs. To obtain 129

additional insight into how glutelin mRNAs are transported to the ER, we performed 130

immunoprecipitation-mass spectrometry (IP-MS) studies using affinity-purified anti-131

RBP-P antibody (Figure 1A). Tandem mass spectrometry of a major polypeptide band 132

observed in the IP generated with anti-RBP-P but not with control anti-GFP identified a 133

major interacting protein as N-ethylmaleimide sensitive fusion protein (NSF). 134

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To determine whether RBP-P interacts directly with NSF, yeast two-hybrid studies 135

were carried out. In such two-hybrid screening, the two proteins of interest were fused to 136

activating domain (AD) and DNA-binding domain (BD) domain of yeast GAL4 137

transcription factor, respectively. Interaction of these proteins restore GAL4 that, in turn, 138

activate the transcription of histidine and adenine reporter genes. While no background 139

interaction of RBP-P and NSF with complementary empty vector was observed, yeast 140

cells carrying both RBP-P and NSF genes activated the reporter genes and survived in 141

selection medium lacking histidine and adenine (Figure 1B). These results indicate that 142

RBP-P and NSF interact under stringent binding conditions. 143

To further substantiate the interaction between RBP-P and NSF, bimolecular 144

fluorescence complementation (BiFC) analyses using tobacco (Nicotiana tabacum) BY-2 145

cells was performed. In addition to supporting an interaction between protein pairs, BiFC 146

also provides intracellular information on where this interaction occurs. RBP-P and NSF 147

were fused to two complementary non-fluorescent fragments of enhanced yellow 148

fluorescent protein (EYFP), nEYFP and cEYFP, respectively, and co-transformed into 149

live BY-2 cells. If the two proteins interact, the two EYFP fragments are brought in close 150

proximity to reform the native protein structure enabling emission of yellow fluorescence 151

and, in turn, direct visualization of the protein complex in live cells (Kerppola, 2006; 152

Miller et al., 2015). EYFP fragments lacking one of the putative protein partners were 153

used as negative control. As shown in Figures 2A-2C, while no interaction was detected 154

between RBP-P or NSF with the empty EYFP fragment controls, cells expressing both 155

RBP-P and NSF fused to EYFP fragments emitted bright yellow fluorescence, indicating 156

that RBP-P interacted with NSF. Closer examination reveals that RBP-P/NSF complexes 157

were distributed to the cytoplasm as intensely bright clusters together with more loosely 158

diffuse structures (Figure 2C). 159

NSF is a soluble hexameric ATPase commonly found in the cytoplasm of eukaryote 160

cells (Mastick and Falick, 1997; Zhao et al., 2007; Zhao et al., 2010; Guo et al., 2017), 161

which predominantly plays a major role as a chaperone in intracellular membrane fusion 162

events. Through its interaction with the adaptor protein, soluble NSF attachment protein 163

(SNAP), NSF binds to SNARE (soluble NSF activating protein receptor) complexes and 164

utilizes the energy of Mg2+

-dependent ATP hydrolysis to disassemble the SNARE 165

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protein complex, and thus facilitating the recycling of SNARE proteins for further cycles 166

of membrane fusion (Zhao et al., 2007; Chang et al., 2012; Ryu et al., 2015). During this 167

process, SNAP serves as chaperone by stimulating the ATPase activity of NSF and 168

dissociating from NSF after ATP hydrolysis (Zhao et al., 2007). This transient interaction 169

between NSF and SNAP is only detected under conditions where a nonhydrolyzable ATP 170

is used as a substrate (Hanson et al., 1995; Barnard et al., 1997; Chang et al., 2012). 171

Alternatively, the addition of EDTA to chelate Mg2+

and thus inhibit ATPase activity has 172

been used to detect the transient interaction between NSF and SNAP (Hanson et al., 1995; 173

Barnard et al., 1997; Chang et al., 2012; Li et al., 2018). 174

Based on the established properties of NSF, we performed Co-IP experiments to 175

assess the formation of RBP-P/NSF complexes in rice endosperm cells. Rice seed lysates 176

supplemented with 1 mM ATP and 8 mM MgCl2 or EDTA were incubated with affinity 177

purified anti-RBP-P and NSF antibodies (Figure 2D) or anti-GFP antibody, the latter 178

used as a negative control. No proteins were captured by the control GFP antibody, 179

revealing the reliability of the IP experiments. Interestingly, RBP-P and NSF were co-180

precipitated in the presence of Mg2+

-ATP but not when EDTA was added to the seed 181

lysates (Figure 2D). This result indicates that formation of the RBP-P/NSF complex in 182

rice endosperm cells requires Mg2+

-ATP, a condition distinct from those complexes 183

involving the stable interaction between NSF and SNAP (Hanson et al., 1995; Barnard et 184

al., 1997; Chang et al., 2012; Li et al., 2018). The differences in binding properties, as 185

well as the absence of SNAP in RBP-P IPs (Figure 2D), indicate that the events of 186

membrane fusion requiring NSF-SNAP are not required for formation of the RBP-P/NSF 187

complex. On the other hand, the interaction between NSF with RBPs suggests that NSF 188

may function in mRNA metabolism by its interaction with RBP-P. 189

190

RBP-P indirectly interacts with Rab5a through NSF 191

The interaction of the glutelin zipcode trans-factor RBP-P with NSF supports a close 192

relationship between glutelin mRNA transport with membrane trafficking. Previous 193

studies demonstrated that Rab5a, an evolutionarily conserved key GTPase involved in the 194

biogenesis of early endosomes and membrane trafficking in the cytoplasm (Woodman, 195

2000; Saito and Ueda, 2009; Ito et al., 2018), is also required for glutelin mRNA 196

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localization (Doroshenk et al., 2010) and vesicular membrane transport between the 197

Golgi and protein storage vacuole in rice endosperm cells (Wang et al., 2010; Fukuda et 198

al., 2011). Therefore, we investigated whether the abovementioned RBP-P/NSF complex 199

is associated with Rab5a to regulate the endosomal transport of mRNAs. 200

We first performed a transient expression study to confirm the association of rice 201

Rab5a (Figures 2E-2F, Supplemental Figure 1) with endosomes. FM4-64 is a lipophilic 202

dye that initially labels the plasma membrane and, subsequently, internalizes with 203

membrane vesicles transported along the endocytic pathway (Vida and Emr, 1995; Ueda 204

et al., 2001). In this experiment, BY-2 protoplasts expressing GFP-Rab5a were treated 205

with FM4-64 for 15 min before observation. As shown in Figure 2F, red fluorescence 206

derived from FM4-64 was evident on the plasma membrane and in internalized 207

endosomes. GFP-Rab5a displayed a similar distribution pattern and co-localized with the 208

internal FM4-64 labeled vesicles (Figure 2F), an observation confirming that Rab5a is 209

associated with endosomes. 210

We then studied the relationship of Rab5a with the abovementioned RBP-P/NSF 211

complex. Co-IP studies were carried out using affinity-purified antibodies to Rab5a, 212

RBP-P and NSF. All three proteins were found in IPs generated by anti-Rab5a, anti-RBP-213

P and anti-NSF (Figure 2D), suggesting that they form a multi-protein complex in rice 214

endosperm cells. Protein complex formation apparently required active ATPase 215

hydrolysis as all three protein interactions could only be simultaneously detected in the 216

presence of Mg2+

and ATP and not when EDTA and ATP were included (Figure 2D). 217

To further characterize this protein interactome and the formation of their complexes, 218

BiFC (Figures 2G-2J) studies were performed. Interaction between NSF and Rab5a was 219

observed in the tested cells, and their complexes existed in the cytoplasm. By contrast, 220

Rab5a does not interact with RBP-P (Figure 2J) and, therefore, the association of RBP-P 221

with Rab5a in the Co-IPs (Figure 2D) is mediated through the interaction between NSF 222

and Rab5a. 223

Rab GTPases are highly conserved small proteins and share two conserved regions 224

that are required for conformational switching between active and inactive states of GTP 225

hydrolysis (Figure 2E, Supplemental Figure 1). The switch regions are disordered in an 226

inactive conformation and adopt a well-defined conformation in the active stage 227

8

(Stenmark and Olkkonen, 2001). Amino acid substitutions within the switch regions 228

affects their conformational switching. While the G45D mutation in switch 1 inhibits the 229

conformation switching to an active GTPase stage and restricts Rab5a in a GDP-bound 230

form, a Q70L mutation in switch 2 locks it in a constitutively active GTP bound form 231

