248:	Carrot cell wall invertase, a model to study protein retention in the ER

Authors:	Pagny, Sophie(A)Cabanes , Marion(A)Lerouge, Patrice(A)Gomord, Veronique(A)Faye, Loic(A)
Affiliations:	(A): LTI-CNRS ESA 6037
Presenter:	Pagny, Sophie , vgomord@crihan.fr

In order to analyse the ER retention mechanism in plant cells, we are using the extracellular invertase from carrot as a model glycoprotein. Carrot invertase has three N-glycans. Two of them are of complex type and are matured in the Golgi apparatus during the transport from the ER to the cell wall. In this study, our goal is a comparison of the structure of invertase glycans when the recombinant glycoprotein is transported to the cell wall (inv) or retained in the ER (invHDEL) of transgenic tobacco cells. The structural analysis of these glycans will give a good indication as to wether or not ER resident proteins recyle between the ER and the Golgi in plants. To solve the main problems generally encountered in this kind of approach we have decided a multiple tagging of our reporter protein not only for targeting (HDEL) but also for purification and immunodetection. Indeed, our selected method of purification uses an affinity tag consisting of six histidine residues (HIS). To analyse wether or not the addition of HIS is an efficient method to purify recombinant invertase from wild type proteins of tobacco, we fused this HIS tag to the N or C-terminal end of carrot invertase (invHIS or HISinv). In addition, to facilitate the immunodetection of the recombinant invertase, which cross-reacts with the endogenous tobacco invertase, we have fused a short FLAG epitope at the C-terminal end of carrot invertase. These forms of the same recombinant glycoprotein have been expressed in tobacco cells and a screening of transformed cells was performed with antibodies directed against the invertase or the HISFLAG sequence. Our preliminary results dealing with the purification, the immunolocalization and the glycosylation of the recombinant protein will be discussed in the poster.