Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retro-translocation complex mediating protein transport for ER degradation

Abstract

The endoplasmic reticulum contains a quality control system that subjects misfolded or unassembled secretory proteins to rapid degradation via the cytosolic ubiquitin proteasome system. This requires retrograde protein transport from the endoplasmic reticulum back to the cytosol. The Sec61 pore, the central component of the protein import channel into the endoplasmic reticulum, was identified as the core subunit of the retro-translocon as well. As import of mutated proteins into the endoplasmic reticulum lumen is successfully terminated, a new targeting mechanism must exist that mediates re-entering of misfolded proteins into the Sec61 pore from the lumenal side de novo. The previously identified proteins Der3p/Hrd1p and, as we show here, Hrd3p of the yeast Saccharomyces cerevisiae, are localised in the endoplasmic reticulum membrane and are essential for the degradation of several substrates of the endoplasmic reticulum degradation machinery. Based on genetic studies we demonstrate that they functionally interact with each other and with Sec61p, probably establishing the central part of the retro-translocon. In the absence of Hrd3p, the otherwise stable protein Der3p/Hrd1p becomes rapidly degraded. This depends on a functional ubiquitin proteasome system and the presence of substrate molecules of the endoplasmic reticulum degradation system. When overexpressed, Der3p/Hrd1p accelerates CPY* degradation in Delta(hrd3) cells. Our data suggest a recycling process of Der3p/Hrd1p through Hrd3p. The retro-translocon seems to be build up at least by the Sec61 pore, Der3p/Hrd1p and Hrd3p and mediates both retrograde transport and ubiquitination of substrate molecules.

ProteinN-Glycosylation along the Secretory Pathway: Relationship to Organelle Topography and Function, Protein Quality Control, and Cell Interactions

“…The mode of action of these noncatalytic, mannosidase-like proteins, in particular the way they distinguish the Man 8 GlcNAc 2 isomer B bearing native from non-native glycoproteins remains elusive at present. Likewise, any possible interaction of these mannosidase-like proteins with other components of ERAD ,− that direct misfolded glycoproteins for retrotranslocation remains to be elucidated.…”

“…The mode of action of these noncatalytic, mannosidase-like proteins, in particular the way they distinguish the Man 8 GlcNAc 2 isomer B bearing native from non-native glycoproteins remains elusive at present. Likewise, any possible interaction of these mannosidase-like proteins with other components of ERAD 189,[226][227][228][229][230][231][232][233][234][235] that direct misfolded glycoproteins for retrotranslocation remains to be elucidated. Some complementary results have been obtained by the immunoelectron microscopic localization of glucosidase II, 48,236 calreticulin, 48 and glucosyltransferase.…”

Viruses Hijack ERAD to Regulate Their Replication and Propagation

“…Several analyses in yeast show that to a large extent, the Hrd1-SEL1L complex itself is the retrotranslocation channel through which the substrate is brought back from the ER to the cytoplasm [ 54 ]. The self-ubiquitination of HRD1 is considered the trigger of substrate retrotranslocation, which is similar to the “door” of the substrate retrotranslocation channel [ 45 , 55 , 56 , 57 , 58 ]. Recently, the structural analysis of the HRD1 complex by cryo-EM from Saccharomyces cerevisiae showed that Hrd1 and Der1 were connected on the cytoplasmic side of the membrane through Usa1.…”

Lipid Biosynthesis Perturbation Impairs Endoplasmic Reticulum-Associated Degradation

“…In ERAD, ubiquitin ligases transfer ubiquitin from ubiquitin-conjugating enzymes to aberrant or overabundant proteins, which are subsequently degraded by the 26S proteasome. The two major ERAD ubiquitin ligases in Saccharomyces cerevisiae are the highly conserved multipass transmembrane enzymes, Hrd1 and Doa10 (HAMPTON et al 1996;PLEMPER et al 1999;SWANSON et al 2001). Hrd1 functions with the soluble ubiquitin-conjugating enzyme Ubc7 and, to a lesser extent, Ubc1 and Ubc6 (PLEMPER et al 1999;BAYS et al 2001;LIPS et al 2020).…”

“…The two major ERAD ubiquitin ligases in Saccharomyces cerevisiae are the highly conserved multipass transmembrane enzymes, Hrd1 and Doa10 (HAMPTON et al 1996;PLEMPER et al 1999;SWANSON et al 2001). Hrd1 functions with the soluble ubiquitin-conjugating enzyme Ubc7 and, to a lesser extent, Ubc1 and Ubc6 (PLEMPER et al 1999;BAYS et al 2001;LIPS et al 2020). Doa10 functions with two ubiquitin-conjugating enzymes, Ubc7 and the transmembrane protein Ubc6 (SWANSON et al 2001).…”