Figure 4.

PDI deletion mutants are defective in export of misfolded secretory proteins from the ER to the cytosol for degradation. a, Export and degradation are dependent on ATP and cytosolic proteasomes. Mutant pΔgpαf was translocated into wild-type microsomes for 50 min at 24°C. Membranes were washed and incubated in the presence or absence of ATP, an ATP-regenerating system, and 6 mg/ml wild-type or proteasome mutant (pre1 pre2) yeast cytosol for the indicated periods of time at 24°C. At each time point, proteins were precipitated with trichloro-acetic acid, and resolved on an 18% polyacrylamide gel containing 4 M urea. Radiolabeled protein was visualized and quantified by PhosphorImaging. A fraction of pΔgpαf (upper band) remains aggregated on the cytosolic face on the microsomes, cannot be removed by buffer washes, and is partially susceptible to proteolysis by cytosolic proteasomes. b, Microsomes were prepared from wild-type cells and pdi1 mutants. Mutant pΔgpαf was translocated into wild-type and mutant microsomes and membranes were incubated in the presence of ATP, an ATP-regenerating system, and 6 mg/ml wild-type yeast cytosol for the indicated periods of time at 24°C. Samples were analyzed as for a. c, The Δgpαf bands from two (wild-type) or four (mutants) degradation experiments performed as in b were quantified using a PhosphorImager (BioRad). Variation at each time point was <10% for wild-type and <5% for the mutants. d, PDI1 wild-type and mutant cells expressing prc1-1 were pulse-labeled for 10 min, chased, and CPY* immunoprecipitated and analyzed as described for CPY in Fig. 3 a. Note that in Δ222–302 and 252–277, an increased proportion of CPY* is transported to the Golgi complex (p2CPY*). p2CPY* is not secreted, and is rapidly degraded in PEP4 wild-type cells, but relatively stable in pep4::URA3 cells (shown here).