Subcellular localization of plant vacuolar sorting receptor proteins and their roles in mediating protein degradation during seed germination

Prevacuolar compartments (PVCs) are membrane-bound organelles that mediate protein traffic between the Golgi and vacuoles in the plant secretory pathway. Here I identify and define organelles as the lytic prevacuolar compartments in pea and tobacco cells using confocal immunofluorescence. I use five different antibodies specific for a vacuolar sorting receptor (VSR) BP-80 and its homologs to detect the location of VSR proteins. In addition, I use well-established Golgi-markers to identify Golgi organelles. I further compare VSR-labeled organelles to Golgi organelles so that the relative proportion of VSR proteins in Golgi vs. PVCs can be quantitated. More than 90% of the BP-80-marked organelles are separate from Golgi organelles; thus, BP-80 and its homologs are predominantly concentrated on the lytic PVCs. Additionally, organelles marked by anti-AtPep12p (AtSYP21p) and anti-AtELP antibodies are also largely separate from Golgi apparatus, whereas VSR and AtPep12p (AtSYP21p) are largely colocalized. I have thus demonstrated for the first time in plant cells that VSR proteins are predominantly present in the lytic PVCs and have provided additional markers for defining plant PVCs using confocal immunofluorescence. Additionally, my approach will provide a rapid comparison between markers to quantitate protein distribution among various organelles.; Upon seed germination, storage proteins are degraded by hydrolytic enzymes to provide nutrients for embryo/seedling growth. However, the molecular mechanism by which storage proteins are degraded within PSV upon seed germination remains unclear. Here I test the hypothesis that vacuolar sorting receptor (VSR) proteins play roles in mediating protein degradation via transporting proteases to sites where protein degradation occurs. Towards this goal, I have produced new anti-VSRat-1 antibodies that cross-react specifically with VSR proteins of various plant seeds. I demonstrate that the amounts of both VSR proteins and tested proteases gradually increase upon seed germination. In contrast, the amounts of marker proteins of storage function decrease coincidently. Confocal double-labeling studies demonstrate that VSR and aleurain colocalize in the same cytosolic organelles of germinating seeds. These putative PVC organelles were further identified through immuno-EM. To identify ligands interacting with VSR in germinating seeds, microsome fractions were prepared and chemically cross-linked, the VSR-ligand complex is then purified using affinity column conjugated with anti-VSRat-1 antibody. Purified proteins were further analyzed via Western blot and MALDI-TOF. In addition to aleurain, a putative novel protein was identified to interact with VSR proteins specifically. Thus, I have identified novel VSR ligands and PVCs that may involve in protein degradation in germinating seeds. Further isolation of PVCs from germinating seeds and their subsequent analysis will allow me to study their involvement in protein degradation of germinating seeds.