INITIAL DOCKING OF ER-DERIVED VESICLES REQUIRES USO1P AND YPT1P BUT IS INDEPENDENT OF SNARE PROTEINS

ER-to-Golgi transport in yeast may be reproduced in vitro with washed membranes, purified proteins (COPII, Uso1p and LMA1) and energy COPII coated vesicles that have budded from the ER are freely diffusible but then dock to Golgi membranes upon the addition of Uso1p, LMA1 and Sec 18p are required for vesicle fusion after Uso1p function. sere, we rep ort that the docking reaction is sensitive to process levels of Sec19p (GDI), a treatment that removes the GTPase, Ypt1p, Once docked, howev er, vesicle fusion is no longer sensitive to GDI. In vitro binding exp eriments demonstrate that the amount of Uso1p associated with membrane s is reduced when incubated with GDI and correlates with the level of membrane-bound Ypt1p, suggesting that this GTPase regulates Uso1p bind ing to membranes, To determine the influence of SNARE proteins on the vesicle docking step, thermosensitive mutations in Sed5p, Bet1p, Bos1p and Sly1p that prevent ER-to-Golgi transport in vitro at restrictive temperatures were employed, These mutations do not interfere with Uso1 p-mediated docking, but block membrane fusion, We propose that an init ial vesicle docking event of ER-derived vesicles, termed, tethering, d epends on Uso1p and Ypt1p but is independent of SNARE proteins.