Vacuolar Transport in Tobacco Leaf Epidermis Cells Involves a Single Route for Soluble Cargo and Multiple Routes for Membrane Cargo

Abstract

We tested if different classes of vacuolar cargo reach the vacuole via distinct mechanisms by interference at multiple steps along the transport route. We show that nucleotide-free mutants of low molecular weight GTPases, including Rab11, the Rab5 members Rha1 and Ara6, and the tonoplast-resident Rab7, caused induced secretion of both lytic and storage vacuolar cargo. In situ analysis in leaf epidermis cells indicates a sequential action of Rab11, Rab5, and Rab7 GTPases. Compared with Rab5 members, mutant Rab11 mediates an early transport defect interfering with the arrival of cargo at prevacuoles, while mutant Rab7 inhibits the final delivery to the vacuole and increases cargo levels in prevacuoles. In contrast with soluble cargo, membrane cargo may follow different routes. Tonoplast targeting of an a-TIP chimera was impaired by nucleotide-free Rha1, Ara6, and Rab7 similar to soluble cargo. By contrast, the tail-anchored tonoplast SNARE Vam3 shares only the Rab7-mediated vacuolar deposition step. The most marked difference was observed for the calcineurin binding protein CBL6, which was insensitive to all Rab mutants tested. Unlike soluble cargo, a-TIP and Vam3, CBL6 transport to the vacuole was COPII independent. The results indicate that soluble vacuolar proteins follow a single route to vacuoles, while membrane spanning proteins may use at least three different transport mechanisms.

Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

“…During the preparation of this report, Batistič et al [46] reported that S-acylation of three cysteine residues in the Nterminus is required for vacuolar targeting of CBL2 in a Brefeldin A-insensitive pathway. Particularly relevant to this study, it was also shown that the CBL6-GFP chimera with a similar TTS at the N-terminus was targeted to tonoplast independently from the typical secretory pathway [47]. In other words, TTS is distinct from a typical signal peptide that mediates protein translocation through the secretory pathway.…”

Trafficking of Vacuolar Proteins: The Crucial Role of Arabidopsis Vacuolar Protein Sorting 29 in Recycling Vacuolar Sorting Receptor

“…However, GFP-VSR2(Y612A) accumulates to the TGN and GFP-BP80-Y612A trafficked to the plasma membrane Foresti et al, 2010). Moreover, a vacuolar cargo Aleu-RFP was trapped in the ER when COPIImediated ER export was inhibited by overexpression of Sec12 (Bottanelli et al, 2011). This alternative model was proposed based on the results of a study showing the localization of SNX2 at the TGN and redistribution of VSRs to the TGN in the presence of a mutant form of SNX (Niemes et al, 2010b).…”

ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN, an Interacting Partner of RAB5 GTPases, Regulates Membrane Trafficking to Protein Storage Vacuoles in Arabidopsis

“…The unique organization of endosomal trafficking in plants is also demonstrated by the observation that the trans-Golgi network (TGN) acts as an early endosome in plants (Dettmer et al, 2006;Lam et al, 2007;Viotti et al, 2010), from which multivesicular endosomes are proposed to be directly generated by maturation (Niemes et al, 2010;Scheuring et al, 2011). The RAB11 group (also known as RABA in Arabidopsis), which is localized to TGNrelated secretory compartments and is involved in cell plate formation and tip growth (Preuss et al, 2004;Chow et al, 2008;Szumlanski and Nielsen, 2009;Kang et al, 2011;Feraru et al, 2012;Asaoka et al, 2013;Berson et al, 2014;Kirchhelle et al, 2016), is also implicated in endosomal/vacuolar trafficking (Bottanelli et al, 2011;Choi et al, 2013). These lines of evidence indicate that endosomal trafficking in plant cells operates in a different manner from that in non-plant organisms.…”

“…Canonical RAB5s and ARA6 are localized to distinct but overlapping populations of multivesicular endosomes Haas et al, 2007;Ebine et al, 2011), suggesting diversification of their functions. ARA6 acts in the endosome-to-plasma membrane trafficking pathway (Ebine et al, 2011) and may also be involved in a vacuolar trafficking pathway (Bottanelli et al, 2011). ARA7 and RHA1, in contrast, redundantly regulate endocytic and vacuolar trafficking pathways (Sohn et al, 2003;Kotzer et al, 2004;Dhonukshe et al, 2006;Ebine et al, 2011Ebine et al, , 2014Beck et al, 2012).…”