GLUTELIN PRECURSOR ACCUMULATION3 encodes a regulator of post-Golgi vesicular traffic essential for vacuolar protein sorting in rice endosperm.

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.