The Z variant of human ␣-1 proteinase inhibitor (A1PiZ) is a substrate for endoplasmic reticulum-associated protein degradation (ERAD). To identify genes required for the degradation of this protein, A1PiZ degradation-deficient (add) yeast mutants were isolated. The defect in one of these mutants, add3, was complemented by VPS30/ATG6, a gene that encodes a component of two phosphatidylinositol 3-kinase (PtdIns 3-kinase) complexes: complex I is required for autophagy, whereas complex II is required for the carboxypeptidase Y (CPY)-to-vacuole pathway. We found that upon overexpression of A1PiZ, both PtdIns 3-kinase complexes were required for delivery of the excess A1PiZ to the vacuole. When the CPY-to-vacuole pathway was compromised, A1PiZ was secreted; however, disruption of autophagy led to an increase in aggregated A1PiZ rather than secretion. These results suggest that excess soluble A1PiZ transits the secretion pathway to the trans-Golgi network and is selectively targeted to the vacuole via the CPY-to-vacuole sorting pathway, but excess A1PiZ that forms aggregates in the endoplasmic reticulum is targeted to the vacuole via autophagy. These findings illustrate the complex nature of protein quality control in the secretion pathway and reveal multiple sites that recognize and sort both soluble and aggregated forms of aberrant or misfolded proteins. INTRODUCTIONCell function and survival depend on protein quality control to identify and remove aberrant proteins. Although two cellular sites of proteolysis are known, the lysosome/vacuole and cytoplasmic 26S proteasome, the recognition of aberrant proteins and mechanisms for delivery to these sites are still being defined. Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control process in which aberrant or misassembled proteins in the secretory pathway are identified and removed (reviewed in Hampton, 2002;Tsai et al., 2002;Kostova and Wolf, 2003;McCracken and Brodsky, 2003). After entering the endoplasmic reticulum (ER), a nascent protein that fails to fold or assemble properly can be "recognized" by the ER quality control machinery, retained within the ER, and then retrotranslocated to the cytoplasm where it is degraded by the proteasome.The recognition of ERAD substrates must exhibit flexibility to distinguish slowly folding proteins from aberrant proteins. Molecular chaperones play a critical role in this selection process; for example, the ER heat-shock protein (Hsp70), BiP, is vital for the selection of soluble substrates and is believed to hold the substrates in an unfolded state competent for retrotranslocation (Nishikawa et al., 2001;Kabani et al., 2003).The complexity of the ERAD pathway has emerged with the identification of new ERAD substrates and the components required for their selection and degradation in yeast. For example, the analysis of topologically distinct ERAD substrates indicates that molecular chaperones in the cytoplasm and in the ER lumen distinguish membrane and soluble substrates, respectively (Huyer et al., 20...

Polypeptide import into the yeast endoplasmic reticulum (ER) requires two hsp70s, Ssa1p in the cytosol and BiP (Kar2p) in the ER lumen. After import, aberrant polypeptides may be exported to the cytoplasm for degradation by the proteasome, and defects in the ER chaperone calnexin (Cne1p) compromise their degradation. Both import and export require BiP and the Sec61p translocation complex, suggesting that import and export may be mechanistically related. We now show that the cne1⌬ and two kar2 mutant alleles exhibit a synthetic interaction and that the export and degradation of pro-␣ factor is defective in kar2 mutant microsomes. Pulse-chase analysis indicates that A1PiZ, another substrate for degradation, is stabilized in the kar2 strains at the restrictive temperature. Because two of the kar2 mutants examined are proficient for polypeptide import, the roles of BiP during ER protein export and import differ, indicating that these processes must be mechanistically distinct. To examine whether Ssa1p drives polypeptides from the ER and is also required for degradation, we assembled reactions using strains either containing a mutation in SSA1 or in which the level of Ssa1p could be regulated. We found that pro-␣ factor and A1PiZ were degraded normally, indicating further that import and export are distinct and that other cytosolic factors may pull polypeptides from the ER.

