Hepatic cytochrome P450 endoplasmic reticulum-associated degradation (ERAD): Topological determinants and cellular partnerships that dictate the preferential P450 proteolytic sorting into macroautophagy rather than UPS.

Many N-terminally endoplasmic reticulum (ER)-anchored cytochrome P450 proteins (P450s) turn over proteolytically via ER-associated degradation (ERAD), others via ER-to-lysosomal-associated degradation (ERLAD), and yet others via both pathways. What precisely dictates their differential proteolytic turnover is unknown. Herein, we employed rabbit liver CYPs 1A1 and 1A2, which reportedly reside in liquid-disorded (ld)- and detergent-resistant, liquid-ordered (lo)-ER-microdomains, respectively, governed by their specific N-terminal (NT) signal-anchor (SA) subdomains. We now report that this precise SA-dependent ER-topology not only determines the proclivity of CYP1A1 towards ERAD and CYP1A2 towards ERLAD, but also their differential lifespans. We further document that the detergent-resistant lo-ER-membranes (DRMs) are morphologically quite similar to mitochondria-associated ER-membranes (MAMs), documented cellular platforms for autophagic-initiation complexes. DRMs and MAMs, composed of saturated fatty-acids, glycosphingolipids and cholesterol, harbor many common morphological markers including the ER-specific prohibitin, erlin-1. Herein employing SURF, a split fluorogenic bifunctional complementation assay, we show that intracellularly, erlin-1 and CYP1A2 interact closely via their ER NT-SAs. siRNA-knockdown (KD) of erlin-1 in HepG2-cells, not only relocated CYP1A2 from DRMs to non-DRMs, but also impaired its ERLAD, resulting in insoluble cellular CYP1A2 aggregates. Upon erlin-1 KD, CYP1A2 ERLAD could be rescued by co-expression of a siRNA-resistant intact erlin-1 or just its NT-1-30 residue SA-domain. Our findings are the first to reveal that the CYP1A2 lifespan and preferential proclivity towards ERLAD is determined by its close association with erlin-1 within DRMs/MAMs. As proof of concept, we document that the ERLAD-proclivity of CYP2B1 is also similarly dependent upon erlin-1-DRM-association.