Sequential assistance of molecular chaperones and transient formation of covalent complexes during protein degradation from the ER

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A stringent quality control process selects misfolded polypeptides generated in the endoplasmic reticulum (ER) for ER-associated degradation (ERAD). Here we assessed the maintenance of efficient glycoprotein folding in cells with defective ERAD caused by lack of adaptation of the intralumenal level of ER degradationenhancing ␣-mannosidase-like protein (EDEM) to an increase in the ER cargo load. When these cells were converted into factories for production of high levels of human ␤-secretase, maturation of this N-glycosylated aspartic protease progressed as in wild-type cells initially to gradually become less efficient. Up-regulation of EDEM to strengthen the ERAD machinery (but not up-regulation of calnexin to reinforce the folding machinery) was instrumental in maintaining folding efficiency and secretory capacity. Our data underscore the important role that the degradation machinery plays in maintaining a functional folding environment in the ER.Only a fraction of the polypeptide chains expressed in the ER 1 is transported to its final destination. A stringent quality control process prevents export of orphan subunits of oligomeric complexes, folding-incompetent products of mutated genes, and by-products of protein biosynthesis that do not acquire the native conformation (1-3). Polypeptides that do not pass quality control are either retained by the ER chaperone network to be used for further folding attempts or are sorted out for cytosolic degradation. The mechanisms regulating ER quality control are better understood for N-glycosylated polypeptides. N-glycans are added to asparagines in nascent chains as pre-assembled tri-antennary oligosaccharides composed of three terminal glucoses, nine mannoses, and two Nacetylglucosamines. The two outermost glucoses are rapidly trimmed by ER glucosidases I and II. Polypeptides exposing mono-glucosylated intermediates of N-glycan trimming associate with the ER lectins calnexin (Cnx) and calreticulin (Crt).The opposing actions of glucosidase II that trim the innermost glucose residue (thereby releasing the newly synthesized polypeptide from Cnx/Crt) and of UDP-glucose-glycoprotein glucosyltransferase that adds back one glucose to non-native conformers sending them back to Cnx/Crt, drive the so-called Cnx cycle (1, 2). For folding-incompetent and terminally misfolded glycopolypeptides, termination of unproductive folding attempts coincides with their definitive release from the Cnx cycle and deviation into the ERAD machinery. Interruption of unproductive folding attempts is a crucial step of ER quality control because it prevents clogging of the ER chaperone system.Emerging evidence underscores the importance of a tightly controlled extraction of folding-incompetent glycopolypeptides from the Cnx cycle to promote their degradation. Extraction is regulated by the ER ␣-mannosidase I (4) and by EDEM (ER degradation enhancing ␣-mannosidase-like protein (5)), an enzymatically inactive mannosidase-like protein. The ER ␣-mannosidase I cleaves one (6) or more (7) mannose residues...

supporting

confidence: 62%

mentioning

confidence: 96%

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EDEM Contributes to Maintenance of Protein Folding Efficiency and Secretory Capacity

Eriksson¹,

Vago

Calanca³

et al. 2004

Self Cite

“…Upon association with CNX and CRT (17,18), ERp57 acts as a thiol-disulfide oxidoreductase for proteins carrying N-linked glycans. Transient disulfide-linked intermediates between ERp57 and nascent and newly synthesized glycoproteins have been observed in vivo (19). In vitro, the ERp57-enhanced oxidative refolding of RNase B, the glycosylated variant of RNase A, is critically dependent on its interaction with CNX and CRT (20).…”

