Membrane Topology of Human Insig-1, a Protein Regulator of Lipid Synthesis

Abstract

Insig-1 is an intrinsic protein of the endoplasmic reticulum (ER) that regulates the proteolytic processing of membrane-bound sterol regulatory element-binding proteins (SREBPs), transcription factors that activate the synthesis of cholesterol and fatty acids in mammalian cells. When cellular levels of sterols rise, Insig-1 binds to the membranous sterol-sensing domain of SREBP cleavage-activating protein (SCAP), retaining the SCAP/SREBP complex in the ER and preventing it from moving to the Golgi for proteolytic processing. Under conditions of sterol excess, Insig-1 also binds to the ER enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, facilitating its ubiquitination and proteasomal degradation. Here, we use protease protection, glycosylation site mapping, and cysteine derivitization to define the topology of the 277-amino acid human Insig-1. The data indicate that short segments at the N and C termini of Insig-1 face the cytosol. Most of the protein is buried within the membrane, forming six transmembrane segments separated by five short luminal and cytosolic loops that range from ϳ5 to 16 amino acids. The membranous nature of Insig-1 is consistent with its sterol-dependent binding to hydrophobic sterolsensing domains in SCAP and HMG CoA reductase.

Regulation of Cholesterol and Fatty Acid Synthesis

“…Both proteins are polytopic membrane proteins with six transmembrane helices (Feramisco et al 2004) that bind to cholesterol-loaded Scap to retain the Scap/SREBP complex in the ER Yang et al 2002). Insig proteins also play an important role in oxysterolregulated cleavage of SREBPs.…”

Isolation of Mutant Cells Lacking Insig-1 through Selection with SR-12813, an Agent That Stimulates Degradation of 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase

“…Sterol-induced binding of Insigs to reductase leads to its ubiquitination and consequent degradation by proteasomes, thus slowing the rate-limiting reaction in cholesterol synthesis (3,4). The two human Insig proteins are 59% identical and are predicted to contain six membrane-spanning helices (5). Insig-1 and Insig-2 appear to have overlapping functions in mediating sterol regulation of SCAP and reductase, but the proteins differ in their mode of regulation (2,6).…”

Dislocation of HMG-CoA Reductase and Insig-1, Two Polytopic Endoplasmic Reticulum Proteins, En Route to Proteasomal Degradation

“…This metabolically regulated traffic of the SREBP precursors critically depends on SREBP cleavage-activating protein (SCAP), a membrane protein with eight transmembranes spans (TMs), which tightly associates with the SREBP precursors and enables the packaging of both proteins in the COP-II vesicles and their transport from the ER to the Golgi Sakai et al, 1997;Nohturfft et al, 1998b). In the presence of sterols, SCAP interacts with either one of two related resident ER membrane proteins, each with six TMs, This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08 -09 -0953) on May 20, 2009. designated Insig-1 and Insig-2 (Feramisco et al, 2004). This sterol-stimulated binding to Insig(s) masks the transport signal in SCAP and abrogates its packaging into the transport vesicles, thus indirectly immobilizes the SREBP precursor in the ER and prevents its movement to the Golgi (Yabe et al, 2002a;Yang et al, 2002).…”

“…When sterols are abundant and demand for MVA-derived products declines, the transport of the SREBP precursor out from the ER is prevented and transcription ceases (Nohturfft et al, 1999(Nohturfft et al, , 2000. This metabolically regulated traffic of the SREBP precursors critically depends on SREBP cleavage-activating protein (SCAP), a membrane protein with eight transmembranes spans (TMs), which tightly associates with the SREBP precursors and enables the packaging of both proteins in the COP-II vesicles and their transport from the ER to the Golgi Sakai et al, 1997;Nohturfft et al, 1998b Feramisco et al, 2004). This sterol-stimulated binding to Insig(s) masks the transport signal in SCAP and abrogates its packaging into the transport vesicles, thus indirectly immobilizes the SREBP precursor in the ER and prevents its movement to the Golgi (Yabe et al, 2002a;Yang et al, 2002).…”