(Lee et al., 2009). Only the GTP-bound form of Rab5 are membrane-bound during 232

endosomal trafficking (Yuan and Song, 2020). 233

To investigate whether the NSF-Rab5a complex is associated with active endosomes, 234

the interaction of NSF with the two structural forms of Rab5a containing G45D or Q70L, 235

was analyzed by BiFC (Figures 2K-2L). Although NSF directly interacts with both GDP- 236

(Rab5aG45D

) and GTP- (Rab5aQ70L

) forms of Rab5a, their complexes are observed as two 237

distinct populations. When bound to the GTP-fixed Rab5a (Q70L) form (Figure 2L), the 238

NSF complex was distributed as endosomal punctate structures. By contrast, the NSF 239

complex constituted by GDP-fixed Rab5a (G45D) showed a diffuse distribution 240

throughout the cytoplasm (Figure 2K). These results are consistent with the view that the 241

GTP-fixed Rab5a (Q70L) form is membrane bound while the GDP-fixed Rab5a (G45D) 242

is mainly soluble. 243

The direct interaction between NSF and Rab5a provides an indirect link to RBP-P 244

and, in turn, RNA localization. To confirm the association of the RBP-P/NSF complex to 245

Rab5a-positive endosomes, we performed a three-way co-localization test consisting of 246

RBP-P and NSF as BiFC interacting partners in BY-2 cells expressing Rab5a tagged with 247

the red fluorescence protein (RFP) (Figures 2M-2N). Florescence analysis of BY-2 cells 248

showed that RFP-Rab5aWT

was distributed as diffuse signals throughout the cytoplasm 249

and as punctate structures (endosomes) near the plasma membrane (Figure 2M, middle 250

panel). By contrast, RFP-Rab5aQ70L

was present only as punctate structures (Figure 2N, 251

middle panel) indicating that the activated GTPase form of Rab5a is associated with 252

endosomes. The fluorescence distribution pattern seen for RFP-Rab5aWT

is consistent 253

with membrane-free Rab5a being distributed in the cytoplasm and active Rab5a 254

associated with endosomes (Yuan and Song, 2020). 255

As earlier seen in Fig. 2C, BiFC-linked RBP-P/NSF complexes are distributed as 256

fluorescent punctate structures in BY-2 cells (Figure 2M and N). These RBP-P/NSF 257

complexes co-localized with RFP-Rab5a-associated endosomes in the cytoplasm, 258

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especially with membrane-associated GTP-fixed (Q70L) Rab5a (Figures 2M-2N). To 259

further confirm that the RBP-P/NSF complexes are associated with endosomes, we used 260

FM4-64, which specifically labels these small membrane compartments (Figure 2O). The 261

fluorescent signals from the RBP-P/NSF complexes co-localized with endosomal 262

compartments labeled by FM4-64, providing direct evidence that RBP-P/NSF complexes 263

co-localize with endosomes. Consistent with the abovementioned Co-IP analysis (Figure 264

2D) where RBP-P, NSF and Rab5a may co-assemble into a multi-protein complex in rice 265

endosperm cells, the BiFC results suggest that RBP-P is associated with endosomes 266

through a NSF-mediated interaction with Rab5a. 267

It should be noted that not all RBP-P/NSF complexes co-localized with Rab5a-linked 268

endosomes (Figures 2M-2N, open arrowheads). Similarly, the location of several Rab5a-269

active endosomes (Figures 2M-2N, open arrowheads) do not coincide with RBP-P/NSF 270

complexes. The lack of overlap in the distribution of a population of RBP-P/NSF 271

complexes and Rab5 endosomes suggests the multiple roles of these proteins in RNA 272

localization and membrane trafficking. 273

274

RBP-L is involved in the RBP-P/NSF/Rab5a complex 275

We had previously demonstrated that RBP-P co-assembles with RBP-L to form a 276

protein complex that is essential for storage protein mRNA localization (Doroshenk et al., 277

2014; Tian et al., 2018; Tian et al., 2019a). To determine whether RBP-L is also 278

involved in interacting with NSF or Rab5a, BiFC studies were conducted (Figures 3A-F). 279

Such analysis showed that RBP-L interacts directly with Rab5a but not with NSF 280

(Figures 3A-3C). The RBP-L/Rab5aWT

complex is observed as two distinct populations. 281

One population is distributed as endosomal punctate structures with the bulk located 282

close to the plasma membrane. The co-localization of RBP-L/ Rab5aWT

with endosomes 283

is supported by their close association with FM4-64 labeled endosomal compartments 284

(Figure 3D). A second population of RBP-L/ Rab5aWT

is viewed as a diffuse cloud 285

around the nucleus. Similar to the RBP-P/NSF/Rab5a complex, the interaction of RBP-L 286

with Rab5a was not dependent on the functional state of Rab5a as it interacts with both 287

the GTP-bound Rab5aQ70L

and GDP-bound Rab5aG45D

(Figures 3E and 3F), although the 288

distribution patterns are distinct. RBP-L/ Rab5aG45D

is distributed mainly as a diffuse 289

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cloud around the nucleus and near the plasma membrane while RBP-L/ Rab5aQ70L

are 290

observed predominantly as discrete endosomal punctate structures. 291

To further determine whether RBP-L is associated with the RBP-P/NSF/Rab5a 292

complex, we performed Co-IP with paraformaldehyde-fixed seed extracts to optimize the 293

capture of any potential dynamic endosome-associated complexes formed in vivo. Such 294

results showed that, irrespective of the antibody used in the initial immunoprecipitation, 295

RBP-P, RBP-L, NSF and Rab5a were detected in IPs generated by all four antibodies 296

(Figure 3G). By contrast, SNAP is only present in IPs obtained with anti-NSF and anti-297

Rab5 but not in IPs generated by antibodies to RBP-P and RBP-L (Figure 3G). These 298

results are consistent with the Co-IP results depicted in Figure 2D where SNAP is present 299

in IPs generated with antibodies to NSF and Rab5a in the presence of EDTA but not in 300

the presence of MgCl2. Overall, these results are consistent with the view that NSF-301

Rab5a complexes exist as two separate populations. One NSF-Rab5a population together 302

with SNAP participates in Rab5-mediated endosomal fusion (Woodman, 2000; Zhao et 303

al., 2007), where NSF and SNAP disassemble the SNARE complex formed by Rab5-304

mediated membrane fusion. A second NSF-Rab5a complex contains RBP-P and RBP-L 305

and is independent of SNAP. 306

To further address whether RBP-P and RBP-L are attached to endosomes together, 307

sequential IPs were performed (Figure 3H). In this analysis, the RBP-P/NSF/Rab5a 308

complexes precipitated by anti-RBP-P antibodies were next subjected to an additional IP 309

using anti-RBP-L. All four proteins were detected in the second IP generated by anti-310

RBP-L, suggesting that RBP-P, RBP-L, NSF and Rab5 co-assemble to form a quaternary 311

protein complex. 312

313

The quaternary complex attaches to active endosomes carries glutelin mRNAs 314

To investigate whether the quaternary complex binds glutelin mRNA, we performed 315

RNA-IP analysis to detect the in vivo association of the complex with glutelin mRNAs 316

(Figure 3I). In this analysis, antibodies to RBP-P, RBP-L, NSF and Rab5a were utilized 317

to capture the associated RNA-protein complexes, and the RNA was subsequently 318

isolated from the IP fractions and subjected to RT-PCR using specific primers to amplify 319

glutelin transcripts. Compared with the negative empty-resin control and anti-GFP 320

11

antibody, glutelin mRNAs were highly enriched in IPs generated by all four antibodies 321

(Figure 3I). The mRNA amount associated with NSF and Rab5a was lower than that of 322

RBP-P and RBP-L, a result consistent with the roles of these proteins in membrane 323

fusion events. Overall, these results support the view that the quaternary complex 324

contains glutelin mRNAs. 325

Based on these results, a working model of cytosolic glutelin mRNA transport is 326

proposed in Figure 4. The mRNP complex containing glutelin mRNA, RBP-P and RBP-L 327

is bound to Rab5a-associated endosomes through a 4-way interactome, i.e. the direct 328

interaction of RBP-P with RBP-L, of RBP-L with Rab5a, of NSF with RBP-P, and of 329

Rab5a with NSF. The GTP-bound Rab5a is associated with endosomes (Yuan and Song, 330

2020) as suggested by distribution of this activated Rab5a form as endosomal punctate 331

structures (Figures 2N and 3F). 332

333

Loss-of-function of Rab5a results in mis-targeting of glutelin mRNAs 334

To provide evidence in support of this model, we analyzed a rab5a mutant EM960 335

