Protein Trafficking to the Plant Vacuolar Membrane
Research in our lab is focused on targeting mechanisms for plant tonoplast proteins using both classical genetic and chemical genetic approaches. We have been involved in using these two approaches to uncover novel developmental roles of the endomembrane system (Sohn et al.2007) and novel inhibitors of tonoplast protein trafficking (Surpin et al. 2005, Rojas-Pierce et al. 2007).
Tonoplast membrane transporters carry out the movement of molecules in and out of the plant vacuole including lipids, ions, sugars, hormones and defense molecules. The transport of molecules across the tonoplast is essential for plant vacuolar function, including the maintenance of cellular turgor and ion sequestration, which ultimately control osmotic potential of the cytosol. To improve plant stress tolerance and ultimately increase the nutritional value of plants for human consumption, it is essential that we understand how to regulate vacuolar trafficking of membrane proteins. We use the family of tonoplast intrinsic proteins (TIPs) as models for tonoplast proteins. Two pathways have been proposed for delivery of protein trafficking to the tonoplast, a Golgi-dependent and a Golgi-independent pathway. Our work has shown that, in Arabidopsis, TIP3;1 and TIP2;1 are likely to use a Golgi-independent pathway, while TIP1;1 is likely to use a Golgi-dependent pathway.
Novel inhibitors of tonoplast protein trafficking
In order to characterize trafficking pathways specific to vacuolar membrane proteins, we developed a new screen for chemical inhibitors that affect protein trafficking to the vacuole. A dual-label marker line where a tonoplast marker protein is labeled with GFP (green) and an endoplasmic reticulum (ER) marker is labeled with mCherry (red fluorescence) was used for this purpose. Using this strategy, we have identified five inhibitors that induce the mis-localization of the tonoplast marker at the ER. Our new inhibitors are specific to tonoplast proteins and one is specific for a subset of these (TIP2;1 and TIP3;1 but not TIP1;1), indicating that we can indeed uncouple multiple pathways of membrane protein trafficking with our chemical probes. The effect of C834 on the localization of TIP2;1 is shown below.
C834 inhibits the targeting of the GFP-TIP2;1 marker protein to the tonoplast.
Genetic screen for tonoplast protein trafficking regulators
With the goal of identifying proteins that regulate the trafficking pathway for TIP2;1 and TIP3;1, we have completed a genetic screen for mutants in which these proteins are mis-localized. Current efforts include the identification of mutations in these plants and characterization of the function of encoded genes on protein trafficking. One example of these mutants is shown below.
Phenotype of a impaired traficking to the tonoplast (itt) mutants