The Dfm1 Derlin Is Required for ERAD Retrotranslocation of Integral Membrane Proteins

Abstract

Endoplasmic reticulum (ER)-associated degradation (ERAD) removes misfolded proteins from the ER membrane and lumen by the ubiquitin-proteasome pathway. Retrotranslocation of ubiquitinated substrates to the cytosol is a universal feature of ERAD that requires the Cdc48 AAA-ATPase. Despite intense efforts, the mechanism of ER exit, particularly for integral membrane (ERAD-M) substrates, has remained unclear. Using a self-ubiquitinating substrate (SUS), which undergoes normal retrotranslocation independently of known ERAD factors, and the new SPOCK (single plate orf compendium kit) micro-library to query all yeast genes, we found the rhomboid derlin Dfm1 was required for retrotranslocation of both HRD and DOA ERAD pathway integral membrane substrates. Dfm1 recruited Cdc48 to the ER membrane with its unique SHP motifs, and it catalyzed substrate extraction through its conserved rhomboid motifs. Surprisingly, dfm1Δ can undergo rapid suppression, restoring wild-type ERAD-M. This unexpected suppression explained earlier studies ruling out Dfm1, and it revealed an ancillary ERAD-M retrotranslocation pathway requiring Hrd1.

Endosome and Golgi‐associated degradation ( EGAD ) of membrane proteins regulates sphingolipid metabolism

“…To better define the Orm2 degradation process, we tested whether ubiquitinated Orm2 became soluble prior to proteasomal degradation or whether it was directly degraded while associated with membranes (Mayer et al , ; Lee et al , ; Smith et al , ). Therefore, cells were lysed (without detergent) after proteasome inhibition and the insoluble membrane fraction (P100) was separated from the soluble cytosolic fraction (S100) by centrifugation at 100,000 × g (Neal et al , , ) (Fig A). Orm2 was immunoprecipitated from these fractions after denaturing and its ubiquitination was analyzed.…”

“…The subcellular fractionation protocol was adapted from Neal et al (). Briefly, 20 OD 600 nm (40 OD 600 nm of MG‐132‐untreated cells in Fig C) of logarithmically growing yeast were collected and resuspended in 1 ml ice‐cold water with phosphatase inhibitors (10 mM NaF, 10 mM β‐glycerophosphate, 0.1× PhosSTOP (Roche)).…”

Peroxisome biogenesis, membrane contact sites, and quality control

“…Because of the potential of mis‐sorting peroxisomal proteins to the wrong subcellular compartment or having misfolded, aberrant, or overexpressed peroxisomal proteins residing in the wrong compartment, the question of quality control pathways to prevent the detrimental consequences of such events has arisen. Although not shown explicitly, any PMPs that are missorted to, or misfolded in, the ER, are likely to be subjected to the ERAD (ER‐associated degradation) pathway .…”

“…As described earlier, the PTS receptors/co‐receptors shuttle relevant PTS cargos from the cytosol to the peroxisome, prior to their return to the cytosol for additional rounds of peroxisomal matrix protein import. An interesting ubiquitin–proteasome system (UPS)‐dependent, QC system similar to the ERAD pathway also exists on peroxisomes.…”

BK channel density is regulated by endoplasmic reticulum associated degradation and influenced by the SKN-1A/NRF1 transcription factor

“…Derlin shares significant homology with rhomboid family intramembrane proteases but has no enzymatic activity. For this reason, Derlin has been proposed to recognize substrates and transfer them to HRD1 for ERAD [44][45][46][47]. While mammals have three different Derlin proteins (Der1, Der2 and Der3), C. elegans possesses two Derlin homologs cup-2 and der-2.…”