Use of Modular Substrates Demonstrates Mechanistic Diversity and Reveals Differences in Chaperone Requirement of ERAD

Taxis, Christof; Hitt, Reiner; Park, Sae-Hun; Deák, Péter; Kostova, Zlatka; Wolf, Dieter H.

doi:10.1074/jbc.m301080200

Cited by 176 publications

(171 citation statements)

References 79 publications

Supporting

Mentioning

166

Contrasting

Order By: Relevance

“…Moreover, CD3␦ is highly ubiquitinated in gp78⌬C49-transfected cells, suggesting an absence of the necessary step to target ubiquitinated CD3␦ for degradation. It is known that multiple pathways operate to degrade unwanted proteins from the ER (45,(47)(48)(49)(50). In agreement with this, a recent study describes the discovery of a novel ER membrane-anchored protein VIMP that interacts with p97/VCP and recruits p97/VCP to Derlin1, 4 Y. Shen, P. Ballar, and S. Fang, unpublished observation.…”

Section: Discussionsupporting

confidence: 54%

AAA ATPase p97/Valosin-containing Protein Interacts with gp78, a Ubiquitin Ligase for Endoplasmic Reticulum-associated Degradation

Zhong

Shen

Ballar

et al. 2004

Journal of Biological Chemistry

191

194

View full text Add to dashboard Cite

Endoplasmic reticulum-associated degradation (ERAD)is a protein quality control mechanism that eliminates unwanted proteins from the endoplasmic reticulum (ER) through a ubiquitin-dependent proteasomal degradation pathway. gp78 is a previously described ER membrane-anchored ubiquitin ligase (E3) involved in ubiquitination of ER proteins. AAA ATPase (ATPase associated with various cellular activities) p97/valosincontaining protein (VCP) subsequently dislodges the ubiquitinated proteins from the ER and chaperones them to the cytosol, where they undergo proteasomal degradation. We now report that gp78 physically interacts with p97/VCP and enhances p97/VCP-polyubiquitin association. The enhanced association correlates with decreases in ER stress-induced accumulation of polyubiquitinated proteins. This effect is abolished when the p97/VCP-interacting domain of gp78 is removed. Further, using ERAD substrate CD3␦, gp78 consistently enhances p97/VCP-CD3␦ binding and facilitates CD3␦ degradation. Moreover, inhibition of endogenous gp78 expression by RNA interference markedly increases the levels of total polyubiquitinated proteins, including CD3␦, and abrogates VCP-CD3␦ interactions. The gp78 mutant with deletion of its p97/VCP-interacting domain fails to increase CD3␦ degradation and leads to accumulation of polyubiquitinated CD3␦, suggesting a failure in delivering ubiquitinated CD3␦ for degradation. These data suggest that gp78-p97/VCP interaction may represent one way of coupling ubiquitination with retrotranslocation and degradation of ERAD substrates.

show abstract

Section: Discussionsupporting

confidence: 54%

AAA ATPase p97/Valosin-containing Protein Interacts with gp78, a Ubiquitin Ligase for Endoplasmic Reticulum-associated Degradation

Zhong

Shen

Ballar

et al. 2004

Journal of Biological Chemistry

191

194

View full text Add to dashboard Cite

show abstract

“…Recently, it was reported that a membrane protein and a soluble luminal protein in the ER were degraded by distinct cellular mechanisms (Taxis et al, 2003;Huyer et al, 2004;Vashist and Ng, 2004). In addition, two ER surveillance mechanisms have been proposed: ERAD-L, which monitors the folded state of luminal domains; and ERAD-C, which monitors that of cytosolic domains (Ahner and Brodsky, 2004;Vashist and Ng, 2004).…”

Section: Discussionmentioning

confidence: 99%

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

Fujita

Yoko-o

Jigami

2006

MBoC

View full text Add to dashboard Cite

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...

