MARCH6 and TRC8 facilitate the quality control of cytosolic and tail‐anchored proteins

Abstract

Misfolded or damaged proteins are typically targeted for destruction by proteasome‐mediated degradation, but the mammalian ubiquitin machinery involved is incompletely understood. Here, using forward genetic screens in human cells, we find that the proteasome‐mediated degradation of the soluble misfolded reporter, mCherry‐CL1, involves two ER‐resident E3 ligases, MARCH6 and TRC8. mCherry‐CL1 degradation is routed via the ER membrane and dependent on the hydrophobicity of the substrate, with complete stabilisation only observed in double knockout MARCH6/TRC8 cells. To identify a more physiological correlate, we used quantitative mass spectrometry and found that TRC8 and MARCH6 depletion altered the turnover of the tail‐anchored protein heme oxygenase‐1 (HO‐1). These E3 ligases associate with the intramembrane cleaving signal peptide peptidase (SPP) and facilitate the degradation of HO‐1 following intramembrane proteolysis. Our results highlight how ER‐resident ligases may target the same substrates, but work independently of each other, to optimise the protein quality control of selected soluble and tail‐anchored proteins.

Parallel genome-wide CRISPR analysis identifies a role for heterotypic ubiquitin chains in ER-associated degradation

“…2D and E, Figs. 3A and D), recently linked to TRC8 and MARCH6-dependent ERAD (Stefanovic-Barrett et al, 2018;van de Weijer et al, 2017). In agreement with these genetic phenotypes, we observed that individual knockouts of the membrane and cytosolic UPS components led to increased steady-state levels (Figs.…”

“…Our study also included GFP u* , a variant of a widely-used reporter of cytosolic UPS activity (Bence et al, 2001) that consists of GFP with a C-terminal 16 amino acid CL1 degron (Gilon et al, 1998) with its single lysine residue mutated to glutamic acid. CL1-containing proteins are clients of ERAD-C in yeast (Metzger et al, 2008;Ravid et al, 2006) and mammalian cells (Stefanovic-Barrett et al, 2018).…”

A BAP31 intrabody induces gastric cancer cell death by inhibiting p27kip1 proteasome degradation

“…However, based on several previously published results, it is likely that the combination of BAP31 and p27 kip1 facilitates some ubiquitin ligases (E3s) on the ER to recognize and promote p27 kip1 degradation. As prior studies have reported, some ER‐resident E3s, such as Doa10p (MARCH 6, also known as TEB4), HRD1, TRC8 and TMEM129, are involved in the proteasome degradation of proteins at the cytosolic face of the ER membrane …”

“…As prior studies have reported, some ER-resident E3s, such as Doa10p (MARCH 6, also known as TEB4), HRD1, TRC8 and TMEM129, are involved in the proteasome degradation of proteins at the cytosolic face of the ER membrane. 41,42 The intrabody approach is a gene-based strategy that relies on the expression of recombinant antibodies in different intracellular compartments to block or modulate the function of target molecules. 27,43 Therefore, we hypothesized that an ERtargeted intrabody against BAP31 might have the specificity to block the interaction of BAP31 with p27 kip1 , which could be beneficial for the suppression of tumor proliferation.…”

Proteolytic ectodomain shedding of membrane proteins in mammals—hardware, concepts, and recent developments

“…More recently, SPP has also been recognized as an ERAD factor that may under certain circumstances act as a non-canonical sheddase, where-different to its common role in regulated intramembrane proteolysis of signal peptides-it does not require initial activatory cleavage by signal peptidase (Boname et al, 2014;Chen et al, 2014;Hsu et al, 2015). Consistent with this dual role, SPP assembles as a homo-tetramer that processes signal peptides (Schrul et al, 2010) and-for its sheddase function-as higher molecular weight assembly with the ERAD factor Derlin1 and ERAD E3 ubiquitin ligases ( Fig 3B) (Stagg et al, 2009;Chen et al, 2014;Stefanovic-Barrett et al, 2018). While SPP had initially been hypothesized to contribute to non-proteolytic dislocation of certain ERAD substrates (Loureiro et al, 2006;Lee et al, 2010), heme oxigenase-1 and XBP1u were recently shown to be shed by SPP, leading to their rapid degradation by the proteasome (Boname et al, 2014;Chen et al, 2014).…”