(Fukuda et al., 2011) expressing a GDP-fixed (G45D) Rab5a (Figure 5A). Similar to the 336

phenotype shown in the EM956 mutant lacking Rab5a (Fukuda et al., 2011) or a mutant 337

line expressing a defective Rab5a effector GEF (Wen et al., 2015), normal endosomal 338

trafficking is disrupted in the endosperm cells of GDP-fixed rab5a mutant and leads to 339

the formation of PMBs (Figures 5B-5C), an aborted endosome complex containing mis-340

sorted endomembrane proteins. These extracellular PMBs, which contain numerous 341

electron-dense vesicles, are located in the space between the invaginating plasma 342

membrane and the cell wall in the mutant endosperm cells (Figures 5B-5C). 343

To investigate the co-localization of RBP-P, RBP-L, and NSF with Rab5a and the 344

subcellular localization of their complex in rice endosperm cells, we performed double 345

immuno-fluorescence labeling on thin sections of rice developing seeds using antibodies 346

raised against each of the four proteins. Although the bulk of these proteins were 347

evidently independent of Rab5, there was ample evidence for co-localization of RBP-P, 348

RBP-L, and NSF with Rab5a. The co-localization of these proteins with Rab5a was 349

apparent as punctate structures in the cytoplasm, particularly in the cortical region 350

underneath the plasma membrane (Figures 6A, 6C and 6E), an intracellular location 351

12

enriched in Rab5a-mediated endosome activity (Chavrier et al., 1990; Fischer von 352

Mollard et al., 1994). To directly assess the co-localization of these proteins, the 353

fluorescence intensity profiles of these proteins were quantified along a specific linear 354

distance (Figure 6, right panels). The fluorescence signals for the proteins examined 355

overlapped substantially indicating that RBP-P, RBP-L and NSF co-localized to Rab5a-356

labeled endosomal compartments in rice endosperm cells. The independent distribution 357

of RBP-P, RBP-L and NSF with Rab5a was also evident in the BiFC/RFP double 358

labeling (Figures 2M-N), which is indicative of their roles in other cellular processes. 359

This view is also supported by the Co-IP results (Figures 2F, 3H) where IPs by antibodies 360

to RBP-P, RBP-L and NSF contained only a small proportion of the total Rab5a amounts. 361

The RBPs retain their co-localization with Rab5a in the rab5a mutant (Figures 6B 362

and 6D), an expected observation as RBP-L as well as NSF interact with both the GDP- 363

and GTP-fixed Rab5a forms (Figures 2K-2L, Figures 3E-3F). These protein complexes 364

exist as punctate structures within the PMBs in the rab5a mutant, (Figures 6B, 6D and 365

6F). Hence, disruption of membrane trafficking in the rab5a mutant displaces not only 366

endosomal proteins to the extracellular PMBs but also RBP-mRNA complexes. 367

Transmission electron microscopy (TEM) analysis further confirmed the co-368

localization of RBP-P and RBP-L with Rab5a on endosomes in wildtype and aborted 369

endosome vesicles within PMBs in rab5a mutant (Figure 7). In wild type, Rab5a-370

mediated endosomes were observed as electron-dense vesicles with an irregular shape 371

likely due to endosomal fusion (Figures 7A-7E). Co-localization of RBP-P and RBP-L 372

with Rab5a was observed on those endosomes, further suggesting that mRNP complexes, 373

carrying glutelin mRNAs bound by RBP-P and RBP-L, are transported on endosomes. In 374

the rab5a mutant line, normal endosomal trafficking is disrupted resulting in the 375

displacement of mRNA-associated endosomes to the extracellular PMBs (Figures 7F-7J). 376

To further address whether glutelin mRNAs are associated with the Rab5a-mediated 377

endosomes, in situ reverse transcription (RT)-PCR on developing endosperm sections 378

was performed to locate glutelin mRNAs. In this experiment, Rhodamine B dye was used 379

to specifically stain the PB-ER (Muench et al., 2000) while glutelin mRNAs were labeled 380

with Alexa488-dUTP by in situ RT-PCR using glutelin-specific primers (Figure 8). In 381

wild type, glutelin mRNAs are localized on the cisternal-ER separate from PB-ER 382

13

(Figure 8). In rab5a mutant cells, however, they were mainly distributed to the PMBs and 383

with smaller amounts associated with the PB-ER. These results indicate that normal 384

glutelin mRNA localization is disrupted in the rab5a mutant. This result resonates with 385

the retention of the complexes formed by RBP-P, RBP-L, NSF and Rab5a in PMBs 386

(Figures 6 and 7), further supporting a Rab5a-dependent endosome transport of glutelin 387

mRNAs. 388

Taken all together, the results described here support the view that glutelin mRNAs 389

are transported to the cisternal-ER membrane via their hitchhiking on endosomes. In the 390

rab5a mutant, however, glutelin mRNA-containing mRNP complexes are not transported 391

to their normal location but are mis-targeted to the PB-ER and displaced to the 392

extracellular PMBs because of the disruption in normal endosomal trafficking. 393

394

395

DISCUSSION 396

The study of rice storage protein mRNA localization as a model has provided 397

considerable information on why and how plant mRNAs are localized to specific ER 398

compartments. Although zipcode cis-elements of glutelin and prolamine mRNAs and 399

several key RNA-binding proteins have been identified (Hamada et al., 2003; Washida et 400

al., 2009; Doroshenk et al., 2012; Washida et al., 2012; Tian et al., 2018; Tian et al., 401

2019a), the mechanism by which glutelin and prolamine mRNAs are transported to 402

distinct subdomains of the ER membrane has yet to be established. Here, we report that 403

rice endosperm cells employ Rab5a and NSF, proteins traditionally known for their 404

essential roles in endosomal trafficking and membrane fusion events, as adaptor proteins 405

linking mRNA-protein complexes to endosomes for active transport of glutelin mRNAs. 406

Active transport of mRNAs along cytoskeleton networks by hitchhiking on motile, 407

membrane-bound organelles has been reported in mammalian and fungi cells. In human 408

axons, the Ras GAP SH3 domain binding protein 1 (G3BP1)-labeled RNA granules were 409

found to co-localize and co-traffic with moving lysosomes along microtubules (Liao et al., 410

2019). The membranous amyotrophic lateral sclerosis (ALS)-associated 411

phosphoinositide-binding protein, annexin A11 (ANX11), functions as a molecular tether 412

between RNA granules and lysosomes (Liao et al., 2019). In addition to mRNAs, 413

14

neuronal precursor miRNAs are transported to the tip of growing axons by hitchhiking on 414

late endosomes/lysosomes (Corradi et al., 2020). 415

Endosomal transport of mRNAs has been defined in the filamentous fungus Ustilago 416

maydis, where mRNAs are transported throughout the growing hyphae via endosome 417

trafficking on microtubules (Baumann et al., 2012; Gohre et al., 2012; Vollmeister et al., 418

2012; Pohlmann et al., 2015; Niessing et al., 2018). mRNP complexes carrying cdc3 419

mRNAs are attached to endosomes via the adaptor protein Upa1, which associates with 420

the endosomal membrane through its FYVE domain and two RNA binding proteins, 421

Pab1 and Rrm4, through its PABP-associated motif 2 (PAM2) and PAM2-like (PAM2L) 422

domains, respectively (Pohlmann et al., 2015). 423

Unlike the abovementioned examples in mammalian and fungi cells, higher plants 424

apparently adapted the two membrane trafficking factors, NSF and Rab5a, to mediate 425

transport of storage protein mRNAs. The linking of glutelin mRNP complexes to 426

endosomes is mediated by the four-way interactions of NSF and Rab5a with the two 427

glutelin zipcode RNA-binding proteins, RBP-P and RBP-L. These interacting proteins 428

likely form a quaternary complex enabling endosomal transport of glutelin mRNAs. Such 429

protein to protein interactions are accomplished through a gain in binding properties by 430

Rab5a and NSF, and the glutelin zipcode mRNA binding proteins, RBP-P and RBP-L, 431

which allow for the heterotypic interaction of a RNA binding protein with a membrane 432

fusion factor (RBP-P/NSF and RBP-L/Rab5a). The interaction of an RBP with a 433

membrane trafficking factor, i.e. between RBP-P with NSF and between RBP-L and 434

