Secretory bulk flow of soluble proteins is efficient and COPII dependent

COPII-coated vesicles, first identified in yeast and later characterized in mammalian cells, mediate protein export from the endoplasmic reticulum (ER ) to the Golgi apparatus within the secretory pathway. In these organisms, the mechanism of vesicle formation is well understood, but the process of s oluble cargo sorting has yet to be resolved. In plants, functional compleme nts of the COPII-dependent protein traffic machinery were identified almost a decade ago, but the selectivity of the ER export process has been subjec t to considerable debate. To study the selectivity of COPII-dependent prote in traffic in plants, we have developed an in vivo assay in which COPII ves icle transport is disrupted at two distinct steps in the pathway. First, ov erexpression of the Sari p-specific guanosine nucleotide exchange factor Se c12p was shown to result in the titration of the GTPase Sar1p, which is ess ential for COPII-coated vesicle formation. A second method to disrupt COPII transport at a later step in the pathway was based on coexpression of a do minant negative mutant of Sar1p (H74L), which is thought to interfere with the uncoating and subsequent membrane fusion of the vesicles because of the lack of GTPase activity. A quantitative assay to measure ER export under t hese conditions was achieved using the natural secretory protein barley a-a mylase and a modified version carrying an ER retention motif. Most importan tly, the manipulation of COPII transport in vivo using either of the two ap proaches allowed us to demonstrate that export of the ER resident protein c alreticulin or the bulk flow marker phosphinothricin acetyl transferase is COPII dependent and occurs at a much higher rate than estimated previously. We also show that the instability of these proteins in post-ER compartment s prevents the detection of the true rate of bulk flow using a standard sec retion assay. The differences between the data on COPII transport obtained from these in vivo experiments and in vitro experiments conducted previousl y using yeast components are discussed.