Figure 8.

Inositol starved semi-intact cells assemble Sec23p-GFP at tER sites but require cytosol to bud vesicles. (A) Wild-type semi-intact cells grown with or without 75 μM inositol, were assessed in COPII-budding assays in the presence of COPII proteins alone or COPII plus cytosol. Note efficient budding requires cytosol. (B) Wild-type (CBY740) cytosol was fractionated on a Superose 6 column, and 12 μl of each fraction was added to COPII proteins to test stimulation of vesicle budding from inositol-starved membranes. (C) Assembly of Sec23-GFP at tER sites using inositol-starved and unstarved semi-intact cell membranes plus purified Sec23p-GFP/Sec24p complex, Sar1p, and Sec13/Sec31 complex. Reactions were assembled on ice, warmed up to 23°C, and then imaged continuously for ∼9 min. Note that both starved and unstarved membranes recruit Sec23p-GFP to tER structures with the minimal set of COPII proteins. (D) Plot showing quantification of total florescence intensity per semi-intact cell from the images obtained in C. At least seven cells were analyzed per condition at each time point.