A vacuolar sorting domain may also influence the way in which proteins leave the endoplasmic reticulum

Protein sorting to plant vacuoles is known to be dependent on a considerabl e variety of protein motifs recognized by a family of sorting receptors. Th is can involve either traffic from the endoplasmic reticulum (ER) through t he Golgi apparatus or direct ER-to-vacuole transport. Barley aspartic prote ase (Phytepsin) was shown previously to reach the vacuole via trafficking t hrough the Golgi apparatus. Here we show that Phytepsin normally exits the ER in a COPII-mediated manner, because the Phytepsin precursor accumulates in the ER upon specific inhibition of the formation of COPII vesicles in vi vo. Phytepsin differs from its yeast and mammalian counterparts by the pres ence of a saposin-like plant-specific insert (PSI). Deletion of this domain comprising 104 amino acids causes efficient secretion of the truncated mol ecule (Phytepsin Delta PSI) without affecting the enzymatic activity of the enzyme. Interestingly, deletion of the PSI also changes the way in which P hytepsin exits the ER. Inhibition of COPII vesicle formation causes accumul ation of the Phytepsin precursor in the ER but has no effect on the secreti on of Phytepsin Delta PSI. This suggests either that vacuolar sorting comme nces at the ER export step and involves recruitment into COPII vesicles or that the PSI domain carries two signals, one for COPII-dependent export fro m the ER and one for vacuolar delivery from the Golgi. The relevance of the se observations with respect to the bulk flow model of secretory protein sy nthesis is discussed.