Post-Golgi trafficking of rice storage proteins requires the small GTPase Rab7 activation complex MON1–CCZ1

Abstract

Protein storage vacuoles (PSVs) are unique organelles that accumulate storage proteins in plant seeds. Although morphological evidence points to the existence of multiple PSV-trafficking pathways for storage protein targeting, the molecular mechanisms that regulate these processes remain mostly unknown. Here, we report the functional characterization of the rice (Oryza sativa) glutelin precursor accumulation7 (gpa7) mutant, which over-accumulates 57-kD glutelin precursors in dry seeds. Cytological and immunocytochemistry studies revealed that the gpa7 mutant exhibits abnormal accumulation of storage pre-vacuolar compartment-like structures, accompanied by the partial mistargeting of glutelins to the extracellular space. The gpa7 mutant was altered in the CCZ1 locus, which encodes the rice homolog of Arabidopsis (Arabidopsis thaliana) CALCIUM CAFFEINE ZINC SENSITIVITY1a (CCZ1a) and CCZ1b. Biochemical evidence showed that rice CCZ1 interacts with MONENSIN SENSITIVITY1 (MON1) and that these proteins function together as the Rat brain 5 (Rab5) effector and the Rab7 guanine nucleotide exchange factor (GEF). Notably, loss of CCZ1 function promoted the endosomal localization of Vacuolar Protein Sorting-associated protein 9 (VPS9), which is the GEF for Rab5 in plants. Together, our results indicate that the MON1-CCZ1 complex is involved in post-Golgi trafficking of rice storage protein through a Rab5 and Rab7-dependent pathway.

Endomembrane‐mediated storage protein trafficking in plants: Golgi‐dependent or Golgi‐independent?

“…Instead, sPVC (200–500 nm in diameter) has been postulated to transport glutelins in rice endosperm [54]. Mutation of CCZ1 induces the accumulation of numerous sPVC‐like structures in rice endosperm [109], suggesting that the MON1/SAND‐CCZ1 complex functions in mediating sPVC fusion with PSVs in the indirect DV‐to‐PSV pathway. How Rab5 recruits different effectors to control SSP trafficking in plant seeds remains to be investigated.…”

“…Small GTPase Rab5 cycling between active and inactive variants via GEF regulation functions as the master regulator for two different post-Golgi trafficking pathways. One pathway corresponding to the DVmediated direct fusion with PSVs requires Rab5 and its effector GPA5, whereas the other pathway corresponding to DV-to-sPVC/MVB route is mediated by Rab5 and Rab7 [66,[106][107][108][109] (Fig. 1).…”

Rice seed storage proteins: Biosynthetic pathways and the effects of environmental factors

“…In dicots such as Arabidopsis and pea, once storage proteins have been exported from the Golgi in DVs, the DVs fuse with small vesicles to form prevacuolar compartments (PVCs)/ multivesicular bodies (MVBs) before being delivered to the PSVs, where they initiate the proteolytic processing of storage proteins (Otegui et al, 2006). Although PVCs/MVBs have occasionally been observed in developing rice endosperm (Shen et al, 2011;Pan et al, 2021), DVs dominate the entire process of SSP transport from the TGN to the PSV in rice.…”

“…Indeed, a collateral proglutelin trafficking pathway in which storage PVCs act as intermediate compartments in TGN-to-PSV transport has recently been revealed (Shen et al, 2011;Pan et al, 2021). CALCIUM CAFFEINE ZINC1 (CCZ1/GPA7) interacts with MONENSIN SENSITIVITY1 (MON1) to form a dimeric complex that functions as the effector of Rab5 (such as Rab5a) and the GEF of Rab7 (such as Rab7b3) to participate in the PVC/MVBmediated post-Golgi trafficking of proglutelins in rice (Pan et al, 2021). Thus, it appears that Rab5 is the core regulator of storage protein trafficking from the TGN to PSV and participates in different post-Golgi transport pathways by employing different downstream effectors, such as GPA5, the MON1-CCZ1 complex and other unknown effectors.…”

“…Notably, the loss of GPA5 function leads to only a moderate sorting defect among proglutelins, suggesting that another pathway also contributes to the trafficking of proglutelins to the PSVs in rice endosperm. Indeed, a collateral proglutelin trafficking pathway in which storage PVCs act as intermediate compartments in TGN-to-PSV transport has recently been revealed (Shen et al, 2011;Pan et al, 2021). CALCIUM CAFFEINE ZINC1 (CCZ1/GPA7) interacts with MONENSIN SENSITIVITY1 (MON1) to form a dimeric complex that functions as the effector of Rab5 (such as Rab5a) and the GEF of Rab7 (such as Rab7b3) to participate in the PVC/MVBmediated post-Golgi trafficking of proglutelins in rice (Pan et al, 2021).…”