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

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.Quality control of newly synthesized proteins in the endoplasmic reticulum (ER) ensures that only correctly folded, processed, and completely assembled proteins exit this compartment for further transport through the secretory pathway. Most proteins that fail to reach this transport competent state are degraded (1-7). The ER-associated protein degradation pathway (ERAD) is highly selective for specific unassembled and͞or aberrant proteins, while the majority of ER resident and secreted proteins are quite stable. Evidence that the ER chaperone calnexin has a role in ERAD indicates that molecular chaperones might be required for this remarkable substrate selectivity (8).Although previous studies suggested that ERAD involves unidentified proteases localized in the ER, more recent evidence indicates that ERAD may require cytosolic proteases. Studies of ERAD in vitro suggest that degradation of aberrant ER-lumenal proteins occurs in the cytoplasm (8), and there is also evidence that the degradation of ER-retained forms of the cystic fibrosis transmembrane conductance regulator is ubiquitin-and proteasome-dependent (9, 10). In addition, cytomegalovirus-induced down-regulation of major histocompatibility complex class I molecules involves the rapid transport of the unassembled major histocompatibility complex I heavy chains from the ER to the cytoplasm for degradation by the proteasome complex (11). In this report we show that the cytosolic proteasome complex is the proteolytic component for the ER-associated degradation of two ER-lumenal proteins in yeast. Furthermore, we provide evidence that indicates ERAD substrates are exported to the cytosol for degradation by the proteasome. MATERIALS AND METHODSMaterials and Strains. Strains used were: RSY607 (Mat␣, pep4::URA3), provided by R. Schekman, University of California, Berkeley; AB122 (Mata, prc1-407, prb1-1122, pep4-3, leu2, ura3-52), provided by A. Brake, University of California, San Francisco; pre 1-1 pre 2-2 proteasome mutant and isogenic wild-type strain, provided by D. H. Wolf (12, 13); ⌬ubc6 ubiquitin-conjugating enzyme mutant and isogenic wild-type strains, provided by S. Jentsch (14); and ubc4-⌬1 u...

Characterization of anERADGene asVPS30/ATG6Reveals Two Alternative and Functionally Distinct Protein Quality Control Pathways: One for Soluble Z Variant of Human α-1 Proteinase Inhibitor (A1PiZ) and Another for Aggregates of A1PiZ

“…This result was unexpected given that A1PiZ is an ERAD substrate in both yeast (McCracken and Kruse, 1993;Werner et al, 1996;Brodsky et al, 1999) and human . Because vacuole morphology is largely unperturbed in vps30 yeast (Raymond et al, 1992), these data suggest that the A1PiZ degradation defect does not arise from gross ultrastructural perturbations.…”

“…The classical form of A1Pi deficiency results from the Z variant (A1PiZ) that contains a K342E substitution (Bathurst et al, 1984;Crystal, 1990). A1PiZ homozygous individuals can develop emphysema via a loss-of-function mechanism because the altered conformation of A1PiZ results in its recognition and degradation by ERAD (Wu et al, 1994;Werner et al, 1996;Teckman et al, 2001). A subset of homozygous individuals (12-15%) also experience liver disease (Sveger, 1988) via a gain-offunction mechanism because A1PiZ accumulates and aggregates within the ER of hepatocytes (Lomas et al, 1992, Lomas andMahadeva, 2002; reviewed in Perlmutter, 2003).…”

Alpha-1-antitrypsin deficiency: A new paradigm for hepatocellular carcinoma in genetic liver disease

“…Although retrograde translocation from the ER to the cytoplasm has been demonstrated for some luminal substrates of the proteosome, very limited evidence exists for retrograde translocation of ␣1ATZ. Werner et al 17 detected ␣1ATZ in the cytosolic fraction of yeast when the proteosome was inhibited, but it was only a small fraction of the total ␣1ATZ in the ER, and there has been no other evidence for retrotranslocation. This may mean that other mechanisms provide for transport from the ER to the cytoplasm such as proteosomemediated extraction through the ER membrane, as has been demonstrated for model ER degradation substrates.…”

“…Studies in many systems have indicated that the proteosomal system is involved. [17][18][19][20][21] This involvement appears to include both the classical ubiquitin-dependent proteosomal mechanism as well as a ubiquitin-independent proteosomal mechanism. 19 However, exactly how ␣1ATZ on the luminal side of the ER membrane is accessed by the proteosome from the cytoplasm is not clear.…”

Mechanisms Underlying the Cellular Clearance of Antitrypsin Z: Lessons from Yeast Expression Systems

“…We discovered that the mutated protein was exported from the ER vesicles into the cytosolic fraction, an event that required ER lumenal chaperones (9). We and others subsequently found that the degradation of these ''retro-translocated'' ER proteins was mediated by the cytosolic proteasome, a 26S multicatalytic protease that had been previously found to trigger the destruction of misfolded cytoplasmic proteins (10,11). We termed this process ERassociated degradation (ERAD).…”