Rab5a, as well as between NSF and Rab5a are unexpected findings as such interactions 435

have not been reported in other organisms. It remains unclear whether the interactions of 436

these membrane trafficking factors to RBPs and even between themselves is an inherent 437

property or whether they are unique to higher plants. While Upa1-mediated endosomal 438

mRNA transport is proposed as evolutionarily conserved in fungi (Muller et al., 2019), 439

further investigation is needed to assess whether endosomal mRNA transport by the NSF-440

Rab5a-RBP machinery is a widespread phenomenon in transporting mRNAs among 441

higher plants and other eukaryotic organisms. 442

It appears that the recognition of NSF and Rab5a with RBPs is highly selective. 443

These non-random interactions highlight the diverse binding capabilities of NSF, Rab5a, 444

15

as well as for RBP-P and RBP-L in rice endosperm cells. Both RBP-P and RBP-L 445

contain RNA recognition motifs (RRM) with the former having two RRM motifs and the 446

latter having three RRM motifs. Due to the conservation of the RRM motifs, the 447

recognition of NSF and Rab5a to RBP-P and RBP-L, respectively, is likely via the unique 448

N- and C-terminal regions that flank the RRM motifs. Indeed, our preliminary results 449

(Supplemental Figure 2) show that the N-terminal end of RBP-P is essential for its 450

interaction with NSF. Through the selective recognition of NSF and Rab5a to other 451

specific RBPs, these membrane trafficking factors could serve as the core components 452

enabling endosome-coupled mRNA transport with the RBPs specifying the mRNA 453

species. 454

NSF is homo-hexamer with three domains: the N-terminal domain (NSF-N) that is 455

required for SNAP-SNARE binding; the ATP-binding domain 1 (NSF-D1) responsible 456

for ATPase activity; and the ATP-binding domain 2 (NSF-D2) responsible for 457

hexamerization (Tagaya et al., 1993; Zhao et al., 2007). The NSF-N domain is the main 458

protein-protein interaction site for NSF binding to other proteins. NSF-N domain is likely 459

responsible for its interaction with the N-terminus of RBP-P in rice as well 460

(Supplemental Figure 2). Sequence alignment of NSF homologues from rice, Arabidopsis, 461

Drosophila, yeast and human show that the N-terminal region displays considerable 462

sequence diversity among these species (Supplemental Figure 3). Further structural 463

studies are required to investigate their binding mechanism and whether this kind of 464

interaction occurs in other species. 465

Rab5a interacts with a large number of proteins, including its regulators (activating 466

effectors and inhibitors), linkers to molecular motors, membranous factors, components 467

of membrane fusion complexes, protein kinases and phosphatases (Herve and 468

Bourmeyster, 2018; Pylypenko et al., 2018). Many of these interacting proteins function 469

as Rab5a effectors to activate endosomal transport and tightly control the specific 470

functions of Rab5a in membrane composition and modification, cytoskeleton regulation, 471

and intracellular trafficking (Herve and Bourmeyster, 2018; Pylypenko et al., 2018). 472

Although NSF has been reported to interact with several Rab5a effectors to drive 473

membrane fusion during endosomal docking (McBride et al., 1999; Grosshans et al., 474

2006), no direct relationship between Rab5a with NSF or with a RNA-binding protein 475

16

had been established until now. These findings will stimulate future research efforts on 476

identifying new alternative roles of Rab5a and NSF beyond membrane fusion. 477

Although the GTPase activity of Rab5a is not required for the apparent formation of 478

the quaternary protein complex as the four proteins still co-localized in the GDP-fixed 479

(G45D) Rab5a line (Figures 6-7), Rab5 GTPase activity is required for proper mRNA 480

transport. Endosome formation and membrane trafficking are dependent on Rab5 GTPase 481

activity (Woodman, 2000; Zeigerer et al., 2012). Null Rab5a activity results in aborted 482

endosomal transport in the mutant, which, in turn, disrupts glutelin mRNA transport on 483

endosomes and causes its mis-localization to the PB-ER as well as displacement to the 484

extracellular PMBs (Figure 8). Therefore, the active GTPase activity of Rab5a is essential 485

for the transport of glutelin mRNAs on endosomes. This also raises the question on 486

whether NSF and RBP-L act as Rab5a effectors to modulate the role of Rab5a in mRNA 487

transport and localization. While this hypothesis needs further examination, other 488

effectors, functionally equivalent to Rabaptin-5 and EEA1 that interact with both NSF 489

and Rab5a (McBride et al., 1999; Grosshans et al., 2006), may also be involved. These 490

effectors, including the abovementioned GEF, may allow further stabilization of the 491

linkage of mRNP complex to active endosomes and regulate endosomal mRNA transport 492

via the cytoskeleton (Figure 4). 493

The identification and study of rice lines expressing mutations in Rab5a were 494

instrumental in establishing its involvement in both RNA and membrane trafficking in 495

developing rice endosperm. While Rab5 is an essential growth factor, loss of Rab5a only 496

had a slight effect on rice growth and development as its activity is complemented by 497

other Rab5 activities. Rice expresses three other Rab5 genes, Rab5b, Rab5c, and Rab5d 498

(Supplemental Figure 4). Like Rab5a, Rab5c is a conventional type, while Rab5b and 499

Rab5d are plant-specific type homologous to the Arabidopsis plant-specific 500

Rab5F1/ARA6. During rice growth and development, the loss of Rab5a is offset by 501

Rab5c and possibly by Rab5b and Rab5d. Although the other Rab5 isoforms can 502

compensate for Rab5a, they are only able to partially fill this role as rab5a rice lines 503

grow slower and flower later than normal, likely due to their significant lower expression 504

compared to Rab5a (Supplemental Figure 5). At the grain filling stage where there is 505

massive protein transport from the ER via the Golgi to the storage vacuole, the reduced 506

17

expression of these other Rab5 isoforms fails to maintain normal membrane trafficking 507

resulting in the secretion of storage proteins, the formation of PMBs, and mis-localization 508

of glutelin mRNAs. 509

Efforts to identify rice lines expressing defective NSF have not been productive, 510

likely because of the importance of its ATPase activity in membrane fusion. Mutations in 511

NSF were reported to induce severe defect and cell lethality in several organism 512

(Boulianne and Trimble, 1995; Golby et al., 2001; Mohtashami et al., 2001; Horsnell et 513

al., 2002; Zhao et al., 2007). For example, Drosophila expresses two NSF isoforms, 514

dNSF1 and dNSF2. dNSF1 is dominant in the adult central nervous system while dNSF2 515

is broadly distributed at the larval/adult stages of development, respectively (Boulianne et 516

al., 1995). While dNSF1 null mutants perish as pharate adults, dNSF2 deletion mediates a 517

recessive lethal phenotype, which is not even rescued by the addition of a dNSF2 518

transgene (Golby et al., 2001; Mohtashami et al., 2001). In yeast, loss-of-function 519

mutation of the NSF gene, Sec18, resulted in a dominant lethal phenotype (Horsnell et al., 520

2002). These studies suggest that NSF mutations are pleiotropic and cause severe growth 521

problems. In Arabidopsis, even a subtle mutation of NSF caused severe abnormal Golgi 522

morphology (Tanabashi et al., 2018). Given that NSF is coded by a single gene copy in 523

the rice genome, mutations that affect NSF activity will likely confer a strong lethal 524

phenotype in rice. 525

Although beyond the scope of this study, NSF mutations that affect binding to RBP-P 526

would be a viable approach to obtain further insight on its role in mRNA trafficking. As 527

discussed earlier, the NSF-N domain is likely responsible for its interaction with the N-528

terminus of RBP-P. Selected residues in the NSF-N region can be replaced by amino 529

acids that alter charge or conformation and then tested for its protein-interactive 530

properties by yeast 2-hybrid analysis. Mutations in NSF that abolish its interaction with 531

RBP-P but not with Rab5a would be potential sites for genetic alteration by CRISPR 532

technology and, thereby, disrupting glutelin mRNA transport but maintaining normal 533

function in membrane vesicle transport. 534

Overall, this study provides evidence on how glutelin mRNPs are able to 535

hitchhike on trafficking endosomes in rice endosperm cells by exploiting the binding 536

properties of RBPs and membrane trafficking factors, NSF and Rab5a. These findings 537

18

will provide the basis for future research on membrane trafficking-mediated mRNA 538

transport and the unique functions of NSF and Rab5a in this cellular process. 539

540

MATERIALS AND METHODS 541 542 Plant materials and growth conditions. The rice wildtype (Oryza sativa japonica 543

variety TC65) and rab5a mutant line EM960 (Fukuda et al., 2011) were potted in 544

