EXPO, exocyst recruitment, and unconventional protein secretion

Exocyst is an evolutionary conserved multisubunit tethering factor composed of 8 proteins: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84. It was originally described as a protein complex which captures and guides secretory vesicles to the plasma membrane (PM) prior to cognate SNARE-mediated membrane fusion. Exocyst homologs have also been found in plants, but in contrast to yeast and animals, the Arabidopsis genome contains 23 paralogs of Exo70 (AtExo70). It has been proposed that plant cells do not have a single exocyst complex and that different exocyst complexes participate in different physiological processes. Using AtExo70E2-GFP as a probe, we have recently identified a novel double-membrane organelle termed EXPO (exocyst-positive organelle) that mediates an unconventional protein secretion in plant cells. EXPO can also be recognized in normal cells by immunofluorescence and immunogold electron microscopy using Exo70E2 antibodies. EXPO are present as discrete structures in the cytoplasm but have also been visualized fusing to the PM. Structurally, EXPO is morphologically similar to the double-membrane autophagosome However, under normal conditions, immunofluorescent labelling with the EXPO marker antiAtExo70E2 and the autophagosomal markers anti-ATG8e or anti-SH3P2 in transgenic Arabidopsis cell lines expressing either GFP-tagged EXPOs or autophagosomes shows that autophagosomes and EXPO are distinct organelles. Nevertheless, upon autophagy induction via starvation in Arabidopsis cells expressing GFP-tagged EXPO or autophagosomal markers, EXPO signals are found to colocalize with autophagic bodies in the vacuoles. By performing transient expression in Arabidopsis protoplasts we have established that a number of exocyst subunits (especially the members of the Sec family) are unable to be recruited to EXPO in the absence of AtExo70E2. The paralog AtExo70A1 is unable to substitute for AtExo70E2 in this regard. FRET and BiFC analyses have confirmed the interaction between AtExo70E2 and Sec6 and Sec10. AtExo70E2, but not its yeast counterpart, is also capable of inducing EXPO formation in an animal cell line (HEK293A cells). Electron microscopy confirmed the presence of double membraned EXPO-like structures in HEK293A cells expressing AtExo70E2. Inversely, neither human nor yeast Exo70 homologs are able to cause the formation of EXPO in Arabidopsis protoplasts. These results point to a specific and crucial role for AtExo70E2 in EXPO formation. L9.2