Membrane Topology of UbiA Prenyltransferase Domain-Containing Protein-1 (UBIAD1), a Novel Regulator of Cholesterol Homeostasis

Abstract

UbiA prenyltransferase domain containing protein-1 (UBIAD1) is a polytopic membrane-bound enzyme that synthesizes the vitamin K2 subtype menaquinone-4 (MK-4) by conjugating the prenyl group of geranylgeranyl pyrophosphate (GGpp) to the aromatic acceptor menadione. The enzyme moonlights as a regulator endoplasmic reticulum (ER)-localized 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate limiting enzyme in the synthesis of sterol and nonsterol isoprenoids. When cells are replete with isoprenoids, UBIAD1 constitutively cycles between membranes of the medial-trans Golgi and the ER. When ER membranes become depleted of GGpp, UBIAD1 becomes trapped in the organelle where it binds to and inhibits the ER-associated degradation (ERAD) of HMGCR. This inhibition permits continued synthesis of nonsterol isoprenoids, even when sterols are abundant. The resultant accumulation of GGpp in the ER causes dissociation of the HMGCR-UBIAD1 complex, which allows maximal ERAD of HMGCR and translocation of UBIAD1 to the Golgi. These findings disclose a novel GGpp sensing mechanism that allows for metabolically-regulated, intracellular trafficking of UBIAD1. However, the mechanism for this GGpp-induced transport remains to be determined. In the current study, we use cysteine derivatization and protease protection assays to determine the membrane topology of UBIAD1. These findings are key to the determination of mechanisms through which GGpp modulates the intracellular trafficking of UBIAD1 and ultimately, the ERAD of HMGCR.