Sunshine Brand #1 soil (Sungro Horticulture) and grown in walk-in growth chambers 545

with a diurnal cycle of 12 h light/12 h dark at 27°C and a lighting intensity of 400 to 700 546

μmol m-2

s-1

using a combination of metal halide and high pressure sodium lamps . 547

Antibodies. Full length cDNAs encoding RBP-P, RBP-L, NSF, SNAP, BiP and GFP 548

were cloned into pET30a for His-tagged fusion protein expression. The His-tagged 549

proteins were then purified and used to immunize New Zealand White rabbits for 550

antibody production. Anti-Rab5a rabbit and mouse antibodies were obtained from an 551

earlier study (Fukuda et al., 2011). For immunofluorescence labeling, Alexa Fluor-488 552

labeled goat anti-mouse IgG antibody (Invitrogen, Cat # A32723) and Alexa Fluor-594 553

labeled goat anti-rabbit IgG antibody (Invitrogen, Cat # A32740) were used as secondary 554

antibodies. For immunogold labeling, EM-grade 10 nm-gold-conjugated goat anti-mouse 555

IgG (Electron Microscopy Science, Cat # 25128) and EM-grade 15 nm-gold-conjugated 556

goat anti-rabbit IgG (Electron Microscopy Science, Cat # 25112) were used as secondary 557

antibodies. 558

Immunoprecipitation (IP) analysis. Most IP experiments were conducted as previously 559

described (Doroshenk et al., 2014) except that incubations with antibody were conducted 560

in the presence/absence of 1mM ATP/GTP, 8 mM MgCl2 or EDTA. To maximize 561

capture of transient mRNP complexes on endosomes, developing seeds were treated with 562

1% paraformaldehyde (PFA). Detailed procedures for IP-MS, the identification of NSF 563

protein, and PFA fixation for the enhanced IP are described below. 564

IP-MS analyses. Affinity purified RBP-P or GFP antibodies were prepared using 565

immobilized metal affinity chromatography based on irreversibly oxidized Co(III)-IDA 566

resin as described previously (Crofts et al., 2010) and crosslinked to Protein A/G agarose 567

resin using the Pierce Crosslink Immunoprecipitation Kit according to the manufacturer’s 568

instructions (Thermo Fisher Scientific). Protein extraction and co-IP experiments were 569

19

performed at 4oC unless indicated. Two g of dehulled mid-developing wild type rice seed 570

harvested 12-14 days after flowering were frozen in liquid nitrogen and grounded to a 571

powder. Proteins were extracted in 6 ml of IP buffer (20 mM Tris-HCl pH 7.5, 0.15 M 572

NaCl, 1 mM EDTA, 0.5% v/v NP40) containing 1X protease inhibitor cocktail and 0.5X 573

phosphatase inhibitor (Sigma-Aldrich). The crude extract was clarified by twice 574

centrifuging at 12,000 g for 10 min. The resulting supernatant was gently rotated 575

overnight with agarose resin to eliminate non-specific interactions. 700 µL of the 576

unbound protein fraction were added to columns containing RBP-P or GFP antibodies 577

crosslinked to Protein A/G resin or naked resin and incubated approximately 7 hours with 578

rotation. The columns were washed 5 times with IP buffer and once with 1X conditioning 579

buffer, and bound proteins were eluted with 50 µL IgG elution buffer (Thermo Fisher 580

Scientific). The antibody conjugated Protein A/G resin columns were regenerated by 581

washing with 1X Coupling Buffer (Thermo Scientific) followed by IP buffer. Fresh, pre-582

cleared rice seed extract was added and incubated overnight. The columns were washed 583

and proteins eluted as above. 584

Eluted proteins from a total of five co-IP experiments using anti-RBP-P, anti-GFP 585

antibodies, or naked resin were pooled, precipitated with addition of 100% trichloroacetic 586

acid (TCA) to a 20% final concentration. The TCA precipitate was washed with acetone 587

and then resuspended in 30 µL SDS sample buffer containing 4 M urea and 5% v/v β-588

mercaptoethanol. Protein samples were resolved on 10% SDS-PAGE gels and stained 589

using a silver nitrate staining protocol (Chevallet et al., 2006). Because the protein profile 590

of the minus antibody control IP looked very similar to the GFP Co-IP, proteins from 591

only the GFP and RBP-P co-IPs were analyzed by mass spectrometry. Gel slices 592

corresponding to similar positions within each lane were excised, in-gel trypsin digested, 593

and subjected to liquid chromatography-tandem mass spectrometry as described 594

previously (Doroshenk et al., 2009). Proteins were identified by searching the Oryza 595

sativa NCBI non-redundant database (134548 sequences) using Mascot 596

(www.matrixscience.com) as previously described (Doroshenk et al., 2009). 597

IP with 1% PFA fixation. Antibodies raised against RBP-L, RBP-P, NSF, or Rab5a 598

were affinity purified as abovementioned. 20µL of resuspended protein A Magbeads 599

slurry (25% w/v slurry, GenScript) in a 1.5 mL tube was washed twice with 1X PBS and 600

20

then incubated overnight at 4 ºС under rotation with 30 µg affinity-purified antibody 601

(diluted to 500 µL by 1x PBS). Antibodies were cross-linked to protein A Magbeads 602

using the above-mentioned Pierce Crosslink Immunoprecipitation Kit. 603

Dehulled developing rice seeds were fixed with 1% paraformaldehyde (PFA) in 604

PBSM (1.76 mM KH2PO4, 10 mM Na2HPO4, 136 mM NaCl, 2.6 mM KCl, 5 mM 605

MgCl2, and 10% glycerol) under vacuum for 30 min. The reaction was stopped by 606

vacuuming in quenching buffer (0.333 M Tris and 10% glycerol) for 10 min. Ten to 607

twenty aleurone-layer peels collected from the fixed seeds were ground to a fine powder 608

using an ice cold mortar and pestle in 800 µL of lysis buffer (50 mM Tris-HCl pH 7.5, 609

150 mM NaCl, 10 mM EDTA, 0.5% NP-40, 0.1% Triton X-100, 1x proteinase inhibitor 610

cocktail, 100 µg/ml cycloheximide, 20 units/mL of RiboLock RNase Inhibitor (Thermo 611

Fisher Scientific) and then centrifuged at 1000 g for 5 min to remove starch followed by 612

centrifugation at 12,000 g for 10 min. The clear supernatant was added to the prepared 613

Magbeads for incubation overnight at 4ºС with gentle rotation. After washing twice with 614

lysis buffer, the bound fractions were eluted with IgG elution buffer (ThermoFisher 615

Scientific) and neutralized by addition of 1/10 volume of 1 M Tris-HCl (pH 8.8). The 616

neutralized elution samples were analyzed by immunoblotting. 617

In situ RT-PCR. In situ RT-PCR on developing rice seed sections was performed as 618

previously described (Washida et al., 2009). Specific primers Glutelin-F 5’-619

CCCTCAAGCATACAGGCGTG-3’ and Glutelin-R 5’-620

CGCTCTCTTGATTGCACTTGTCC-3’ were used in the PCR to amplify glutelin RNAs. 621

Construction of vectors. Gene sequences of RBP-P, NSF and their truncated forms were 622

cloned into pGAD T7 and pGBK T7 vectors and used as preys and baits, respectively, in 623

Y2H analyses for detection of protein-protein interaction. BiFC vectors of pSAT1-624

nEYFP-C1 and pSAT1-cEYFP-C1-B for N- and C-terminal EYFP fusion, respectively, 625

were obtained from the Arabidopsis Biological Resource Center 626

(https://www.arabidopsis.org). The cDNA sequences of RBP-P, RBP-L, NSF, Rab5a and 627

its mutant forms (Rab5aQ70L

and Rab5aG45D

) were cloned into pSAT1-nEYFP-C1 or 628

pSAT1-cEYFP-C1-B vectors for BiFC analysis. GFP or RFP-fusion vector driven by 629

double 35S promoters was constructed by replacing the N-terminal EYFP in pSAT1-630

21

nEYFP-C1 with GFP or RFP cDNA sequences. The cDNA sequences of NSF and Rab5a 631

were then cloned into GFP or RFP-fusion vector to obtain RFP protein fusions. 632

RNA-IP, Yeast two hybrid (Y2H), and BiFC assays. Experimental procedures for 633

these studies were performed as previously described (Doroshenk et al., 2014; Tian et al., 634

2018; Tian et al., 2019a). Briefly, for RNA-IP analysis, developing rice seeds, collected 635

