Poster: Organelle Biogenesis
Abs #
1153: Molecular characterization of plant prevacuolar compartments
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Presenter: |
jiang, liwen , ljiang@cuhk.edu.hk |
Authors | jiang, liwen (A) Tse, Yu Chung (A) Lo, Sze Wan (A) | | Affiliations: |
(A): Department of Biology and Molecular Biotechnology Program, The Chinese University of Hong Kong
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Little is known about dynamics and molecular components of plant prevacuolar compartments (PVCs). We recently demonstrated that vacuolar sorting receptor (VSR) proteins concentrated on PVCs (Plant Cell & Physiol. 43:726). Here we develop PVC markers in living cells based on our previous studies on traffic of integral membrane reporter proteins (J. Cell Biol. 143:1183). Toward this goal, we have generated transgenic tobacco BY-2 cell lines expressing two YFP-fusions (termed YFP1 and YFP2 fusions) that mark distinct PVCs for lytic vacuole and protein storage vacuole respectively. As a control, we have also generated cell lines expressing a Golgi marker GONST1-YFP. All transgenic cell lines exhibit punctuating YFP signals. Confocal immunofluorescence and uptake studies demonstrate that both YFP1 and YFP2 fusions colocalize with VSR- and FM4-64-marked PVC organelles, but do not colocalize with Man1- or JIM84-marked Golgi. In contrast, GONST1-YFP do not colocalize with VSR-marked PVCs. The dynamics of YFP-marked PVCs are studied in living cells. We demonstrate that the physiological culture conditions and some drugs affect the biogenesis and change sizes of YFP-marked PVCs in transgenic cells. ImmunoEM study is performed to identify the morphology of normal and enlarged YFP-marked PVCs. In addition, studies using transgenic cell lines co-expressing two GFP/YFP fusions that mark Golgi and PVCs demonstrate that PVC is distinct from Golgi, and that two distinct PVCs coexist in the same living cells. Current efforts focus on PVC proteomics via purification of YFP-marked PVC organelles and their subsequent analysis of protein components. Our study will contribute novel insight into molecular mechanism of PVC biogenesis and protein sorting. Supported by RGC (CUHK 4260/02M).
