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Fig. 7. Glucosidase II is not packaged into COPII vesicles in vitro and strains lacking a cycling Erv41p-Erv46p complex have a glycoprotein processing defect. (A) Microsomes from a strain expressing Rot2p-3HA (SOY01117) were incubated without (–) or with (+) purified COPII subunits in the presence of GTP and an ATP regeneration system. Membranes from 10% of the total reaction (T) and vesicle fractions were collected by centrifugation, resolved on a polyacrylamide gel and immunoblotted. (B) ³⁵S-labelled ${alpha}$ factor pheromone precursor was translocated into semi-intact cells. Membranes were solubilized and glycoproteins precipitated on concanavalin A Sepharose beads. Bound radiolabelled protein was eluted from the beads, resolved on polyacrylamide gels and detected by phosphorimaging. Fully core glycosylated ${alpha}$ factor pheromone precursor is indicated (gp ${alpha}$ f). The lower bands represent partially glycosylated species. Arrowheads mark the band representing incompletely trimmed gp ${alpha}$ f. Semi-intact cells were prepared from wild-type (wt, FY834), rot2 ${Delta}$ (CBY1087), cwh41 ${Delta}$ (CBY1086), an erv41 ${Delta}$ strain containing empty vector pRS314 (CBY1036), or an erv41 ${Delta}$ strain containing the ERV41 gene in vector pRS314 (CBY1037). (C) Same procedure as in B, but semi-intact cells were prepared from an erv41 ${Delta}$ strain containing a plasmid which encodes a truncated version of ERV41 lacking C-terminal residues 340-352 (CBY1038), or from erv41 ${Delta}$ strains transformed with pRS314 plasmids containing alanine scan ERV41 mutants (CBY1074-1080, CBY1089-1096, CBY1158 and CBY1159, respectively).

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