Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate - PubMed (original) (raw)

Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate

E D Werner et al. Proc Natl Acad Sci U S A. 1996.

Abstract

Until recently, the degradation of aberrant and unassembled proteins retained in the endoplasmic reticulum (ER) was thought to involve unidentified ER-localized proteases. We now show that the ER-associated degradation (ERAD) of two mutant proteins that accumulate in the ER lumen is inhibited in a proteasome-defective yeast strain and when cytosol from this mutant is used in an in vitro assay. In addition, ERAD is limited in vitro in the presence of the proteasome inhibitors, 3,4-dichloroisocoumarin and lactacystin. Furthermore, we find that an ERAD substrate is exported from ER-derived microsomes, and the accumulation of exported substrate is 2-fold greater when proteasome mutant cytosol is used in place of wild-type cytosol. We conclude that lumenal ERAD substrates are exported from the yeast ER to the cytoplasm for degradation by the proteasome complex.

PubMed Disclaimer

Figures

Figure 1

The proteasome complex is required for ER-associated degradation of A1PiZ. (a) Phosphorimage of pulse-chase radiolabeling of A1PiZ in the proteasome mutant (pre 1-1 pre 2-2), the isogenic wild-type strain (wild type), the proteasome mutant expressing a wild-type copy of PRE1(pre 1-1 pre 2-2 + PRE1), and the proteasome mutant expressing a wild-type copy of PRE2(pre 1-1 pre 2-2 + PRE2). Immunoprecipated products of chase incubations (0, 60, 90, or 120 min) were treated with (+) or without (−) endoglycosidase H (Endo H) to demonstrate removal of the three carbohydrate chains added to A1PiZ in the ER and to convert glycosylated forms of A1PiZ to a single species. Endo H digestion was incomplete in the 0 chase samples. (b) First-order decay curves generated with averaged values from at least three experiments to determine the half-life of A1PiZ. Relative amounts of A1PiZ were determined from the phosphorimages using a Bio-Rad

phosphor analyst

program.

Figure 2

In vitro assay demonstrates ATP and proteasome-dependent ERAD and reveals export of pαF from ER-derived microsomes. (a) Phosphorimage of a 30-min posttranslocation chase incubation performed in the presence (+) or absence (−) of ATP with either proteasome mutant (pre 1-1 pre 2-2) or isogenic wild-type (wild type) cytosol. Radiolabeled ΔGppαF was translocated into wild-type microsomes and the signal sequence cleaved to generate unglycosylated pαF, a substrate for ERAD_in vivo_ (20) and in vitro (8). (b) First-order decay curves generated with averaged values from at least three independent experiments indicate that pαF is stabilized in the presence of pre 1-1 pre 2-2 proteasome mutant cytosol. Posttranslocation chase incubations (0, 10, 20, and 40 min) in the presence of either wild-type cytosol (wild type), mutant cytosol (pre 1-1 pre 2-2 cytosol), or buffer (buffer 88 + ATP) were treated with or without trypsin before trichloroacetic acid precipitation, and samples were resolved on 18% SDS/urea-PAGE and analyzed as in Fig. 1. Degradation of pαF by trypsin treatment (0.25 g/ml) indicates that the protein substrate has been exported from microsomes, while protease protection indicates membrane-occluded pαF. All reactions were performed in the presence of ATP.

Figure 3

DCI and lactacystin inhibit ER-associated degradation of pαF. (a) Phosphorimage of in vitro ERAD chase reactions (0, 10, 20, and 40 min) in the presence of 10 μM lactacystin, 10nM DCI, or dimethyl sulfoxide. All reactions were performed in the presence of ATP. (b) First-order decay curves generated with averaged values from at least three experiments indicate that pαF is stabilized in the presence of proteasome inhibitors. Relative amounts of pαF were determined from the phosphorimages as in Fig. 1.

Figure 4

Mutations in specific ubiquitin-conjugating enzymes do not inhibit ERAD in vitro. Microsomes and cytosol from either the ubc6 ubc7 (ubc) or an isogenic wild-type strain (WT) were used in an in vitro ERAD assay as previously described (8). Addition of ubiquitin-aldehyde (UA) to wild-type cytosol to a final concentration of 1 μM, did not inhibit degradation of pαF in vitro. Results represent the means of at least two independent determinations.

References

1. Chen C, Bonifacino J S, Yaun L C, Klausner R D. J Cell Biol. 1988;107:2149–2161. - PMC - PubMed
1. Amara J F, Lederkremer G, Lodish H F. J Cell Biol. 1989;109:3315–3324. - PMC - PubMed
1. Hurtley S M, Helenius A. Annu Rev Cell Biol. 1989;5:277–307. - PubMed
1. Bonifacino J S, Lippincott-Schwartz J. Curr Opin Cell Biol. 1991;3:592–600. - PubMed
1. Finger A, Knop M, Wolf D H. Eur J Biochem. 1993;218:565–574. - PubMed

Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate - PubMed (original) (raw)