A Guanine Nucleotide Exchange Factor for Rab5 Proteins Is Essential for Intracellular Transport of the Proglutelin from the Golgi Apparatus to the Protein Storage Vacuole in Rice Endosperm1[C][W][OA]

GLUP6/GEF is the activator of Rab5 GTPase, and the cycling of GTP- and GDP-bound forms of this regulatory protein is essential for the intracellular transport of proglutelin and α-globulin from the Golgi to PSV and in the maintenance of the general structural organization of the endomembrane system in rice seeds. Rice ( Oryza sativa ) glutelins are synthesized on the endoplasmic reticulum as a precursor, which are then transported via the Golgi to protein storage vacuoles ( PSVs ), where they are proteolytically processed into acidic and basic subunits. The glutelin precursor mutant6 ( glup6 ) accumulates abnormally large amounts of proglutelin. Map-base cloning studies showed that glup6 was a loss-of-function mutant of guanine nucleotide exchange factor (GEF), which activates Rab GTPase, a key regulator of membrane trafficking. Immunofluorescence studies showed that the transport of proglutelins and α-globulins to PSV was disrupted in glup6 endosperm. Secreted granules of glutelin and α-globulin were readily observed in young glup6 endosperm, followed by the formation of large dilated paramural bodies ( PMBs ) containing both proteins as the endosperm matures. The PMBs also contained membrane biomarkers for the Golgi and prevacuolar compartment as well as the cell wall component, β-glucan. Direct evidence was gathered showing that GLUP6/GEF activated in vitro GLUP4/Rab5 as well as several Arabidopsis ( Arabidopsis thaliana ) Rab5 isoforms to the GTP-bound form. Therefore, loss-of-function mutations in GEF or Rab5 disrupt the normal transport of proglutelin from the Golgi to PSVs , resulting in the initial extracellular secretion of these proteins followed, in turn, by the formation of PMBs . Overall, our results indicate that GLUP6/GEF is the activator of Rab5 GTPase and that the cycling of GTP- and GDP-bound forms of this regulatory protein is essential for the intracellular transport of proglutelin and α-globulin from the Golgi to PSVs and in the maintenance of the general structural organization of the endomembrane system in rice seeds.