Figure 3.

(A) Experimental setup to analyze Erd2p-mediated uptake and retrograde transport of the HDEL-cargo Kar2p into the ER of a yeast kar2^ts mutant. (B) Dissolved oxygen content in the culture supernatant of spheroplasts from KAR2 wild-type and kar2^ts cells at 37 °C. (C) Dissolved oxygen content in the culture supernatant of spheroplasts from kar2^ts cells (37 °C) in the presence of increasing amounts of Kar2p or bovine serum albumin (BSA, negative control). Each experiment in (B,C) was performed in triplicate (n = 3); shown is the mean average. (D) Immunoblot of internalized Kar2p (containing an N-terminal (His)₆ tag and detected with anti-His) in cell lysates of spheroplasts from wild-type strain SEY6210 and HDEL receptor defective Δerd2 cells after treatment with recombinant Kar2p (9 μg/ml) for 2 h at 30 °C. Rpn12p, a regulatory subunit of the 26S proteasomal lid, served as loading control. Cropped blots shown in (D) originate from the same gel/blot after stripping and antibody reprobing.