10-14 days after flowering, were subjected to 1% PFA fixation and extracts prepared and 636

used for IP as mentioned above. The elution samples obtained from IP were incubated at 637

70 °C for 45 min to reverse the RNA-protein crosslinks followed by RNA extraction 638

using TRIzol (Invitrogen), cDNA synthesis using M-MLV reverse transcriptase 639

(Promega), and PCR with 20-25 cycles of amplification using glutelin and ACTIN 640

specific primers (Glutelin-F and Glutelin-R as mention in in situ RT-PCR, Actin-F 5’- 641

TCCATCTTGGCATCTCTCAG-3’, and Actin-R 5’- GTACCCGCATCAGGCATCT-642

3’). The antibodies used in the RNA-IP were affinity-purified as aforementioned. In Y2H 643

analysis, synthetic dropout (SD) growth media without leucine and tryptophan (SD/-644

Leu/-Trp) was used to screen positive transformants and selection media without leucine, 645

tryptophan, histidine, and adenine (SD/-Leu/- Trp/-His/-Adenine medium) with 646

supplement of 3 mM 3-amintriazole (3-AT) (SD/-Leu/-Trp/-His/-Adenine/+ 3-AT) or 40 647

mg/L X-α-Gal was used to verify protein-protein interaction. 648

BY-2 suspension cells were used for BiFC analyses. In brief, BY-2 cells were 649

treated with cell wall digestion buffer (1% cellulase (Onozuka RS, PhytoTechnology 650

Laboratories), 0.05% pectolyase (Seishin Pharmaceutical, Japan), 0.2% Driselase 651

(Sigma-Aldrich), 20 mM KCl, 10 mM CaCl2, 20 mM MES hydrate, and 0.5M sucrose, 652

pH 5.7) at room temperature for 3 h. After washing with W5 solution (154 mM NaCl, 653

125 mM CaCl2, 5 mM KCl, 5 mM glucose, pH 5.8-6.0), the BY-2 protoplasts were 654

subject to PEG-mediated transformation with the abovementioned vectors of pSAT1-655

nEYFP-C1 and pSAT1-cEYFP-C1-B as described previously (Tian et al., 2018). After 656

culture at 26oC for 16 hours, the BiFC fluorescence images were observed using a Leica 657

SP-8 confocal microscope. Negative controls using empty vectors were also examined to 658

check the reliability of the transformation procedure. The localization pattern of target 659

proteins or complexes was determined by examining at least 5 different protoplast cells. 660

To confirm the involvement of Rab5a and the corresponding complexes in endocytic 661

22

pathway, protoplast incubation was treated with the endocytic tracer FM4-64 (Invitrogen) 662

at a final concentration of 10 μM for 15-30 mins before observation. 663

Microscopy. Light microscopy was performed on 10 μm thick sections of developing 664

rice seed samples embedded in LR-white resin. The sections were positioned on Leica X-665

tra slides, stained by 1% Toluidine blue and observed using an Olympus BH-2 Light 666

microscope. Co-localization test of RBP-P, RBP-L, and NSF with Rab5a in rice 667

endosperm cells was performed through double-immunolabeling using the rabbit anti-668

RBP-P, RBP-L, NSF antibodies and mouse anti-Rab5a antibodies (see Section of 669

Antibodies) on 1 μm thick LR-white sections as described previously (Fukuda et al., 670

2011), and observed under a Leitz Epi-Fluorescent Microscope with Leica DFC425C 671

Camera. Fluorescence intensity of green and red signals was analyzed by plot profile tool 672

in FUJI (ImageJ) software. Transmission electron microscopy analysis was performed as 673

previously described (Tian et al., 2018). 674

675

ACCESSION NUMBERS 676

Sequence data from this article can be found in the GenBank/EMBL data libraries under 677

NCBI accession numbers shown in the legends of Supplemental Figures 3 and 4. 678

679

SUPPLEMENTAL DATA 680

Supplemental Figure 1. Sequence information of Rab5a in rice. 681

Supplemental Figure 2. Possible binding domains of RBP-P with NSF revealed by yeast 682

two hybrid (Y2H) analysis. 683

Supplemental Figure 3. Protein sequence alignment of NSF. 684

Supplemental Figure 4. Protein sequence alignment of Rab5 isoforms. 685

Supplemental Figure 5. Expression profile of Rab5 isoforms in rice plants. 686

687

ACKNOWLEDGEMENTS 688

This work was financially supported by grants from the National Science Foundation 689

(MCB-1444610 and IOS-1701061), from the USDA National Institute of Food and 690

23

Agriculture, Hatch umbrella project 899 1015621 and project WNP00119, and from the 691

Japan Society for the Promotion of Science (M.F. and T.K.). We thank Ai Nagamine for 692

her help to construction of BiFC plasmids and technical support provided by the 693

Franceschi Microscopy and Imaging Center at Washington State University. 694

695

AUTHOR CONTRIBUTIONS 696

L.T. designed the study; K.A.D. identified NSF as interacting partner of RBP-P 697

through IP-MS; L.T. discovered interaction of RBPs and NSF with Rab5a and conducted 698

BiFC, RNA-IP, yeast two hybrid, light microscopy and TEM analyses; L.T. and L.Z. 699

conducted co-IP analysis; M.F. conducted immunofluorescence microscopy; L.T. 700

constructed vectors; L.T. and H.W. conducted in situ RT-PCR; T.K. provided rab5a 701

mutant; T.W.O. supervised the project; L.T. and T.W.O. wrote the manuscript. 702

703

24

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917 918 919

920

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921 FIGURE LEGENDS 922 923 Figure 1. Identification of NSF as an interacting partner of RBP-P. (A) Precipitation 924 of NSF by RBP-P antibody as revealed by IP-MS. Left panel, immunoblot (IB) analysis 925 to test the IP reliability; right panel, silver stained SDS- polyacrylamide gel of eluted 926 samples from ()-GFP and RBP-P IPs. Input, starting material of rice lysate; Ub, 927 unbound fraction from IPs; B, bound fraction (eluted samples) from IPs. Blue asterisk (*) 928 indicates a modified form of RBP-P. The bands indicated by red and black arrows were 929 excised for MS analysis and NSF was identified as a specific protein precipitated by anti-930 RBP-P but not by anti-GFP. (B) Interaction between RBP-P and NSF revealed by yeast 931 two hybrid. Yeast colonies co-transfected with pGBK and pGAD constructs were labeled 932 1-4 as described in the upper Table. --, empty vector. Yeast cells carrying the 933 corresponding genes were grown on SD/-Leu/-Trp medium as growth control and SD/-934 Leu/-Trp/-His/-Ade/+ 3-AT selection medium to detect their interaction. Note that only 935 the yeast cells carrying both NSF and RBP-P survived on the strict selection medium 936 (lower panel), suggesting that RBP-P interacts with NSF. 937 938 Figure 2. RBP-P interacts indirectly with Rab5a through NSF, forming a complex 939 attached to endosomes. 940 (A-C) RBP-P interacts with NSF as revealed by BiFC studies. BF, bright field; Merge, 941 the merged images of BF and BiFC (middle panel); ---, empty vector as control. (D) 942 SNAP-independent formation of a protein complex consisting of RBP-P, NSF and Rab5a 943 as revealed by Co-IP. IP studies were conducted with ()-GFP, RBP-P, NSF, or Rab5a 944 antibodies in the presence of ATP and MgCl2 or with EDTA. GFP antibody was used as a 945 negative control. Input, the starting rice lysate. Note that the association of RBP-P with 946 NSF and Rab5a requires MgCl2 and ATP, unlike the interaction of SNAP with NSF and 947 Rab5a, which requires EDTA. (E) Schematic structure of Rab5a in rice. The two switch 948 regions are indicated as Switch 1 and 2. The two mutation sites (G45D and Q70L) to 949 generate GDP- and GTP-fixed forms of Rab5a, respectively, are indicated in red. (F) 950 GFP-Rab5a (green) co-localized with FM4-64 (magenta) labeled plasma membrane and 951 endosomal compartment. Co-localization of GFP-Rab5a with FM4-64 endosomes are 952 denoted by arrows. (G-J) Rab5a interacts with NSF (I), but not to RBP-P (J) as shown 953 by BiFC. (K-L) NSF interacts with both GDP- (panel J, Rab5a

