Figure 1.
A misfolded variant of Wsc1p. (A) Schematic representation of HA-tagged Wsc1p and Wsc1-L63R. The asterisk marks the position of the L63R mutation. S.S., signal sequence; TM, transmembrane domain; Cyt, cytoplasmic domain. (B) Wild-type (WT) and sec12-4 cells expressing Wsc1p or Wsc1-L63R were grown to log phase at 23°C and shifted to 37°C for 20 min before a 5-min pulse-label with [35S]methionine/cysteine and a 15-min chase. Wsc1p and Wsc1-L63R were immunoprecipitated from detergent lysates using anti-HA antibody and resolved by SDS-PAGE. (C) Wild-type cells incubated in the presence or absence of 10 mM DTT were pulse-labeled for 5 min and chased for 0 and 30 min at 30°C. Proteins were incubated with or without Mal-PEG after TCA precipitation. CPY and CPY* were immunoprecipitated from detergent lysates using anti-CPY and anti-HA antisera, respectively. The positions of ER (p1), Golgi (p2), and mature vacuolar form (mCPY) of CPY are indicated. (D) The sec12-4 strain expressing Wsc1p or Wsc1-L63R was labeled for 5 min and chased for 15 min as described in B. PEGylation-based folding assay was performed as described in C. Immunoprecipitated Wsc1p and Wsc1-L63R were resolved by SDS-PAGE. The arrowhead denotes the position of a nonspecific protein that cross-reacts with the anti-HA antibody.