The endoplasmic reticulum (ER) quality control processes recognize and remove aberrant proteins from the secretory pathway. Several variants of the plasma protein fibrinogen are recognized as aberrant and degraded by ER-associated protein degradation (ERAD), thus leading to hypofibrinogenemia. A subset of patients with hypofibrinogenemia exhibit hepatic ER accumulation of the variant fibrinogens and develop liver cirrhosis. One such variant named Aguadilla has a substitution of Arg375 to Trp in the ␥-chain. To understand the cellular mechanisms behind clearance of the aberrant Aguadilla ␥-chain, we expressed the mutant ␥D domain in yeast and found that it was cleared from the ER via ERAD. In addition, we discovered that when ERAD was saturated, aggregated Aguadilla ␥D accumulated within the ER while a soluble form of the polypeptide transited the secretory pathway to the trans-Golgi network where it was targeted to the vacuole for degradation. Examination of Aguadilla ␥D in an autophagy-deficient yeast strain showed stabilization of the aggregated ER form, indicating that these aggregates are normally cleared from the ER via the autophagic pathway. Fibrinogen, a large plasma protein synthesized in hepatocytes, plays a critical role in blood coagulation. Mature circulating fibrinogen is a symmetric dimeric molecule in which each half consists of three polypeptide chains: A␣, B␤, and ␥. The two halves of the molecule associate into a trinodal D-E-D structure with the N termini of all six chains in the central E domain, the C termini of B␤ and ␥ forming the two globular D domains, and the C termini of the A␣-chains forming a less compact structure associated with the E domain.

Previously published online as an Autophagy E-publication:

Human alpha-1-proteinase inhibitor (AlPI) deficiency, associated with the Z-variant AlPI (AlPI/Z) gene, results from defective secretion of the inhibitor from the liver. The AlPI/Z gene exhibits two point mutations which specify amino acid substitutions, Val-213 to Ala and Glu-342 to Lys. The functional importance of these substitutions in AlPI deficiency was investigated by studying the secretion of AlPI synthesized in COS cells transfected with AlPI genes altered by site-directed mutagenesis. This model system correctly duplicates the secretion defect seen in individuals homozygous for the AlPI/Z allele and shows that the substitution of Lys for Glu-342 alone causes defective secretion of AlPI. The substitution of Lys for eliminates the possibility for a salt bridge between residues 342 and 290, which may decrease the conformational stability of the molecule and thus account for the secretion defect. However, when we removed the potential to form a salt bridge from the wild-type inhibitor by changing Lys-290 to Glu (AIPI/SB-290Glu), secretion was not reduced to the 19% of normal level seen for AlPI/Z-342Lys; in fact, 75% of normal secretion was observed. When the potential for salt bridge formation was returned to AlPI/Z-342Lys by changing Lys-290 to Glu, only 46% of normal secretion was seen. These data indicate that the amino acid substitution at position 342, rather than the potential to form the 290-342 salt bridge, is the critical alteration leading to the defect in AlPI secretion.Alpha-1-proteinase inhibitor (AlPI), an inhibitor of serine proteases, is one of the major serum glycoproteins synthesized in and secreted by the liver. Individuals homozygous for the Z-variant AlPI (AlPI/Z) gene have about 15% of the normal circulating level of this protease inhibitor and as a result are predisposed to pulmonary emphysema and hepatic cirrhosis (6). The decreased serum levels of AlPI in affected individuals are caused by impaired secretion of AlPI from the liver rather than by defective synthesis of the protein (5). This conclusion is supported by the observations that the levels of AiPI mRNA in affected and normal livers are essentially the same (30), the efficiencies of translation of these mRNAs are not significantly different (1,25,30), and AlPI/Z accumulates in the endoplasmic reticulum (ER) of the liver (4). The only demonstrated differences between the normal (AlPI/M) and variant (AlPI/Z) forms of this protein are two changes in the primary sequence. In A1PI/Z, alanine is substituted for valine at position 213 (23, 27) and lysine is stibstituted for glutamic acid at position 342 (13). These substitutions do not appear to cause major changes in the conformation of A1PI/Z, since the specific trypsin-inhibitory capacities, circulating half-lives, sedimentation coefficients (1, 21), immunological activities (34), and oligosaccharide structures (1, 35) of the serum forms of AlPI/Z and AlPI/M appear to be identical. Furthermore, after solubilization, the AlPI/Z that accumulates in inclusion bodies in the ER of...