INSIG: a broadly conserved transmembrane chaperone for sterol-sensing domain proteins

Flury, Isabelle; Garza, Renee M.; Shearer, Alexander G.; Rosén, Johanna; Cronin, Stephen; Hampton, Randolph Y.

doi:10.1038/sj.emboj.7600855

Cited by 64 publications

(80 citation statements)

References 32 publications

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“…Conversely, the E3 ligase, the substrate, and Cue1p, the required anchor for Ubc7p, are integral membrane proteins. Our in vitro assay uses two distinct strains as sources of ER membranes and cytosol that are mixed to initiate the in vitro reaction (21,22). The microsome strain expresses epitope-tagged ligase 3HA-Hrd1p, substrate Hmg2p-GFP, Cue1p, and any other membranebound proteins required for the process.…”

Section: Resultsmentioning

confidence: 99%

“…In Vitro Ubiquitination-In vitro reactions were prepared and analyzed as described previously (21,22), and are delineated in detail in the supplemental material. Briefly, cytosols from strains overexpressing Ubc7p or an otherwise identical ubc7⌬ null strain were prepared similarly to those from the Schekman laboratory (24).…”

Section: Methodsmentioning

confidence: 99%

“…We have reconstituted Hrd1p-mediated ubiquitination and retrotranslocation of Hmg2p-GFP in vitro (21,22). We have now directly demonstrated that the entire 8-spanning Hmg2p-GFP protein is removed from the membrane by this process, remaining intact yet soluble after retrotranslocation.…”

mentioning

confidence: 87%

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In Vitro Analysis of Hrd1p-mediated Retrotranslocation of Its Multispanning Membrane Substrate 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase

Garza

Sato

Hampton

2009

Journal of Biological Chemistry

104

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Endoplasmic reticulum (ER)-associated degradation (ERAD)is responsible for the ubiquitin-mediated destruction of both misfolded and normal ER-resident proteins. ERAD substrates must be moved from the ER to the cytoplasm for ubiquitination and proteasomal destruction by a process called retrotranslocation. Many aspects of retrotranslocation are poorly understood, including its generality, the cellular components required, the energetics, and the mechanism of transfer through the ER membrane. To address these questions, we have developed an in vitro assay, using the 8-transmembrane span ER-resident Hmg2p isozyme of HMG-CoA reductase fused to GFP, which undergoes regulated ERAD mediated by the Hrd1p ubiquitin ligase. We have now directly demonstrated in vitro retrotranslocation of full-length, ubiquitinated Hmg2p-GFP to the aqueous phase. Hrd1p was rate-limiting for Hmg2p-GFP retrotranslocation, which required ATP, the AAA-ATPase Cdc48p, and its receptor Ubx2p. In addition, the adaptors Dsk2p and Rad23p, normally implicated in later parts of the pathway, were required. Hmg2p-GFP retrotranslocation did not depend on any of the proposed ER channel candidates. To examine the role of the Hrd1p transmembrane domain as a retrotranslocon, we devised a self-ubiquitinating polytopic substrate (Hmg1-Hrd1p) that undergoes ERAD in the absence of Hrd1p. In vitro retrotranslocation of full-length Hmg1-Hrd1p occurred in the absence of the Hrd1p transmembrane domain, indicating that it did not serve a required channel function. These studies directly demonstrate polytopic membrane protein retrotranslocation during ERAD and delineate avenues for mechanistic understanding of this general process. The endoplasmic reticulum (ER)2 -associated degradation (ERAD) pathway mediates the destruction of numerous integral membrane or lumenal ER-localized proteins (1, 2). ERAD functions mainly in the disposal of misfolded or unassembled proteins but also participates in the physiological regulation of some normal residents of the organelle. This ER-localized degradation pathway has been implicated in a wide variety of normal and pathophysiological processes, including sterol synthesis (3, 4), rheumatoid arthritis (5), fungal differentiation (6), cystic fibrosis (7, 8), and several neurodegenerative diseases (9). Accordingly, there is great impetus to understand the molecular mechanisms that mediate this broadly important route of protein degradation.ERAD proceeds by the ubiquitin-proteasome pathway, by which an ER-localized substrate is covalently modified by the addition of multiple copies of 7.6-kDa ubiquitin to form a multiubiquitin chain that is recognized by the cytosolic 26S proteasome (10, 11). Ubiquitin is added to the substrate by the successive action of three enzymes. The E1 ubiquitin-activating enzyme uses ATP to covalently add ubiquitin to an E2 ubiquitin-conjugating (UBC) enzyme. Ubiquitin is then transferred from the charged E2 to the substrate or the growing ubiquitin chain by the action of an E3 ubiquitin ligase, resulting in a...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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In Vitro Analysis of Hrd1p-mediated Retrotranslocation of Its Multispanning Membrane Substrate 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase

Garza

Sato

Hampton

2009

Journal of Biological Chemistry

104

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“…2F). Strains carrying the SEC61-2 ts allele, encoding the ERAD substrate Sec61-2, are temperature sensitive because of Hrd1-mediated degradation of essential ER membrane protein Sec61 (23,24). When the HRD pathway is nonfunctional, SEC61-2 ts strains can grow at the normally nonpermissive temperature (37°C).…”

Section: Significancementioning

confidence: 99%

Direct and essential function for Hrd3 in ER-associated degradation

Vashistha

Neal

Singh

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The HRD (HMG-CoA reductase degradation) pathway is a conserved route of endoplasmic reticulum-associated degradation (ERAD), by which misfolded ER proteins are ubiquitinated and degraded. ERAD substrates are ubiquitinated by the action of the Hrd1 RING-H2 E3 ligase. Hrd1 is always present in a stoichiometric complex with the ER membrane protein Hrd3, which is also required for HRD-dependent degradation. Despite its conserved presence, unequivocal study of Hrd3 function has been precluded by its central role in Hrd1 stability. Loss of Hrd3 causes unrestricted self-degradation of Hrd1, resulting in significant loss of the core ligase. Accordingly, the degree to which Hrd3 functions independently of Hrd1 stabilization has remained unresolved. By capitalizing on our studies of Usa1 in Hrd1 degradation, we have devised a new approach to evaluate Hrd3 functions in ERAD. We now show that Hrd3 has a direct and critical role in ERAD in addition to Hrd1 stabilization. This direct component of Hrd3 is phenotypically as important as Hrd1 in the native HRD complex. Hrd3 was required the E3 activity of Hrd1, rather than substrate or E2 recruitment to Hrd1. Although Hrd1 can function in some circumstances independent of Hrd3, these studies show an indispensable role for Hrd3 in living cells.highly conserved quality control pathway responsible for the degradation of both misfolded and normal proteins that limits cellular stress and cytotoxicity caused by an accumulation of misfolded ER proteins (1, 2). ERAD occurs through the ubiquitin proteasome pathway, wherein ER-localized ubiquitin ligases covalently modify substrates by attaching multiple copies of the protein ubiquitin, thus tagging them for degradation by cytosolic 26S proteasome (3-5).In Saccharomyces cerevisiae, the highly conserved HRD (HMGCoA † reductase degradation) pathway degrades a variety of ERAD substrates, including misfolded luminal proteins (ERAD-L substrates) such as CPY* and integral membrane proteins (ERAD-M substrates) such as Hmg2 (6, 7). Briefly, ERAD-L substrates are ubiquitinated by the HRD complex, which includes the ubiquitin RING-H2 ligase Hrd1, complex members Hrd3, Usa1, and Der1, and luminal factors implicated in substrate selection (3,(8)(9)(10)(11). Conversely, ERAD-M substrates require only Hrd1 and Hrd3 for in vivo degradation (12, 13). Although Hrd1 functions as the core catalytic subunit of the HRD E3 ligase, Hrd3's function has remained enigmatic. Both Hrd1 and Hrd3 are highly conserved, and Hrd1 is rate limiting for degradation of misfolded substrates (3,12,14,15). In yeast, the steady-state levels of Hrd1 strongly depend on Hrd3. At native levels of the HRD components, Hrd3 is required for Hrd1 stability: In a hrd3Δ, Hrd1 half-life drops to less than 15 min, resulting in very low levels (12). This concomitant loss of Hrd1 is due to rapid autodegradation, catalyzed by the RING domain of Hrd1 (16).Because of Hrd3's strong role in maintaining Hrd1 stability, it has remained unclear whether Hrd3 has any other independent ERAD functions. A ...

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“…Reduction of intracellular cholesterol by sterol starvation or treatment with lowering-cholesterol drugs triggers the translocation of the SREBPs from the endoplasmic reticulum to the Golgi by the SREBP cleavage activating protein (SCAP), a sensor of the cholesterol intracellular concentration [5]. This displacement is escorted by 2 chaperons Insig-1 and Insig-2 that are proteins tightly bound to the endoplasmic reticulum [6,7].…”

Section: Introductionmentioning

confidence: 99%

Atorvastatin effects on SREBF1a and SCAP gene expression in mononuclear cells and its relation with lowering-lipids response

Arazi

Genvigir

Willrich

et al. 2008

Clinica Chimica Acta

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INSIG: a broadly conserved transmembrane chaperone for sterol-sensing domain proteins

Cited by 64 publications

References 32 publications

In Vitro Analysis of Hrd1p-mediated Retrotranslocation of Its Multispanning Membrane Substrate 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase

In Vitro Analysis of Hrd1p-mediated Retrotranslocation of Its Multispanning Membrane Substrate 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase

Direct and essential function for Hrd3 in ER-associated degradation

Atorvastatin effects on SREBF1a and SCAP gene expression in mononuclear cells and its relation with lowering-lipids response

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