Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast

Finger, Andreas; Knop, Michael; Wolf, Dieter H.

doi:10.1111/j.1432-1033.1993.tb18410.x

Cited by 194 publications

(179 citation statements)

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“…It is posttranslationally translocated to the ER lumen where it is glycosylated and subsequently matured in the Golgi after which it is exported to the vacuole [7,8]. A point mutant of this protein, CPY\, have been previously demonstrated to be retained in the ER and retrotranslocated back to the cytosol, where it serves as an ERAD substrate [9,10]. Interestingly, Suzuki et al [3] have reported that in a deletion mutant of Png1 a slower rate of CPY\ degradation is observed.…”

Section: Introductionmentioning

confidence: 99%

Misfolding of glycoproteins is a prerequisite for peptide: N‐glycanase mediated deglycosylation

2005

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Peptide:N-glycanase (PNGase) is a deglycosylating enzyme that catalyzes the hydrolysis of the b-aspartylglycosylamine bond of aspargine-linked glycopeptides and glycoproteins. Earlier studies from our laboratory indicated that PNGase catalyzed de-N-glycosylation was limited to glycopeptide substrates, but recent reports have demonstrated that it also acts upon full-length misfolded glycoproteins. In this study, we utilized two glycoprotein substrates, yeast carboxypeptidase and chicken egg albumin (ovalbumin), to study the deglycosylation activity of yeast PNGase and its mutants. Our results provide further evidence that PNGase acts upon full-length glycoprotein substrates and clearly establish that PNGase acts only on misfolded or denatured glycoproteins.

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Section: Introductionmentioning

confidence: 99%

Misfolding of glycoproteins is a prerequisite for peptide: N‐glycanase mediated deglycosylation

2005

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“…In yeast, a mutated carboxypeptidase Y (CPY*) has been used as a model for misfolding of soluble luminal glycoproteins, and the process of CPY* degradation and its effects of N-glycan processing have been investigated in detail previously (Knop et al, 1996;Jakob et al, 1998). Mutant CPY carries an arginine instead of a glycine residue at position 255 of prepro-CPY (Finger et al, 1993). This mutated amino acid is located in a hydrophobic region in CPY.…”

Section: Construction and Characterization Of Misfolded Gas1mentioning

confidence: 99%

Inositol Deacylation by Bst1p Is Required for the Quality Control of Glycosylphosphatidylinositol-anchored Proteins

Fujita

Yoko-o

Jigami

2006

MBoC

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Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytosol, and degraded by the proteasome. A number of proteins are processed and modified by a glycosylphosphatidylinositol (GPI) anchor in the ER, but the quality control mechanisms of GPI-anchored proteins remain unclear. Here, we report on the quality control mechanism of misfolded GPI-anchored proteins. We have constructed a mutant form of the ␤-1,3-glucanosyltransferase Gas1p (Gas1*p) as a model misfolded GPI-anchored protein. Gas1*p was modified with a GPI anchor but retained in the ER and was degraded rapidly via the proteasome. Disruption of BST1, which encodes GPI inositol deacylase, caused a delay in the degradation of Gas1*p. This delay was because of an effect on the deacylation activity of Bst1p. Disruption of genes involved in GPI-anchored protein concentration and N-glycan processing caused different effects on the degradation of Gas1*p and a soluble misfolded version of carboxypeptidase Y. Furthermore, Gas1*p associated with both Bst1p and BiP/Kar2p, a molecular chaperone, in vivo. Our data suggest that GPI inositol deacylation plays important roles in the quality control and ER-associated degradation of GPI-anchored proteins. INTRODUCTIONCells possess several quality control mechanisms for the maintenance of proper protein folding and function. On synthesis, membrane and secretory proteins are inserted into the lumen of the endoplasmic reticulum (ER), where they are folded and undergo oligomerization. There are a number of chaperones and enzymes in the ER required for proper protein folding (Ellgaard et al., 1999). Before exiting from the ER, proteins are monitored by a quality control system that ensures correct folding. Misfolded proteins that fail to pass the quality control checkpoint are transported back to the cytosol and degraded by an ER-associated degradation (ERAD) mechanism that involves the ubiquitinproteasome pathway (Kopito, 1997;Kostova and Wolf, 2003). N-linked oligosaccharide, one of the major posttranslational modifications in the ER, is trimmed and processed by glucosidase I, glucosidase II, and mannosidase I (Jakob et al., 1998;Helenius and Aebi, 2001). The processing of Nlinked oligosaccharides plays important roles in the quality control of glycoprotein folding in the ER Aebi, 2001, 2004).A number of cell surface proteins are posttranslationally modified in the ER with glycosylphosphatidylinositol (GPI). Mechanisms for quality control and degradation of GPIanchored proteins are important in folding diseases, including prion diseases and transmissible spongiform encephalopathies, which are caused by a conformational modification of prion, a GPI-anchored protein (Prusiner, 1998). There have been several biochemical studies on both the degradation of mutated prions and the quality control of proteins with mutated GPI attachment signals (Field et al., 1994;Oda et al., 1996;Wainwright and Field, 1997;Jin et al., 2000;Ito et al., 2002;Ishida et al., 2003). In contrast, the molecular mech...