G45D) and GTP-bound 954

(panel K, Rab5aQ70L

) Rab5a. (M-N) RBP-P/NSF complexes labeled by BiFC (yellow) 955 co-localize with RFP-Rab5a (magenta) in the cytoplasm. (M) The punctate RBP-P/NSF 956 structures align with the particle-like, endosome structures of RFP-Rab5a. (N) RBP-957 P/NSF complexes co-localize with the membrane-associated, GTP-fixed Rab5a

Q70L, 958

indicating RBP-P/NSF complexes are associated with active endosomes. In M-N, co-959 localized RBP-P/NSF complexes with endosomal Rab5a are indicated by white arrows. 960 Non-colocalization of RBP-P/NSF complexes and Rab5a-postive endosome are indicated 961 by empty arrowheads. (O) Co-localization of RBP-P/NSF complexes with FM4-64 962 labeled endosomes (magenta) are denoted by arrows. Scale bars, 20 μm. 963 964 Figure 3. RBP-L interacts directly with Rab5a, which collectively forms a multi-965 protein complexes with RBP-P and NSF to transport glutelin mRNAs on endosomes. 966

30

(A-C) RBP-L interacts directly with Rab5a (C) but not to NSF (B). Note that the RBP-967 L/Rab5a complex is detected in both the nucleus and cytoplasm. (D) RBP-L/Rab5a 968 complexes (yellow) co-localized with FM4-64 labeled endosomes (magenta) located 969 close to the plasma membrane. Co-localized signals are indicated by arrows. (E-F) in 970 vivo interactions of RBP-L with GDP- (F, Rab5a

G45D) or GTP-bound (G, Rab5a

Q70L)971

Rab5a. (G) Association of RBP-P, RBP-L with NSF and Rab5a as revealed by IP. The IP 972 experiment was conducted with developing seeds treated with 1% paraformaldehyde 973 (PFA) to preserve labile protein complexes. Input, the starting rice lysate. Control, GFP 974 antibody. IB, immunoblot. (H) RBP-P, RBP-L, NSF and Rab5a form a ternary complex 975 as revealed by sequential IPs. The first IP was conducted with ()-GFP (negative control) 976 or RBP-P antibodies. Proteins captured by RBP-P antibody were subjected to a 2

nd IP977

using RBP-L antibody. Subsequent immunoblot analysis showed the presence of NSF 978 and Rab5a together with RBP-P and RBP-L. Input, the starting rice lysate; FT, flow-979 through (unbound fraction); E, bound fraction; W1 and W2, washing fractions. (I) The 980 RBP-P/RBP-L/NSF/Rab5a complex contains glutelin mRNAs based on RNA-IP. RNAs 981 extracted from each IP generated by anti-RBP-P, anti-RBP-L, anti-GFP or empty resin (-982 CT) were subjected to RT-PCR using glutelin-specific primers and resolved by agarose 983 gel electrophoresis. GFP antibody (-GFP) and empty resin (-CT) were used as negative 984 controls. Input, PCR products using cDNA synthesized from total RNAs. Actin was used 985 as control genes to verify the specific binding of the complex to glutelin RNAs. Scale 986 bars, 20 μm. 987

988 Figure 4. A working model of glutelin mRNA transport via trafficking endosomes to 989 the cortical ER. 990 In rice endosperm cells, glutelin mRNA is bound by the scaffold complex of RBP-P and 991 RBP-L, forming a mRNP complex. Through direct or NSF-mediated indirect interaction 992 with membrane-associated Rab5a, RBP-L and RBP-P link the mRNP complex to 993 endosomes for active transport via the cytoskeleton. Rab5a effectors (e, light orange) may 994 be involved to stabilize the quaternary complex and modulate GTP-bound active Rab5a-995 drived endosomal trafficking on actin filaments in rice endosperm cells. Other unknown 996 RBPs or factors, showed in light grey shapes, may also be involved to constitute the 997 mRNP complex and define the linkage onto endosomes. 998

999 Figure 5. Rab5a mutation leads to abnormal trafficking of endosomes and 1000 formation of extracellular paramural bodies (PMBs). 1001 (A) Schematic representation of the Rab5a mutation site in the rab5a mutant. A G134A1002 base substitution within the Rab5a gene resulted in a G45D amino acid replacement. (B)1003 Formation of PMBs (white asterisks) was observed in endosperm cells of rab5a mutant1004 through light microscopy observations on seed sections stained with 1% Toluidine Blue.1005 Scale bar, 25 m. (C) Ultrastructure of PMBs formed in rab5a mutant due to aborted1006 endosomal trafficking in comparison to wildtype (WT) endosperm cells. Cell wall and1007 PMB boundaries are indicated by magenta and green dashed lines, respectively. SG,1008 starch granules; orange *, protein body I; blue *, protein storage vacuoles. Scale bar, 11009 m.1010

1011

31

Figure 6. Rab5a mutation leads to the transport of the RBP-P/RBP-L/NSF/Rab5a 1012 ternary complex to PMBs. 1013 (A-F) intracellular location of RBP-P (A-B, magenta), RBP-L (C-D, magenta), NSF (E-1014 F, magenta) and Rab5a (A-F, green) in wildtype (A, C, and E) and rab5a mutant (B, D, 1015 and F) rice endosperm cells as revealed by immunofluorescence labeling. Co-1016 localization patterns of RBP-P, RBP-L, and NSF with Rab5a are shown in the Merge 1017 panel, with rectangular areas enlarged in the adjacent fourth panels. Fluorescence 1018 intensity graphs on the very right show the relative strength of the magenta and green 1019 fluorescence signals as measured by scanning the region indicated by the white line in the 1020 fourth panel. X and y axes represent the fluorescence intensity and position of the signals 1021 (pixels), respectively, as evaluated by FUJI ImageJ. PMBs are indicated by asterisk (*) 1022 and co-localization signals are highlighted by white arrow heads. Scale bar, 20 m (the 1023 left three panels) and 10 m (the right panels). 1024 1025 Figure 7. Retention of RBP-P and RBP-L on the aborted endosomes in the PMBs of 1026 rab5a mutant as revealed by immunocytochemistry and transmission electron 1027 microcopy. 1028 (A-J) Localization of RBP-P (A-C, F-G) or RBP-L (D-E, H-J) on Rab5a-labeled 1029 endosomes in wildtype (A-E) and rab5a mutant (F-J) endosperm cells. RBPs (RBP-P 1030 and RBP-L) and Rab5a were labeled with 15 nm (blue arrowheads) and 10 nm (red 1031 arrows) gold particles, respectively. Panels B, C, E, G, I and J are the enlarged areas 1032 (rectangle) shown in A, D, F and H. Cell wall and PMB boundaries are indicated by red 1033 and yellow dashed lines, respectively. Scale bar, 2m (A, D, F and H) and 200 nm 1034 (B,C,E,G,I and J). Note that Rab5a-mediated endosomes are observed as electron-dense 1035 vesicles with an irregular-shape likely due to endosomal fusion (A to E) and as aborted 1036 endosome vesicles trapped within the PMBs in the rab5a mutant (F to J). 1037 1038 Figure 8. Rab5a mutation leads to mis-targeting of glutelin mRNAs to PB-ER and 1039 the PMBs as assessed by in situ RT-PCR. 1040 The PB-ER was stained by Rhodamine B dye (magenta), and glutelin mRNAs were 1041 labeled by in situ RT-PCR in the presence of Alexa-488-UTP (green). In wildtype (WT) 1042 endosperm cells (upper panel), glutelin mRNAs are localized on the cisternal-ER 1043 adjacent to the PB-ER. In rab5a mutant (lower panel), glutelin mRNAs are mis-targeted 1044 to the PMBs (asterisk) and PB-ER (arrows). Fluorescence intensity graphs on the very 1045 right show the relative position of magenta and green fluorescence signals in the regions 1046 indicated by rectangle in the Merge panels. X and y axes represent the fluorescence 1047 intensity and position of the signals (pixels), respectively, evaluated by FUJI ImageJ. 1048 Note the extensive overlap in the distributions between PB-ER and glutelin mRNAs in 1049 rab5a but not in wild type. Scale bar, 20m. 1050 1051

Figure 1. Identification of NSF as an interacting partner of RBP-P. (A) Precipitation of NSF by RBP-P antibody as revealed by IP-MS. Left panel, immunoblot (IB) analysis to test the IP reliability; right panel, silver stained SDS- polyacrylamide gel of eluted samples from (a)-GFP and RBP-P IPs. Input, starting material of rice lysate; Ub, unbound fraction from IPs; B, bound fraction (eluted samples) from IPs. Blue asterisk (*) indicates a modified form of RBP-P. The bands indicated by red and black arrows were excised 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 yeast two hybrid. Yeast colonies co-transfected with pGBK and pGAD constructs were labeled 1-4 as described in the upper Table. --, empty vector. Yeast cells carrying the corresponding genes were grown on SD/-Leu/-Trp medium as growth control and SD/-Leu/-Trp/-His/-Ade/+ 3-AT selection medium to detect their interaction. Note that only the yeast cells carrying both NSF and RBP-P survived on the strict selection medium (lower panel), suggesting that RBP-P interacts with NSF.

