Vacuolar sorting receptor (VSR) proteins reach the plasma membrane in germinating pollen tubes

Vacuolar sorting receptors (VSRs) are type I integral membrane proteins that mediate the vacuolar transport of soluble cargo proteins via prevacuolar compartments (PVCs) in plants. Confocal immunofluorescent and immunogold Electron Microscope (EM) studies have localized VSRs to PVCs or multivesicular bodies (MVBs) and trans-Golgi network (TGN) in various plant cell types, including suspension culture cells, root cells, developing and germinating seeds. Here, we provide evidence that VSRs reach plasma membrane (PM) in growing pollen tubes. Both immunofluorescent and immunogold EM studies with specific VSR antibodies show that, in addition to the previously demonstrated PVC/MVB localization, VSRs also localize to PM in lily and tobacco pollen tubes prepared from chemical fixation or high-pressure freezing/frozen substitution. Such a PM localization suggests an additional role of VSR proteins in mediating protein transport to PM and endocytosis in growing pollen tubes. Using a high-speed Spinning Disc Confocal Microscope, the possible fusion between VSR-positive PVC organelles and the PM was also observed in living tobacco pollen tubes transiently expressing the PVC reporter GFP-VSR. In contrast, the lack of a prominent PM localization of GFP-VSR in living pollen tubes may be due to the highly dynamic situation of vesicular transport in this fast-growing cell type.