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

Abstract

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...

Direct and essential function for Hrd3 in ER-associated degradation

“…At endogenous levels of Hrd1, the absence of Hrd3 rendered stable Hrd1 inactive toward all substrates tested. The importance of Hrd3 was also clear at high levels of Hrd1 used to test Hrd1 autonomy in ERAD (17,18). Hrd3 was not required for Hrd1-substrate interaction nor Hrd1 multimerization, but was required for Hrd1-mediated E2-substrate interactions.…”

“…Is Hrd3 simply enhancing the same catalytic process, or is it altering the enzymology of Hrd1? This question is particularly relevant because many studies including our own have capitalized on the dispensability of Hrd3 for examination of Hrd1 in microsomes or reconstituted systems (15,17). Certainly these approaches are useful in studying aspects of the HRD pathway although caution must be exerted in marginalization of Hrd3 in the light of its unveiled importance in Hrd1 action.…”

“…These results indicated that the Hrd3-dependent activation of Hrd1 was specific for ubiquitin transfer to ERAD substrates. We tested the effect of Hrd3 on Hrd1 E3 activity in vitro (13,17). The assay involves a microsome donor strain with overexpressed Hmg2-GFP and Hrd1 in a ubc7Δ strain and a cytosol strain overexpressing Ubc7 in a hrd1Δ strain.…”

Dislocation of HMG-CoA Reductase and Insig-1, Two Polytopic Endoplasmic Reticulum Proteins, En Route to Proteasomal Degradation

“…These results complement reports on the dislocation of luminal ERAD substrates (Rodighiero et al, 2002;Elkabetz et al, 2004;Afshar et al, 2005) and of membrane proteins with a single TMs (Huppa and Ploegh, 1997;Tortorella et al, 1998;Ye et al, 2003). Together with recent in vitro evidence for dislocation of ubiquitinated polytopic Ste6*p and Hmg2p (Garza et al, 2009) from yeast microsomes, it seems that, among the possible models suggested for dislocation of polytopic membrane ERAD substrates , complete extraction to the cytosol of intact polypeptide chains is a general feature of the delivery to the proteasome of this class of proteins.…”

Sequential Actions of the AAA-ATPase Valosin-containing Protein (VCP)/p97 and the Proteasome 19 S Regulatory Particle in Sterol-accelerated, Endoplasmic Reticulum (ER)-associated Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase

“…However, a major difference exists in the mechanism for membrane extraction and cytosolic dislocation of reductase and s: proteolytic activity of proteasomes is required for dislocation of s, whereas reductase dislocation can only be observed upon proteasome inhibition. Proteasome inhibition is known to cause several membrane-bound ERAD substrates in addition to reductase to accumulate in the cytosol of cells (13,(41)(42)(43)(44)(45)(46)(47). It will thus be important to determine in future studies whether membrane extraction and release into the cytosol through sequential actions of VCP/p97 and the proteasome 19 S RP are a general ERAD mechanism or a peculiarity in the sterol-accelerated ERAD of reductase.…”