GLUTELIN PRECURSOR ACCUMULATION3Encodes a Regulator of Post-Golgi Vesicular Traffic Essential for Vacuolar Protein Sorting in Rice Endosperm  

Abstract

In seed plants, a major pathway for sorting of storage proteins to the protein storage vacuole (PSV) depends on the Golgi-derived dense vesicles (DVs). However, the molecular mechanisms regulating the directional trafficking of DVs to PSVs remain largely elusive. Here, we report the functional characterization of the rice (Oryza sativa) glutelin precursor accumulation3 (gpa3) mutant, which exhibits a floury endosperm phenotype and accumulates excess proglutelins in dry seeds. Cytological and immunocytochemistry studies revealed that in the gpa3 mutant, numerous proglutelin-containing DVs are misrouted to the plasma membrane and, via membrane fusion, release their contents into the apoplast to form a new structure named the paramural body. Positional cloning of GPA3 revealed that it encodes a plant-specific kelch-repeat protein that is localized to the trans-Golgi networks, DVs, and PSVs in the developing endosperm. In vitro and in vivo experiments verified that GPA3 directly interacts with the rice Rab5a-guanine exchange factor VPS9a and forms a regulatory complex with Rab5a via VPS9a. Furthermore, our genetic data support the notion that GPA3 acts synergistically with Rab5a and VPS9a to regulate DV-mediated post-Golgi traffic in rice. Our findings provide insights into the molecular mechanisms regulating the plant-specific PSV pathway and expand our knowledge of vesicular trafficking in eukaryotes.

ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN, an Interacting Partner of RAB5 GTPases, Regulates Membrane Trafficking to Protein Storage Vacuoles in Arabidopsis

“…Although it is not clear whether the precursors of these storage proteins are segregated in the ER in wild-type plants, some populations of 12S globulin might be transported from the ER to the vacuole via a distinct pathway from 2S albumin in an EREX-and EREL1-dependent manner. Alternatively, canonical RAB5 may regulate sorting for PSVs on the TGN, which is similar to reports in rice (Oryza sativa) endosperm (Ren et al, 2014;Wen et al, 2015). A subpopulation of canonical RAB5 has been localized to the TGN, which is likely responsible for maturation from the TGN to the multivesicular endosomes (Stierhof and El Kasmi, 2010;Scheuring et al, 2011;Singh et al, 2014).…”

GOLGI TRANSPORT 1B Regulates Protein Export from the Endoplasmic Reticulum in Rice Endosperm Cells

“…The w379/glup3 mutants, both resulting from mutation of VPE1, develop round-shaped PBII (Wang et al, 2009;Kumamaru et al, 2010). The other three mutants defective in post-Golgi trafficking, gpa1/glup4/rab5a, gpa2/glup6/vps9a, and gpa3, all develop large amounts of protein granules and paramural bodies in the apoplast (Wang et al, 2010;Fukuda et al, 2011Fukuda et al, , 2013Liu et al, 2013;Ren et al, 2014). Moreover, PDIL1-1 functions in the ER lumen, and VPE1 plays a role in PSV/PBII, while GPA1, GPA2, and GPA3 form a complex that is present in the trans-Golgi networks and prevacuolar compartments.…”

“…When expressed in N. benthamiana leaf epidermal cells, GFP-GOT1B was localized in dynamically moving punctate structures (Supplemental Movie 1), but faint signals in the ER networks were observed in cells with higher expression levels (Supplemental Figures 5B and 5C). To determine the nature of these punctate structures, GFP-GOT1B was coexpressed with several fluorescent markers characteristic for the ER (mRFP-HDEL), ERES (AtSar1b-mRFP; Hanton et al, 2008), cis-Golgi (GmMan1-mRFP; Ren et al, 2014), trans-Golgi (ST-mRFP; Saint-Jore et al, 2002), and the trans-Golgi networks (mRFP-SYP61; Ren et al, 2014) in N. benthamiana leaf epidermal cells ( Figures 6A to 6E). Confocal microscopy analysis revealed strong correlation between GFP-GOT1B and the ERES marker signals (r s = 0.776) ( Figure 6B; French et al, 2008) and relative weaker correlation with the Golgi markers (r s = 0.532 and 0.524 for cis-Golgi and trans-Golgi, respectively) ( Figures 6C and 6D).…”

“…The prolamins are retained in the ER lumen after synthesis and are pinched off to form spherical protein bodies I (PBI) (Bechtel and Juliano, 1980;Tanaka et al, 1980;Yamagata and Tanaka, 1986). Glutelins are initially synthesized on the rough endoplasmic reticulum (RER) as 57-kD precursors and then transported to the protein storage vacuoles (PSVs; also called protein body II [PBII]) through the Golgi apparatus and the dense vesicle-mediated post-Golgi trafficking pathway (Krishnan et al, 1986;Takemoto et al, 2002;Liu et al, 2013, Ren et al, 2014. The a-globulin is deposited together with glutelins in PBIIs.…”

“…Any defects in the proglutelin trafficking process before reaching the PBII can lead to overaccumulation of the 57-kD precursor proteins (referred to as the 57H phenotype) in the seeds. Through the studies of 57H mutants, several key factors involved in post-Golgi trafficking of storage proteins have been characterized (Wang et al, 2010;Fukuda et al, 2011Fukuda et al, , 2013Liu et al, 2013;Ren et al, 2014). However, how these storage proteins are first exported from the ER remains largely unknown.…”