Arabidopsis dynamin-related proteins, DRP2A and DRP2B, function coordinately in post-Golgi trafficking

Dynamin-related proteins (DRPs) are large GTPases involved in a wide range of cellular membrane remodeling processes. In Arabidopsis thaliana, two paralogous land plant-specific type DRPs, DRP2A and DRP2B, are thought to participate in the regulation of post-Golgi trafficking. Here, we examined their molecular properties and functional relationships. qRT-PCR and GUS assays showed that DRP2A and DRP2B were expressed ubiquitously, although their expressions were strongest around root apical meristems and vascular bundles. Yeast two-hybrid, bi-molecular fluorescent complementation, and co-immunoprecipitation mass spectrometry analyses revealed that DRP2A and DRP2B interacted with each other. In observations with confocal laser scanning microscopy and variable incidence angle fluorescent microscopy, fluorescent fusions of DRP2A and DRP2B almost completely co-localized and were mainly localized to endocytic vesicle formation sites of the plasma membrane, clathrin-enriched trans-Golgi network and the cell plate in root epidermal cells. Treatments with wortmannin, an inhibitor of phosphatidylinositol 3-/4-kinases, latrunculin B, an inhibitor of actin polymerization, and oryzalin, an inhibitor of microtubule polymerization, increased the resident time of DRP2A and DRP2B on the plasma membrane. These results show that DRP2A and DRP2B function coordinately in multiple pathways of post-Golgi trafficking in phosphatidylinositol 3- or 4-kinase and cytoskeleton polymerization-dependent manners.