Overexpression of theArabidopsisSyntaxin PEP12/SYP21 Inhibits Transport from the Prevacuolar Compartment to the Lytic Vacuole in Vivo

Abstract

Golgi-mediated transport to the lytic vacuole involves passage through the prevacuolar compartment (PVC), but little is known about how vacuolar proteins exit the PVC. We show that this last step is inhibited by overexpression of Arabidopsis thaliana syntaxin PEP12/SYP21, causing an accumulation of soluble and membrane cargo and the plant vacuolar sorting receptor BP80 in the PVC. Anterograde transport proceeds normally from the endoplasmic reticulum to the Golgi and the PVC, although export from the PVC appears to be compromised, affecting both anterograde membrane flow to the vacuole and the recycling route of BP80 to the Golgi. However, Golgi-mediated transport of soluble and membrane cargo toward the plasma membrane is not affected, but a soluble BP80 ligand is partially mis-sorted to the culture medium. We also observe clustering of individual PVC bodies that move together and possibly fuse with each other, forming enlarged compartments. We conclude that PEP12/SYP21 overexpression specifically inhibits export from the PVC without affecting the Golgi complex or compromising the secretory branch of the endomembrane system. The results provide a functional in vivo assay that confirms PEP12/SYP21 involvement in vacuolar sorting and indicates that excess of this syntaxin in the PVC can be detrimental for further transport from this organelle.

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

“…Figure 1B shows that none of the total a-amylase activities (medium + cells) is reduced, thus ruling out cell mortality as a cause for induced secretion. By contrast, an increase in the total activity was seen in samples where vacuolar sorting was inhibited, which is explained by a higher stability of the reporter in the culture medium compared with the vacuole (Foresti et al, 2006).…”

“…Vam3 is a tail-anchored transmembrane SNARE that has been extensively used as a tonoplast marker (Saito et al, 2002;Uemura et al, 2002) and a green fluorescent protein (GFP)-Vam3 fusion was previously shown not to interfere with vacuolar sorting (Foresti et al, 2006).…”

Sorting and Anterograde Trafficking at the Golgi Apparatus: Figure 1.

“…In these cases, whether active sorting processes are required for these pathways is not clear, since proteins destined to the central lytic vacuole or the PSV are secreted out of the cell when vacuolar trafficking pathways are inhibited by various treatments, including overexpression of VSR/BP80 and RMR mutants or overexpression of the PVC-localized t-SNARE AtPEP12p (Shimada et al, 2003;Sohn et al, 2003;daSilva et al, 2005;Park et al, 2005;Foresti et al, 2006;Fuji et al, 2007;Craddock et al, 2008). The number of different routes for protein secretion and trafficking of plasma membrane proteins in plant cells is unknown.…”

A SNARE Complex Unique to Seed Plants Is Required for Protein Storage Vacuole Biogenesis and Seed Development ofArabidopsis thaliana

“…However, it is most likely that VAMP727 overexpression increases the incorporating efficiency for other Qa-SNAREs that have functions redundant with SYP22. The SYP21 protein could be a candidate for such a Qa-SNARE because it is also involved in traffic from the PVC (Foresti et al, 2006).…”