Figure 2. RBP-P interacts indirectly with Rab5a through NSF, forming a complex attached to endosomes. (A-C) RBP-P interacts with NSF as revealed by BiFC studies. BF, bright field; Merge, the merged images of BF and BiFC (middle panel); ---, empty vector as control. (D) SNAP-independent formation of a protein complex consisting of RBP-P, NSF and Rab5a as revealed by Co-IP. IP studies were conducted with (a)-GFP, RBP-P, NSF, or Rab5a antibodies in the presence of ATP and MgCl2 or with EDTA. GFP antibody was used as a negative control. Input, the starting rice lysate. Note that the association of RBP-P with NSF and Rab5a requires MgCl2 and ATP, unlike the interaction of SNAP with NSF and Rab5a, which requires EDTA. (E) Schematic structure of Rab5a in rice. The two switch regions are indicated as Switch 1 and 2. The two mutation sites (G45D and Q70L) to generate GDP- and GTP-fixed forms of Rab5a, respectively, are indicated in red. (F) GFP-Rab5a (green) co-localized with FM4-64 (magenta) labeled plasma membrane and endosomal compartment. Co-localization of GFP-Rab5a with FM4-64 endosomes are denoted by arrows. (G-J) Rab5a interacts with NSF (I), but not to RBP-P (J) as shown by BiFC. (K-L) NSF interacts with both GDP- (panel J, Rab5aG45D) and GTP-bound (panel K, Rab5aQ70L) Rab5a. (M-N) RBP-P/NSF complexes labeled by BiFC (yellow) co-localize with RFP-Rab5a (magenta) in the cytoplasm. (M) The punctate RBP-P/NSF structures align with the particle-like, endosome structures of RFP-Rab5a. (N) RBP-P/NSF complexes co-localize with the membrane-associated, GTP-fixed Rab5aQ70L, indicating RBP-P/NSF complexes are associated with active endosomes. In M-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 are indicated by empty arrowheads. (O) Co-localization of RBP-P/NSF complexes with FM4-64 labeled endosomes (magenta) are denoted by arrows. Scale bars, 20 μm.

Figure 3. RBP-L interacts directly with Rab5a, which collectively forms a multi-protein complexes with RBP-P and NSF to transport glutelin mRNAs on endosomes. (A-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/Rab5a complexes (yellow) co-localized with FM4-64 labeled endosomes (magenta) located close to the plasma membrane. Co-localized signals are indicated by arrows. (E-F) in vivo interactions of RBP-L with GDP- (F, Rab5aG45D) or GTP-bound (G, Rab5aQ70L) Rab5a. (G) Association of RBP-P, RBP-L with NSF and Rab5a as revealed by IP. The IP experiment was conducted with developing seeds treated with 1% paraformaldehyde (PFA) to preserve labile protein complexes. Input, the starting rice lysate. Control, GFP antibody. IB, immunoblot. (H) RBP-P, RBP-L, NSF and Rab5a form a ternary complex as revealed by sequential IPs. The first IP was conducted with (a)-GFP (negative control) or RBP-P antibodies. Proteins captured by RBP-P antibody were subjected to a 2nd IP using RBP-L antibody. Subsequent immunoblot analysis showed the 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; W1 and W2, washing fractions. (I) The RBP-P/RBP-L/NSF/Rab5a complex contains glutelin mRNAs based on RNA-IP. RNAs extracted from each IP generated by anti-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) and empty resin (-CT) were used as negative controls. Input, PCR products using cDNA synthesized from total RNAs. Actin was used as control genes to verify the specific binding of the complex to glutelin RNAs. Scale bars, 20 μm.

Figure 4. A working model of glutelin mRNA transport via trafficking endosomes to the cortical ER. In rice endosperm cells, glutelin mRNA is bound by the scaffold complex of RBP-P and RBP-L, forming a mRNP complex. Through direct or NSF-mediated indirect interaction with membrane-associated Rab5a, RBP-L and RBP-P link the mRNP complex to endosomes for active transport via the cytoskeleton. Rab5a effectors (e, light orange) may be involved to stabilize the quaternary complex and modulate GTP-bound active Rab5a-drived endosomal trafficking on actin filaments in rice endosperm cells. Other unknown RBPs or factors, showed in light grey shapes, may also be involved to constitute the mRNP complex and define the linkage onto endosomes.

e ee

RBP-P RBP-L

endosome

AAA

GLUTELIN

AAA

AAA

AAA

AAA

AAA

GLUTELIN mRNA

rice grain endosperm cell

AAAAAA

AAA

AAA

AAA

AAA

GLUTELIN

nucleus

ER

Golgi

Figure 5. Rab5a mutation leads to abnormal trafficking of endosomes and formation of extracellular paramural bodies (PMBs). (A) Schematic representation of the Rab5a mutation site in the rab5a mutant. A G134A base substitutionwithin the Rab5a gene resulted in a G45D amino acid replacement. (B) Formation of PMBs (whiteasterisks) was observed in endosperm cells of rab5a mutant through light microscopy observations onseed sections stained with 1% Toluidine Blue. Scale bar, 25 µm. (C) Ultrastructure of PMBs formed inrab5a mutant due to aborted endosomal trafficking in comparison to wildtype (WT) endosperm cells. Cellwall and PMB boundaries are indicated by magenta and green dashed lines, respectively. SG, starchgranules; orange *, protein body I; blue *, protein storage vacuoles. Scale bar, 1 µm.

Figure 6. Rab5a mutation leads to the transport of the RBP-P/RBP-L/NSF/Rab5a ternary complex to PMBs. (A-F) intracellular location of RBP-P (A-B, magenta), RBP-L (C-D, magenta), NSF (E-F, magenta) and Rab5a (A-F, green) in wildtype (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 panel, with rectangular areas enlarged in the adjacent fourth panels. Fluorescence intensity graphs on the very right show the relative strength of the magenta and green fluorescence signals as measured by scanning the region indicated by the white line in the fourth panel. X and y axes represent the fluorescence intensity and position of the signals (pixels), respectively, as evaluated by FUJI ImageJ. PMBs are indicated by asterisk (*) and co-localization signals are highlighted by white arrow heads. Scale bar, 20 µm (the left three panels) and 10 µm (the right panels).

Figure 7. Retention of RBP-P and RBP-L on the aborted endosomes in the PMBs of rab5a mutant as revealed by immunocytochemistry and transmission electron microcopy. (A-J) Localization of RBP-P (A-C, F-G) or RBP-L (D-E, H-J) on Rab5a-labeled endosomes in wildtype (A-E) and rab5a mutant (F-J) endosperm cells. RBPs (RBP-P and RBP-L) and Rab5a were labeled with 15 nm (blue arrowheads) and 10 nm (red arrows) gold particles, respectively. Panels B, C, E, G, I and J are the enlarged areas (rectangle) shown in A, D, F and H. Cell wall and PMB boundaries are indicated by red and yellow dashed lines, respectively. Scale bar, 2µm (A, D, F and H) and 200 nm (B,C,E,G,I and J). Note that Rab5a-mediated endosomes are observed as electron-dense vesicles with an irregular-shape likely due to endosomal fusion (A to E) and as aborted endosome vesicles trapped within the PMBs in the rab5a mutant (F to J).

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). In wildtype (WT) endosperm cells (upper panel), glutelin mRNAs are localized on the cisternal-ER adjacent to the PB-ER. In rab5a mutant (lower panel), glutelin mRNAs are mis-targeted to the PMBs (asterisk) and PB-ER (arrows). Fluorescence intensity graphs on the very right show the relative position of magenta and green fluorescence signals in the regions indicated by rectangle in the Merge panels. X and y axes represent the fluorescence intensity and position of the signals (pixels), respectively, evaluated by FUJI ImageJ. Note the extensive overlap in the distributions between PB-ER and glutelin mRNAs in rab5a but not in wildtype. Scale bar, 20µm.

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

and Thomas W 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

 This information is current as of August 6, 2020

 

Supplemental Data /content/suppl/2020/06/09/tpc.20.00111.DC1.html

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