The N-myristoylated Rab-GTPase m-Rabmc is involved in post-Golgi trafficking events to the lytic vacuole in plant cells

Abstract

We report on the sub-cellular localisation and function of m-Rabmc, a N-myristoylated plant-specific Rab-GTPase previously characterised at the molecular level and also by structural analysis in Mesembryanthemum crystallinum. By confocal laser scanning microscopy, we identified m-Rabmc predominantly on the prevacuolar compartment of the lytic vacuole but also on the Golgi apparatus in various plant cell types. Two complementary approaches were used immunocytochemistry and cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP)-fusion proteins. Co-localisation studies of m-Rabmc with a salinity stress modulated integral calcium-ATPase suggest involvement of m-Rabmc in a plant-specific transport pathway to the prevacuolar compartment of the lytic vacuole. This hypothesis was strengthened by the inhibition of the transport of aleurain fused to green fluorescent protein (GFP), a marker of the lytic vacuole, in the presence of the dominant negative mutant m-Rabmc(N147I) in Arabidopsis thaliana protoplasts. The inhibitory effect of m-Rabmc(N147I) was specific for the transport pathway to the lytic vacuole, since the transport of chitinase-YFP, a marker for the neutral vacuole, was not hindered by the mutant.

The ATP Binding Cassette Transporter AtMRP5 Modulates Anion and Calcium Channel Activities in Arabidopsis Guard Cells

“…Seal resistances were Ͼ10 G⍀. The liquid junction potential was measured for I Ca analyses and corrected as described previously (33). pClamp software (version 8.0, Axon Instruments) was used to acquire and analyze whole cell currents.…”

“…In case of the vacuole we used the fluorescence dye FM4-64 (37,38). It has been reported that, after 60-min incubation, FM4-64 reaches the vacuolar membrane of BY2 cells and that, between 3 and 10 h, all vacuolar membranes are stained (33,39). As shown in Fig.…”

Evidence for a sorting endosome in Arabidopsis root cells

“…Three other groups have previously shown that the over-expression of dominant negative RABF proteins interferes with the correct sorting of lytic enzymes towards the vacuoles (Bolte et al, 2004a;Kotzer et al, 2004;Sohn et al, 2003). More recently, over-expression of a dominant negative form of RABF2b in Arabidopsis roots was reported to inhibit endocytosis and normal cell plate formation (Dhonukshe et al, 2006).…”

“…In contrast, in tobacco leaf epidermal cells, AtRABF2b was localized to the late endosomal/pre-vacuolar compartment (PVC) (Kotzer et al, 2004). Localization of RABF proteins to the PVC was also seen in other cell types (Bolte et al, 2004a;Haas et al, 2007;Lee et al, 2004;Sohn et al, 2003). The contradiction regarding the localization of RABF1 and RABF2b, which is probably due to the various model systems employed, precludes the use of these proteins as specific markers to distinguish between early and late endosomes/PVC.…”

Divergent functions of VTI12 and VTI11 in trafficking to storage and lytic vacuoles in Arabidopsis

“…Moreover, trafficking to these organelles can be selectively and independently inhibited (4,5), indicating that separate transport pathways to these vacuoles exist in plant cells. It is thought that proteins targeted to LVs are sorted in the Golgi complex by vacuolar sorting receptors that recruit cargo proteins into clathrin-coated vesicles for transport to the vacuole via a prevacuolar compartment (6, 7), a trafficking pathway similar to those present in other eukaryotes.…”