Poster: Protein Targeting & Vesicular Trafficking

Abs # 1184: Molecular mechanism of protein degradation upon seed germination

Presenter:	Li, Yu Bing , ljiang@cuhk.edu.hk
Authors	Li, Yu Bing  (A)   Zhao, Min  (A)   Samuel S.M., Sun  (A)   jiang, liwen  (A)
Affiliations:	(A): Department of Biology, The Chinese University of Hong Kong

Plants accumulate and store proteins in protein storage vacuole (PSV) during seed development. Upon seed germination, storage proteins are degraded by hydrolytic enzymes to provide nutrients for embryo/seedling growth. We demonstrated that PSVs in most seeds are compound organelles with subcompartments of distinct functions (Trends Cell Bio. 12:362; J. Cell Bio. 155:911). However, the molecular mechanism by which storage proteins are degraded within PSV upon seed germination remains unclear. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play role in mediating protein degradation via actively transporting proteases to sites where protein degradation occur. Towards this goal, we have produced new anti-VSR antibodies that cross-react specifically with VSR proteins of various plant seeds. We demonstrate that the amounts of both VSR proteins and proteases gradually increase upon seed germination. In contrast, the detectable amounts of marker proteins of storage function decrease coincidently. Confocal double-labeling studies demonstrate that VSR and specific proteases colocalize in the same organelles of germinating seeds. These VSR-marked transport vesicles are further identified via immunoEM. To identify ligands interacting with VSR in germinating seeds, microsome fractions are prepared and chemically cross-linked, the VSR-complex is then purified using affinity column conjugated with anti-VSR antibodies. Purified proteins are analyzed via Western blot and MALDI-TOF. So far, VSR proteins and two cysteine proteases (one contains the NPIR motif recognized by VSR) are identified. Thus, we have identified novel VSR ligands and transport vesicles involving protein degradation in germinating seeds. Supported by RGC (CUHK4156/01M and 4260/02M).