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

Abstract

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...

The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells

“…Recent evidence indicates that in yeast, when the ERAD system is saturated, autophagy removes both soluble and aggregated forms of unfolded proteins. 37 The second role of autophagy is to help cells maintain a new steady-state level of ER abundance and cell survival even in the face of continuously accumulating unfolded proteins. 28 Interestingly, yeast cell survival upon ER stress and ER-phagy does not require autophagosomal degradation, but rather ER encapsulation.…”

Autophagy in the liver

“…59 Furthermore, in the yeast model of ATZ degradation, it has also been found that aggregated ATZ is targeted to the autophagy pathway for degradation. 60 It is thus conceivable that autophagy can play an important role in the liver pathogenesis of AT deficiency.…”

Endoplasmic Reticulum Stress: A New Pathway to Induce Autophagy

“…To prevent the toxicity, cell degrades accumulated ATZ by ERAD. Recently, proteins involved in autophagy were shown to participate in the removal of ATZ aggregates, indicating that autophagy also is utilized to degrade this protein; [8][9][10] however, the relationship between autophagy and ER stress had not been clear. We recently found that autophagy is indeed induced in response to ER stress.…”