Fig. 4. Vesicles from a vma21 deletion strain are fusion competent, and Erv46p is required only on the acceptor membrane. (A) In the first stage of a transport assay, COPII vesicles were generated from wild-type (BY4742), erv46 (SOY01409) or vma21 (SOY01629) microsomes containing gpf. In the second stage, these vesicles were incubated with washed semi-intact wild-type (BY4742, Recon +wt) or erv46 (SOY01409, Recon +erv46) cells in presence of Uso1p, LMA1 and the ATP regeneration system. NA, mock reaction with wild-type semi-intact cells but no Uso1p or LMA1; Recon –Acc, control containing Uso1p and LMA1 but no semi-intact cells. Percentage fusion was quantified after precipitation of the outer-chain modified forms of gpf. All assays were done in duplicate and error bars represent the range. (B) Overexpression of Bos1p does not suppress the cold-sensitive phenotypes of vma21 or vma21 erv46 strains. Wild-type, single or double deletion strains (SOY163-SOY174) carrying empty vectors or multicopy BOS1 or BET1 overexpression plasmids as indicated were grown in appropriate selective media, spotted on plates and incubated as described in Fig. 1.