Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N

Abstract

SREBP cleavage activating protein (SCAP), a membrane-bound glycoprotein, regulates the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors that control lipid synthesis in animal cells. SCAP-stimulated proteolysis releases active fragments of SREBPs from membranes of the endoplasmic reticulum and allows them to enter the nucleus where they activate transcription. Sterols such as 25-hydroxycholesterol inactivate SCAP, suppressing SREBP proteolysis and turning off cholesterol synthesis. We here report the isolation of Chinese hamster ovary cells with a point mutation in SCAP (Y298C) that renders the protein resistant to inhibition by 25-hydroxycholesterol. Like the previously described D443N mutation, the Y298C mutation occurs within the putative sterol-sensing domain, which is part of the polytopic membrane attachment region of SCAP. Cells that express SCAP(Y298C) continued to process SREBPs in the presence of 25-hydroxycholesterol and hence they resisted killing by this sterol. In wild-type Chinese hamster ovary cells the N-linked carbohydrate chains of SCAP were mostly in the endoglycosidase H-sensitive form when cells were grown in medium containing 25-hydroxycholesterol. In contrast, when cells were grown in sterol-depleted medium, these chains were converted to an endoglycosidase H-resistant form. 25-Hydroxycholesterol had virtually no effect in cells expressing SCAP(D443N) or SCAP(Y298C). The relation between this regulated carbohydrate processing to the SCAP-regulated proteolysis of SREBP remains to be explored.SREBP cleavage-activating protein (SCAP) is a central regulator of lipid synthesis and uptake in animal cells (reviewed in ref. 1). Located in the endoplasmic reticulum (ER), SCAP stimulates the proteolytic cleavage of sterol regulatory element binding proteins (SREBPs), a family of three closely related membrane-bound transcription factors that activate the synthesis of cholesterol and unsaturated fatty acids and their uptake from plasma via the low density lipoprotein receptor (2). The activity of SCAP is abolished by sterols, a regulatory mechanism that maintains a constant lipid composition of cell membranes (1).The SREBPs are tripartite proteins of Ϸ1,100 aa that are bound to membranes of the ER in a hairpin fashion (1). The NH 2 -terminal domain of Ϸ480 aa and the COOH-terminal domain of Ϸ560 aa project into the cytosol. They are joined by a membrane attachment domain consisting of two membranespanning helices and a short hydrophilic loop of 31 aa that projects into the ER lumen. The NH 2 -terminal domain of SREBP is a transcription factor of the basic helix-loop-helix leucine zipper family. In sterol-depleted cells, it is released from the ER membrane by two-step proteolysis, which begins when an enzyme called site-1 protease cleaves SREBP at site 1, which is a leucine-serine bond in the luminal loop (3). This first cleavage separates SREBP into halves and allows a second protease to cleave at site 2, which is a ...

Identification of Luminal Loop 1 of Scap Protein as the Sterol Sensor That Maintains Cholesterol Homeostasis

“…In earlier papers, we referred to the region of Scap containing transmembrane helices 2-6 as the "sterol-sensing domain" (24,27). This nomenclature was based on the observation that evolutionarily related sequences are present in other membrane proteins that are postulated to interact with or be influenced by sterols.…”

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

“…On increased demand for MVA-derived metabolites, the SREBP precursor is transported by COP-II vesicles to the Golgi, where it is sequentially cleaved at two sites by resident site-1 and site-2 proteases. The liberated transcriptionally active domain enters the nucleus and stimulates the transcription of target genes, including HMGR (Sakai et al, 1996;Nohturfft et al, 1998aNohturfft et al, , 2000. 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.…”

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

“…A segment corresponding to helices 2-6 of the SCAP membrane domain demonstrates significant sequence similarities to the corresponding region in reductase, whose membrane domain also contains eight membrane-spanning segments (11), and this region has become known as the sterol-sensing domain (12)(13)(14). Point mutations within the sterol-sensing domain of SCAP prevent it from binding Insigs (1, 2), thus preventing sterolmediated ER retention of SCAP⅐SREBP complexes (8,9,15,16).…”