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 Endosperm      

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-offunction 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 a-globulins to PSV was disrupted in glup6 endosperm. Secreted granules of glutelin and a-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, b-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 a-globulin from the Golgi to PSVs and in the maintenance of the general structural organization of the endomembrane system in rice seeds.

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

“…For example, the esp2/pdi1-1 mutant forms small PBIs, W379/vpe1 develops round PBIIs (Wang et al, 2009), while gpa3 contains normal PBIs but small PBIIs. The observed gpa3 mutant phenotypes and subcellular defects are most reminiscent of the recently reported glup4/gpa1/rab5a and glup6/gpa2/vps9a mutants (Wang et al, 2010;Fukuda et al, 2011Fukuda et al, , 2013Liu et al, 2013). However, the defects in gpa3 appeared to be more severe than those observed in glup4/gpa1/rab5a and glup6/gpa2/ vps9a (Figure 7).…”

“…It has been reported that loss of function of rice GLUP4/GPA1/ Rab5a results in partial secretion of proglutelins and a-globulins to the extracellular space (Wang et al, 2010;Fukuda et al, 2011). Recently, rice VPS9a has also been identified to be the GEF of Rab5a, and its loss-of-function mutant gpa2/glup6 displays similar but more severe phenotypic defects than glup4/gpa1/ rab5a (Fukuda et al, 2013;Liu et al, 2013). These observations support a role for Rab5a and VPS9a in the transport of proglutelins from the Golgi apparatus to PSV.…”

“…Among them, ESP2 encodes PDI1-1 (for PROTEIN DISULFIDE ISOMERASE-LIKE1-1), involved in proglutelin maturation in the ER (Takemoto et al, 2002). GLUP4 and GLUP6 encode the small GTPase Rab5a and its GEF, VPS9a, respectively, and they were shown to work together in regulating post-Golgi trafficking of proglutelins and a-globulins (Fukuda et al, 2011(Fukuda et al, , 2013, while GLUP3 codes for a vacuolar processing enzyme, which proteolytically processes proglutelins into acidic and basic subunits within the PSV . We previously reported three 57H mutants named W379, glutelin precursor accumulation1 (gpa1), and gpa2, which are allelic to glup3, glup4, and glup6, respectively (Wang et al, 2009(Wang et al, , 2010Liu et al, 2013).…”

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.…”

“…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.…”