Abstract
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.
Highlights
Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as a precursor, which are transported via the Golgi to protein storage vacuoles (PSVs), where they are proteolytically processed into acidic and basic subunits
Intracellular transport of glutelins and a-globulins is disrupted in glup4, where the bulk of these storage proteins are secreted and accumulate in the space formed between the invaginating plasma membrane and the cell wall, forming a paramural body (PMB)
The size of the large distended structures in glup6 mutants increased during seed development, unlike protein body type II (PB-II), whose size did not change (Supplemental Fig. S5, C–F and I–L). As this distended structure was common to all three independently derived glup6 lines, its formation can be directly attributed to the loss of function of GLUP6/guanine nucleotide exchange factor (GEF). These results suggest that the normal transport of proglutelin to the PSV is disrupted in the glup6 mutant and that GLUP6/GEF is an essential factor in this transport process
Summary
Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as a precursor, which are transported via the Golgi to protein storage vacuoles (PSVs), where they are proteolytically processed into acidic and basic subunits. Seeds of glup, which belongs to the same class as the glup4/rab mutant, exhibit a protein pattern similar to glup in having elevated proglutelin levels and reduced levels of acidic and basic glutelin subunits, prolamines, and 26-kD a-globulin (Ueda et al, 2010) These observations suggest that glup acts in the same pathway responsible for the intracellular transport of proglutelins from the Golgi to the PSV as glup. PMBs are frequently observed in plant tissues in which exocytosis and endocytosis actively occur (Wang et al, 2005; An et al, 2006a, 2006b) This view is consistent with the condition seen in the rice Rab glup mutant, where glutelin precursors as well as many other components of the endosome system, including b-glucan, assemble to form large PMBs in developing endosperm (Fukuda et al, 2011). Our results indicate that the GDP/GTP recycling of GLUP4/Rab by GLUP6/GEF is a key regulatory process in the trafficking of storage proteins to the PSV and in the general maintenance of the endomembrane system in developing rice endosperm
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