Poster: Protein targeting & vesicular trafficking

Abs # 642: Dynamic of ER protein export in tobacco BY-2 cells

Presenter:	Lu, Shanxiang , biolue@hotmail.com
Authors	Lu, Shanxiang  (A)   Sun, Sai-Ming S. (A)   Jiang, Liwen  (A)
Affiliations:	(A): Department of Biology, the Chinese University of Hong Kong

A fusion protein composed of signal peptide (sp), yellow fluorescent protein (YFP) and a cytosolic lysine-rich protein (LRP) was expressed and found to mainly locate to in the endoplasmic reticulum (ER) of transgenic tobacco BY-2 cells. However, when a control fusion protein composed of LRP and YFP was expressed in BY-2 cells, it located in both nucleus and cytoplasm. Here we use transgenic BY-2 cell lines expressing the sp:YFP:LRP fusion as a tool to study protein export from the ER using both confocal scanning laser microscope (CSLM) and electron microscope (EM). When living cells expressing the sp:YFP:LRP fusion were treated with Brefeldin A (BFA), the ER-located YFP fusions were redistributed from an ER-network pattern into a punctate pattern, and such change is reversible after washing off the BFA. In addition, unique possibly ER-derived ring-like structures were found in cytosol and vacuoles under TEM in BFA-treated transgenic cells. On the other hand, when the cells were incubated at 37 degrees, a temperature promoting protein export from mammalian ER, a time-course induction of novel network containing YFP signals was identified under CSLM and such network was found to contain numerous vesicles adjacent to the nucleus under EM. In addition, Golgi stacks were found to be reduced in these temperature-treated cells, indicating that the temperature treatment may cause the loss of Golgi membrane possibly to be used for the formation of new vesicles involving protein transport. Further studies focus on confirming the origins of ER-derived vesicles via immuno-EM and studying their physiological roles in protein transport. Supported by RGC and AoE to L. Jiang.