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Abstract
During ER-associated degradation (ERAD), misfolded polytopic membrane proteins are ubiquitinated and retrotranslocated to the cytosol for proteasomal degradation. However, our understanding as to how polytopic membrane proteins are extracted from the ER to the cytosol remains largely unclear. To better define the localization and physical properties of ubiquitinated polytopic membrane substrates in vivo, we performed subcellular fractionation analysis of Ste6*, a twelve transmembrane protein that is ubiquitinated primarily by Doa10 E3 ligase in yeast. Consistent with previous in vitro studies, ubiquitinated Ste6* was extracted from P20 (20,000 g pellet) fraction to S20 (20,000 g supernatant) fraction in a Cdc48/p97-dependent manner. Similarly, Ubx2p, which recruits Cdc48/p97 to the ER, facilitated the extraction of Ste6*. By contrast, lipid droplet formation, which was suggested to be dispensable for the degradation of Hrd1-substrates in yeast, was not required for the degradation of Ste6*. Intriguingly, we found that ubiquitinated Ste6* in the S20 fraction could be enriched by further centrifugation at 100,000 g. Although it is currently uncertain whether ubiquitinated Ste6* in P100 fraction is completely free from any lipids, membrane flotation analysis suggested the existence of two distinct populations of ubiquitinated Ste6* with different states of membrane association. Together, these results imply that ubiquitinated Ste6* may be sequestered into a putative quality control sub-structure by Cdc48/p97. Fractionation assays developed in the present study provide a means to further dissect the ill-defined post-ubiquitination step during ERAD of polytopic membrane substrates.
Highlights
Endoplasmic reticulum (ER)-associated degradation (ERAD) is a conserved pathway that mediates the destruction of lumenal and integral transmembrane proteins. [1,2,3,4,5,6]
A recent study showed that quality control of proteins in the inner nuclear membrane, a specialized ER subdomain, depends on a third ER integral membrane E3 ligase complex that is composed of Asi1, Asi2, and Asi3 [17, 18]
To further analyze the retrotranslocation of ubiquitinated polytopic membrane substrates and to define their physical properties in the cytosol, we focused on a model ER-associated degradation (ERAD)-C substrate, Ste6Ã, an integral membrane protein with 12 transmembrane spans [27, 44, 45], and performed a series of subcellular fractionation assays
Summary
Endoplasmic reticulum (ER)-associated degradation (ERAD) is a conserved pathway that mediates the destruction of lumenal and integral transmembrane proteins. [1,2,3,4,5,6]. The degradation of integral membrane substrates that have misfolded domains in the transmembrane domain (ERAD-M substrate) is Hrd dependent [10, 11]. In addition to ERAD-M type substrates, Hrd was proposed to recognize membrane substrates that persistently associate with the Sec translocon. Degradation of structurally complex proteins, those that possess multiple spans, often depends on both Hrd and Doa, likely because they have multiple misfolded domains [15, 16]. A recent study showed that quality control of proteins in the inner nuclear membrane, a specialized ER subdomain, depends on a third ER integral membrane E3 ligase complex that is composed of Asi, Asi, and Asi3 [17, 18]. Degradation of lanosterol 14-alpha-demethylase (Erg in yeast) is mediated by the Asi complex [17, 18]
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