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 Sec18p are required for vesicle fusion after Uso1p function. Here, we report that the docking reaction is sensitive to excess levels of Sec19p (GDI), a treatment that removes the GTPase, Ypt1p. Once docked, however, vesicle fusion is no longer sensitive to GDI. In vitro binding experiments demonstrate that the amount of Uso1p associated with membranes is reduced when incubated with GDI and correlates with the level of membrane-bound Ypt1p, suggesting that this GTPase regulates Uso1p binding 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 Uso1p-mediated docking, but block membrane fusion. We propose that an initial vesicle docking event of ER-derived vesicles, termed tethering, depends on Uso1p and Ypt1p but is independent of SNARE proteins.
Cao
X
X
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Ballew
N
N
Barlowe
C
C
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D018699:Coated Vesicles / Q000378:metabolism*; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000378:metabolism*; D051938:Munc18 Proteins; D010940:Plant Proteins / Q000378:metabolism; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013879:Thioredoxins / Q000378:metabolism; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins
C095309:BET1 protein, Zea mays; C068037:BOS1 protein, S cerevisiae; D002352:Carrier Proteins; D005656:Fungal Proteins; C086796:GDP dissociation inhibitor 1; D006023:Glycoproteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D008565:Membrane Proteins; D051938:Munc18 Proteins; D010940:Plant Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; C072520:SLY1 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C077554:Sed5 protein, S cerevisiae; C086888:USO1 protein, S cerevisiae; D033921:Vesicular Transport Proteins; C018169:yeast proteinase B inhibitor; D013879:Thioredoxins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C060755:YPT1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins