Endoplasmic reticulum-associated degradation (ERAD) disposes of aberrant proteins in the secretory pathway. Protein substrates of ERAD are dislocated via the Sec61p translocon from the endoplasmic reticulum to the cytosol, where they are ubiquitinated and degraded by the proteasome. Since the Sec61p channel is also responsible for import of nascent proteins, this bidirectional passage should be coordinated, probably by molecular chaperones. Here we implicate the cytosolic chaperone AAA-ATPase p97/Cdc48p in ERAD. We show the association of mammalian p97 and its yeast homologue Cdc48p in complexes with two respective ERAD substrates, secretory immunoglobulin M in B lymphocytes and 6myc-Hmg2p in yeast. The membrane 6myc-Hmg2p as well as soluble lumenal CPY*, two short-lived ERAD substrates, are markedly stabilized in conditional cdc48 yeast mutants. The involvement of Cdc48p in dislocation is underscored by the accumulation of ERAD substrates in the endoplasmic reticulum when Cdc48p fails to function, as monitored by activation of the unfolded protein response. We propose that the role of p97/Cdc48p in ERAD, provided by its potential unfoldase activity and multiubiquitin binding capacity, is to act at the cytosolic face of the endoplasmic reticulum and to chaperone dislocation of ERAD substrates and present them to the proteasome.Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control process that selectively eliminates aberrant proteins in the secretory pathway. Protein substrates of ERAD are dislocated from the ER to the cytosol, where they are ubiquitinated and degraded by the proteasome (5). The Sec61p translocon is involved both in the import of nascent proteins into the ER and in dislocation of aberrant proteins from the ER. These two activities of Sec61p are mechanistically different because they involve distinct domains within Sec61p and dislocation-defective mutants of Sec61p are still proficient in protein import (40,50,56).Since nascent and aberrant proteins pass through the same Sec61p translocon, this bidirectional passage requires coordination. Moreover, the Sec61p translocon is a passive conduit; thus, the driving force to move polypeptides across it should be provided by accessory proteins. Indeed, passage through the Sec61p translocon requires molecular chaperones, and their contribution further illustrates that import and dislocation must be mechanistically distinct. For example, of the two hsp70s involved in import in yeast, BiP/Kar2p in the ER lumen and Ssa1p in the cytosol, mutants of BiP/Kar2p that are defective in dislocation are still proficient in import, and mutation in SSA1 does not affect degradation of the ERAD substrates pro-␣-factor and A1PiZ (9). Although the kar2 mutant initially used to link BiP to CPY* dislocation and degradation was also defective in protein import (41), the kar2 mutants that are defective only in dislocation of pro-␣-factor and A1PiZ directly demonstrate the role of Kar2p in dislocation (9). Furthermore, chaperones that are required for ERA...

SummaryAging (senescence) is characterized by the development of numerous pathologies, some of which limit lifespan. Key to understanding aging is discovery of the mechanisms (etiologies) that cause senescent pathology. In C. elegans, a major senescent pathology of unknown etiology is atrophy of its principal metabolic organ, the intestine. Here we identify a cause of not only this pathology but also of yolky lipid accumulation and redistribution (a form of senescent obesity): autophagy-mediated conversion of intestinal biomass into yolk. Inhibiting intestinal autophagy or vitellogenesis rescues both visceral pathologies and can also extend lifespan. This defines a disease syndrome leading to multimorbidity and contributing to late-life mortality. Activation of gut-to-yolk biomass conversion by insulin/IGF-1 signaling (IIS) promotes reproduction and senescence. This illustrates how major, IIS-promoted senescent pathologies in C. elegans can originate not from damage accumulation but from direct effects of futile, continued action of a wild-type biological program (vitellogenesis).

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research.

Abstract. We have raised two monospecific antibodies against synthetic peptides derived from the membrane domain of the ER glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway. This domain, which was proposed to span the ER membrane seven times (Liscum, L., J. FinerMoore, R. M. Stroud, K. L. Luskey, M. S. Brown, and J. L. Goldstein. 1985. J. Biol. Chem. 260:522-538), plays a critical role in the regulated degradation of the enzyme in the ER in response to sterols. The antibodies stain the ER of cells and immunoprecipitate HMG-CoA reductase and HMGal, a chimeric protein composed of the membrane domain of the reductase fused to Escherichia coli/3-galactosidase, the degradation of which is also accelerated by sterols. We show that the sequence Arg TM through Leu 242 of HMG-CoA reductase (peptide G) faces the cytoplasm both in cultured cells and in rat liver, whereas the sequence Thr TM through Glu m (peptide H) faces the lumen of the ER. This indicates that a sequence between peptide G and peptide H spans the membrane of the ER. Moreover, by epitope tagging with peptide H, we show that the loop segment connecting membrane spans 3 and 4 is sequestered in the lumen of the ER. These results demonstrate that the membrane domain of HMG-CoA reductase spans the ER eight times and are inconsistent with the seven membrane spans topological model. The approximate boundaries of the proposed additional transmembrane segment are between Lys 24s and Asp 276. Replacement of this 7th span in HMGal with the first transmembrane helix of bacteriorhodopsin abolishes the sterol-enhanced degradation of the protein, indicating its role in the regulated turnover of HMG-CoA reductase within the endoplasmic reticulum.3-HYDROXY-3-METHYLGLUTARYL coenzyme A (HMGCoA) t reductase catalyzes the conversion of HMG-CoA to mevalonate (Durr and Rudney, 1960), the precursor for a wide variety of metabolites that play central roles in cellular functions. These products of the mevalonate pathway include sterols, ubiquinone, dolichols, isopentenyladenine, heme A and the isoprenyl moieties of proteins (for a recent review see Goldstein and Brown, 1990). In mammalian cells, HMG-CoA reductase is the major regulatory enzyme in this pathway and is subject to complex metabolic control ensuring an adequate supply of intermediates and products of this pathway. This is achieved by regulation of transcription of the HMG-CoA reductase gene (Liscum et al., 1983a;Osborne et al., 1985), regulation of translation of its mRNA (Tanaka et al., 1983; Petfley and Sinensky, 1985; Nakanishi Shoshana Bar-Nun is a visiting Scholar from the

Dislocation of endoplasmic reticulum-associated degradation (ERAD) substrates from the endoplasmic reticulum (ER) lumen to cytosol is considered to occur in a single step that is tightly coupled to proteasomal degradation. Here we show that dislocation of luminal ERAD substrates occurs in two distinct consecutive steps. The first is passage across ER membrane to the ER cytosolic face, where substrates can accumulate as ubiquitin conjugates. In vivo, this step occurs despite proteasome inhibition but requires p97/Cdc48p because substrates remain entrapped in ER lumen and are prevented from ubiquitination in cdc48 yeast strain. The second dislocation step is the release of accumulated substrates to the cytosol. In vitro, this release requires active proteasome, consumes ATP, and relies on salt-removable ERbound components, among them the ER-bound p97 and ER-bound proteasome, which specifically interact with the cytosol-facing substrates. An additional role for Cdc48p subsequent to ubiquitination is revealed in the cdc48 strain at permissive temperature, consistent with our finding that p97 recognizes luminal ERAD substrates through multiubiquitin. BiP interacts exclusively with ERAD substrates, suggesting a role for this chaperone in ERAD. We propose a model that assigns the cytosolic face of the ER as a midpoint to which luminal ERAD substrates emerge and p97/Cdc48p and the proteasome are recruited. Although p97/Cdc48p plays a dual role in dislocation and is involved both in passage of the substrate across ER membrane and subsequent to its ubiquitination, the proteasome takes part in the release of the substrate from the ER face to the cytosol en route to degradation.The endoplasmic reticulum-associated degradation (ERAD) 1 is a quality control process that selectively eliminates malfolded proteins or unassembled subunits of oligomeric proteins in the secretory pathway (1, 2). ERAD substrates are dislocated from the endoplasmic reticulum (ER) back to the cytosol via the Sec61 complex (3-6). In the cytosol, ubiquitin is conjugated to the ERAD substrates that are degraded by the proteasome (7). The proteolytically active proteasome has been implicated in the dislocation of ERAD substrates by virtue of their scarcity in cytosol of proteasome-inhibited cells (8 -11). Although stabilization of proteins in the secretory pathway by proteasome inhibitors is generally accepted as an indication for ERAD, ubiquitination of such proteins is a direct evidence for the access of the substrate to the cytosol (7). In most cases, however, accumulation of the multiubiquitinated ERAD substrates to detectable levels requires inhibition of the proteasome. If the proteasome is indeed required for dislocation (8 -11), then ubiquitination of ERAD substrates would not be observed. This applies especially to luminal ERAD substrates, as membrane ERAD substrates inherently display cytosolic domains, which may be ubiquitinated irrespective of dislocation (11-15). Therefore, it is our view that luminal ERAD substrates, whose dislocation is an ...