show abstract

“…Microsomal membranes containing a green fluorescent protein-tagged transmembrane domain linked to carboxypeptidase yscY (for details, see Ref. 16) were derived from W303-1C ⌬der3 (MAT␣ ade2-1 ura3-1 his3-11, 15 leu2-3, 112 trp1-1 can1-100 prc1-1, ⌬der3::HIS3) harboring the plasmid pMA1 (kindly provided by D. Wolf, University Stuttgart, Germany). Strains were grown at 27°C in liquid YPD medium (1% Bacto yeast extract, 2% Bacto peptone (Difco, 2% glucose).…”

Section: Methodsmentioning

confidence: 99%

Flippase Activity Detected with Unlabeled Lipids by Shape Changes of Giant Unilamellar Vesicles

Papadopulos

Vehring

López‐Montero

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

Transbilayer movement of phospholipids in biological membranes is mediated by energy-dependent and energy-independent flippases. Available methods for detection of flippase mediated transversal flip-flop are essentially based on spin-labeled or fluorescent lipid analogues. Here we demonstrate that shape change of giant unilamellar vesicles (GUVs) can be used as a new tool to study the occurrence and time scale of flippase-mediated transbilayer movement of unlabeled phospholipids. Insertion of lipids into the external leaflet created an area difference between the two leaflets that caused the formation of a bud-like structure. Under conditions of negligible flip-flop, the bud was stable. Upon reconstitution of the energy-independent flippase activity of the yeast endoplasmic reticulum into GUVs, the initial bud formation was reversible, and the shapes were recovered. This can be ascribed to a rapid flip-flop leading to relaxation of the monolayer area difference. Theoretical analysis of kinetics of shape changes provides self-consistent determination of the flip-flop rate and further kinetic parameters. Based on that analysis, the half-time of phospholipid flip-flop in the presence of endoplasmic reticulum proteins was found to be on the order of few minutes. In contrast, GUVs reconstituted with influenza virus protein formed stable buds. The results argue for the presence of specific membrane proteins mediating rapid flip-flop.In lipid bilayers the spontaneous movement of major phospholipids, e.g. of phosphatidylcholine (PC), 6 between the two monolayers is slow, with half-times on the order of hours or even days. However, lipid topology of cellular membranes results from a continuous bidirectional movement (flip-flop) of lipids between the two leaflets in which specific membrane proteins, so called flippases, play an essential role (1, 2). Energyindependent flippases allow phospholipids to equilibrate rapidly between the two monolayers, whereas energy-dependent flippases mediate a net transfer of specific phospholipids to one leaflet of the membrane. Candidates for the latter flippase are members of a conserved subfamily of P-type ATPases (2) as well as ATP binding cassette transporters (3).In eukaryotes the cytoplasmic leaflet of the endoplasmic reticulum (ER) membrane is the major site of phospholipid biosynthesis. To ensure stable membrane growth, energy-independent flippases mediate rapid, bidirectional, and rather unspecific phospholipid flip-flop with half-times of minutes or less (4 -7). A similar flippase activity was also found in the bacterial inner membrane, where lipid synthesis occurs likewise at the cytoplasmic leaflet (8).Techniques to determine transbilayer phospholipid movement as well as the activity of flippases have been critically evaluated (9). Spin-labeled and fluorescent lipid analogues have provided much insight into protein-mediated transbilayer dynamics of phospholipids (9). However, the bulky reporter moieties may affect the absolute values of transbilayer lipid movement.An alternati...

show abstract

Use of Modular Substrates Demonstrates Mechanistic Diversity and Reveals Differences in Chaperone Requirement of ERAD

Cited by 176 publications

References 79 publications

AAA ATPase p97/Valosin-containing Protein Interacts with gp78, a Ubiquitin Ligase for Endoplasmic Reticulum-associated Degradation

AAA ATPase p97/Valosin-containing Protein Interacts with gp78, a Ubiquitin Ligase for Endoplasmic Reticulum-associated Degradation

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

Flippase Activity Detected with Unlabeled Lipids by Shape Changes of Giant Unilamellar Vesicles

Contact Info

Product

Resources

About