De Novo Formation of Plant Endoplasmic Reticulum Export Sites Is Membrane Cargo Induced and Signal Mediated

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Abstract
The plant endoplasmic reticulum (ER) contains functionally distinct subdomains at which cargo molecules are packed into transport carriers. To study these ER export sites (ERES), we used tobacco (Nicotiana tabacum) leaf epidermis as a model system and tested whether increased cargo dosage leads to their de novo formation. We have followed the subcellular distribution of the known ERES marker based on a yellow fluorescent protein (YFP) fusion of the Sec24 COPII coat component (YFP-Sec24), which, differently from the previously described ERES marker, tobacco Sar1-YFP, is visibly recruited at ERES in both the presence and absence of overexpressed membrane cargo. This allowed us to quantify variation in the ERES number and in the recruitment of Sec24 to ERES upon expression of cargo. We show that increased synthesis of membrane cargo leads to an increase in the number of ERES and induces the recruitment of Sec24 to these ER subdomains. Soluble proteins that are passively secreted were found to leave the ER with no apparent up-regulation of either the ERES number or the COPII marker, showing that bulk flow transport has spare capacity in vivo. However, de novo ERES formation, as well as increased recruitment of Sec24 to ERES, was found to be dependent on the presence of the diacidic ER export motif in the cytosolic domain of the membrane cargo. Our data suggest that the plant ER can adapt to a sudden increase in membrane cargo-stimulated secretory activity by signal-mediated recruitment of COPII machinery onto existing ERES, accompanied by de novo generation of new ERES.