mentioning

confidence: 90%

ERp57 Is a Multifunctional Thiol-Disulfide Oxidoreductase

Frickel

Frei

Bouvier

et al. 2004

175

129

The thiol-disulfide oxidoreductase ERp57 is a soluble protein of the endoplasmic reticulum and the closest known homologue of protein disulfide isomerase. The protein interacts with the two lectin chaperones calnexin and calreticulin and thereby promotes the oxidative folding of newly synthesized glycoproteins. Here we have characterized several fundamental structural and functional properties of ERp57 in vitro, such as the domain organization, shape, redox potential, and the ability to catalyze different thiol-disulfide exchange reactions. Like protein disulfide isomerase, we find ERp57 to be comprised of four structural domains. The protein has an elongated shape of 3.4 ؎ 0.1 nm in diameter and 16.8 ؎ 0.5 nm in length. The two redox-active a and a domains were determined to have redox potentials of ؊0.167 and ؊0.156 V, respectively. Furthermore, ERp57 was shown to efficiently catalyze disulfide reduction, disulfide isomerization, and dithiol oxidation in substrate proteins. The implications of these findings for the function of the protein in vivo are discussed.

“…Cells were washed with Met/Cysfree Dulbecco's modified Eagle's medium and then preincubated for 30 min in the same medium containing a proteasome or lysosome inhibitor or the vehicle. After three washes with Met/Cys-free Dulbecco's modified Eagle's medium, the cells were pulsed for 10 min by adding 150 Ci of [ 35 S]Met/Cys in 1 ml of medium and then chased for the times indicated in the figures with complete Dulbecco's modified Eagle's medium containing a proteasome or lysosome inhibitor or the vehicle (34). At the end of a chase, the cells were quickly placed on ice, washed with ice-cold PBS, and then lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 g of aprotinin/ml, and 2 g of leupeptin/ml).…”

Section: Methodsmentioning

confidence: 99%

Proteasomal Degradation of the Nuclear Targeting Growth Factor Midkine

Suzuki

Shibata

Urano

et al. 2004

It is widely held that growth factor signaling is terminated by lysosomal degradation of its activated receptor and the endocytosed growth factor is transported to lysosomes. Nuclear targeting is another important pathway through which signals of growth factors are mediated. However, mechanisms underlying desensitization of nuclear targeting growth factors are poorly understood. Here we report that the nuclear targeting pathway is down-regulated by the proteasome system. Degradation of endocytosed midkine, a heparin-binding growth factor, was suppressed by both proteasome and lysosome inhibitors to similar extents. By contrast, a proteasome inhibitor, but not lysosome ones, accelerated the nuclear accumulation of midkine. An expression vector of signal sequence-less midkine, which is produced in the cytosol, was constructed because endocytosed midkine may be translocated to the cytosol from cellular compartments before entering the nucleus. The cytosol-produced midkine underwent proteasomal degradation and accumulated in the nucleus as did the endocytosed midkine. It was polyubiquitinated, and its nuclear accumulation was enhanced by a proteasome inhibitor. We further dissected the midkine molecule to investigate roles in degradation and trafficking. The Nterminal half-domain of midkine was significantly more susceptible to proteasomal degradation, whereas the Cterminal half-domain was sufficient for nuclear localization. Together, these data highlight the desensitization of nuclear targeting by growth factors and indicate a critical role of the proteasome system in it.Coordinated regulation of "on" and "off" of signaling is essential for life. Upon ligation of a growth factor with its receptor(s), receptor-mediated intracellular signaling starts from the cell surface and/or endosomes. Desensitization (termination of signaling) then takes place via degradation of the activated receptor. Degradation of the activated receptor occurs essentially through the following two mechanisms. First, the intracellular domain of the activated receptor is degraded by the proteasome system as revealed for the ␤c chain, a shared receptor for interleukin-5 and -3 and granulocyte-macrophage colony-stimulating factor (1). Second, endocytosed receptors are transported from endosomes to lysosomes where they are degraded.Because the proteasome system plays a critical role in endosome-to-lysosome trafficking (1-7), it may indirectly regulate the lysosomal degradation of receptors. In addition to the classical desensitization described above, an additional route, namely nuclear targeting by growth factors, should be considered. In this case, signaling is mediated directly by the growth factor and not via the cell surface receptor. Therefore, desensitization occurs only when the growth factor is degraded.Mounting evidence indicates that nuclear targeting by growth factors plays an indispensable role in their biological activities. For example, the nuclear localization of fibroblast growth factor 1 and Schwannoma-derived growth factor is n...