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“…both CPY* (left) (28) and KWW (right) (29) was assayed in wild type, usa1⌬, USA1⌬UBL, and USA1⌬UBL-myc strains (Fig. 4C).…”

Section: Volume 285 • Number 8 • February 19 2010mentioning

confidence: 99%

Usa1p Is Required for Optimal Function and Regulation of the Hrd1p Endoplasmic Reticulum-associated Degradation Ubiquitin Ligase

Carroll

Hampton

2010

Journal of Biological Chemistry

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Usa1p is a recently discovered member of the HRD ubiquitin ligase complex. The HRD pathway is a conserved route of ubiquitin-dependent, endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous lumenal (ERAD-L) and membrane-anchored (ERAD-M) substrates. We have investigated Usa1p to understand its importance in HRD complex action. Usa1p was required for the optimal function of the Hrd1p E3 ubiquitin ligase; its loss caused deficient degradation of both membrane-associated and lumenal proteins. Furthermore, Usa1p functioned in regulation of Hrd1p by two mechanisms. First, Hrd1p self-degradation, which serves to limit the levels of uncomplexed E3, is absolutely dependent on Usa1p and the ubiquitin-like (Ubl) domain of Usa1p. We found that Usa1p allows Hrd1p degradation by promoting trans interactions between Hrd1p molecules. The Ubl domain of Usa1p was required specifically for Hrd1p self-ubiquitination but not for degradation of either ERAD-L or ERAD-M substrates. In addition, Usa1p was able to attenuate the activity-dependent toxicity of Hrd1p without compromising substrate degradation, indicating a separate role in ligase regulation that operates in parallel to stability control. Many of the described actions of Usa1p are distinct from those of Der1p, which is recruited to the HRD complex by Usa1p. Thus, this novel, conserved factor is broadly involved in the function and regulation of the HRD pathway of ERAD. ER3 -associated degradation (ERAD) is a conserved process by which eukaryotic cells target and degrade ER-resident proteins by the ubiquitin-proteasome pathway. ERAD pathways play a major role in the destruction of misfolded or unassembled ER proteins, including both lumenal and integralmembrane substrates. In addition, normal proteins are regulated by this pathway, the most prominent case being the sterol pathway-regulated degradation of HMG-CoA reductase in both yeast and mammals (1, 2). Eukaryotic ERAD is brought about by the action of multiple pathways of ubiquitin-mediated degradation that operate at the ER surface (3, 4).Covalent addition of ubiquitin to proteins brings about their recognition and degradation by the cytosolic 26S proteasome. Protein ubiquitination occurs by a cascade of enzymes that add 7.6-kDa ubiquitin to the targeted protein. The E1 ubiquitinactivating enzyme first forms a high energy bond with ubiquitin in an ATP-dependent reaction, and then the ubiquitin is transferred to an E2, or ubiquitin-conjugating enzyme. E2-bound ubiquitin is next transferred from the charged E2 to the target protein by the action of a ubiquitin ligase, or E3, that ensures specificity of transfer to the proper degradation substrate. The action of the E3 is iterative, causing the construction of a substrate-bound multiubiquitin chain that is recognized by the 26S proteasome (5). Several E3 ubiquitin ligases are involved in the destruction of ER proteins. Thus, ERAD is a composite of ubiquitination pathways with distinct ligases that use both separate and common components to effect recognition,...

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Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast

Cited by 194 publications

References 53 publications

Misfolding of glycoproteins is a prerequisite for peptide: N‐glycanase mediated deglycosylation

Misfolding of glycoproteins is a prerequisite for peptide: N‐glycanase mediated deglycosylation

Inositol Deacylation by Bst1p Is Required for the Quality Control of Glycosylphosphatidylinositol-anchored Proteins

Usa1p Is Required for Optimal Function and Regulation of the Hrd1p Endoplasmic Reticulum-associated Degradation Ubiquitin Ligase

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