Wortmannin induces homotypic fusion of plant prevacuolar compartments*

Abstract

Wortmannin, a specific inhibitor of phosphatidyl-inositol 3-kinase, is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. It has recently been demonstrated that wortmannin at 16.5 μM or 33 μM caused the prevacuolar compartments (PVCs), identified as multivesicular bodies (MVBs) by their enrichment in vacuolar sorting receptor (VSRs) proteins and the BP-80 reporter, to form small vacuoles rapidly. However, the source(s) of the membrane needed for the rapid enlargement of PVCs/MVBs has been unclear. Using both confocal immunofluorescence and immunogold EM with high pressure freeze substitution of plant samples, it has been demonstrated here that wortmannin induces homotypic fusions of PVCs/MVBs thus providing an explanation for the demand for extra membrane. In addition, possible wortmannin-induced fusions between the trans-Golgi network (TGN) and PVC, as well as between the small internal vesicles and PVC membrane, were also observed and they may also contribute to the membranes needed for PVC enlargement. In contrast to mammalian cells and yeast, wortmannin-induced fusion of PVCs appears to be unique to plants.

Activation of the Rab7 GTPase by the MON1-CCZ1 Complex Is Essential for PVC-to-Vacuole Trafficking and Plant Growth in Arabidopsis

“…Statistical analysis showed that the average diameter of PVCs in mon1-2 and RABG3f T22N was larger than that in wild-type cells (Supplemental Figure 18A). Enlarged PVCs were largely derived from homotypic fusion of PVCs (Supplemental Figures 18B and 18C), a result similar to ARA7 Q69L and Wort-induced enlarged PVC structures (Wang et al, 2009;Jia et al, 2013). Golgi stacks and the TGN remained normal in both mon1-2 and RABG3f T22N root cells (Supplemental Figures 18D to 18F).…”

Rapid endocytosis is triggered upon imbibition in Arabidopsis seeds

“…Wortmannin and LY294002 inhibit phosphoinositol-3-kinase an enzyme responsable for the production of phosphoinositol-3-phosphate, (PI-3P), a characteristic lipid of endosome membranes. 42 On the other hand, tyrphostin A23 is a tyrosine analog that competes with type YXXф protein domains for its interaction with clathrin. In consequence, clathrin coated vesicles in tyrphostin A23 treated cells, are not capable to recruit the proteins to be endosytosed.…”

A Recycling-Defective Vacuolar Sorting Receptor Reveals an Intermediate Compartment Situated between Prevacuoles and Vacuoles in Tobacco

“…Interesting parallels can also be drawn from observations with the drug wortmannin, which was shown to inhibit VSR recycling (daSilva et al, 2005), leading to colocalization of TGN and PVC markers in enlarged vacuolated structures (Wang et al, 2009). If PVC-to-LPVC maturation depends on the selective retrieval of VSRs, then it stands to reason that wortmannin would cause a maturation defect, but it is remarkable that it also leads to fusion with the TGN.…”