EV-based secretion in plants. UPS of the cargo lectin Helja does not involve MVB

PINEDO M; DE LA CANAL L; FASSOLARI M

Assisi

Workshop; Molecular mechanisms of unconventional protein secretion in eukaryotic cells; 2019

EMBO

Different UPS pathways have been proposed in plants, mainly based on the information available for other eukaryotes. Among them a vesicular route involving extracellular vesicles (EV) has been suggested for Helja, a lectin that could function as a model cargo. Helja is a signal peptide-lacking protein already detected in sunflower apoplast (plant extracellular compartment) and in EV. Current working models in plant systems assume that multivesicular bodies (MBV) are involved in EV-mediated secretion, although no experimental demonstration of the molecular mechanism involved has yet been presented.Here we analyze the secretion of a fluorescent tagged version of Helja. Transient expression of GFP-Helja in Nicotiana benthamiana shows its secretion to the extracellular compartment, revealing that the lectin exhibits UPS signatures recognized in the heterologous system. Its apoplastic localization was verified by partial colocalization with the protein marker SEC-RFP using confocal microscopy and by immunodetection in extracellular fluids isolated from transformed plants. Notably, GFP-Helja was also detected in EV obtained from N. benthamiana apoplastic fluids, supporting its transport in an EV-dependent UPS pathway. The tagged protein was also observed in intracellular structures which did not colocalize with markers for trans-Golgi and autophagosomes. Besides, neither colocalization with the membrane endocytic marker FM 4-64, nor with two MBV protein markers was detected. These findings contradict the current working model assuming that MBV are involved in Helja secretion. On the other hand, colocalization with the endoplasmic reticulum (ER) marker HDEL was observed. Our results reveal that the UPS secretion of Helja-containing EV uses a yet unidentified vesicular pathway probably deriving from the ER. The participation of EXPO- (exocyst positive organelle) and CUPS- (compartment for unconventional protein secretion) like structures is discussed.