Sterol regulatory element binding proteins (SREBPs) are transcription factors attached to the endoplasmic reticulum. The NH2-segment, which activates transcription, is connected to membranes by a hairpin anchor formed by two transmembrane sequences and a short lumenal loop. Using H-Ras-SREBP-2 fusion proteins, we show that the NH2-segment is released from membranes by two sequential cleavages. The first, regulated by sterols, occurs in the lumenal loop. The second, not regulated by sterols, occurs within the first transmembrane domain. The liberated NH2-segment enters the nucleus and activates genes controlling cholesterol synthesis and uptake. Certain mutant Chinese hamster ovary cells are auxotrophic for cholesterol because they fail to carry out the second cleavage; the NH2-segment remains membrane-bound and transcription is not activated.

A sterol-regulated protease initiates release of the NH 2 -terminal segments of sterol regulatory elementbinding proteins (SREBPs) from cell membranes, thereby allowing them to enter the nucleus and to stimulate transcription of genes involved in the uptake and synthesis of cholesterol and fatty acids. Using SREBP-2 as a prototype, we here identify the site of sterol-regulated cleavage as the Leu 522 -Ser 523 bond in the middle of the 31-residue hydrophilic loop that projects into the lumen of the endoplasmic reticulum and nuclear envelope. This site was identified through use of a vector encoding an SREBP-2/Ras fusion protein with a triple epitope tag that allowed immunoprecipitation of the cleaved COOH-terminal fragment. The NH 2 terminus of this fragment was pinpointed by radiochemical sequencing after replacement of selected codons with methionine codons and labeling the cells with [35 S]methionine. Alanine scanning mutagenesis revealed that only two amino acids are necessary for recognition by the sterol-regulated protease: 1) the leucine at the cleavage site (leucine 522), and 2) the arginine at the P4 position (arginine 519). These define a tetrapeptide sequence, RXXL, that is necessary for cleavage. Cleavage was not affected when the second transmembrane helix of SREBP-2 was replaced with the membrane-spanning region of the low density lipoprotein receptor, indicating that this sequence is not required for regulation. Glycosylation-site insertion experiments confirmed that leucine 522 is located in the lumen of the endoplasmic reticulum. We conclude that the sterol-regulated protease is a novel enzyme whose active site faces the lumen of the nuclear envelope, endoplasmic reticulum, or another membrane organelle to which the SREBPs may be transported before cleavage.Proteolytic processing of sterol regulatory element-binding proteins (SREBPs) 1 controls the metabolism of cholesterol and fatty acids in animal cells (1-3). SREBPs are transcription factors that are bound to membranes of the ER and nuclear envelope. Each SREBP is composed of three segments: 1) an NH 2 -terminal segment of ϳ485 amino acids that is a transcription factor of the basic helix-loop-helix-leucine zipper family, 2) a membrane attachment segment of ϳ75 amino acids composed of two membrane-spanning sequences separated by a short hydrophilic loop of 31 amino acids, and 3) a COOH-terminal segment of ϳ585 amino acids that plays a regulatory role. The proteins are oriented so that the NH 2 -and COOH-terminal segments project into the cytoplasm, and only the short hydrophilic loop projects into the lumen of the ER or nuclear envelope (4).Before it can activate transcription, the NH 2 -terminal segment is released from the membrane in a complex two-step proteolytic sequence (2, 3). First, a protease cleaves the protein at Site-1, which is near an arginine in the lumenal loop, thereby breaking the attachment between the two transmembrane sequences. This allows a second protease to cleave the protein at Site-2, which is near the middle of th...

In response to sterol deprivation, two sequential proteolytic cleavages release the NH 2 -terminal fragments of sterol regulatory element-binding proteins (SREBPs) from cell membranes. The fragments translocate to the nucleus where they activate genes involved in cholesterol and fatty acid metabolism. The SREBPs are bound to membranes in a hairpin fashion. The NH 2 -terminal and COOH-terminal domains face the cytoplasm, separated by two membrane spanning segments and a short lumenal loop. The first cleavage occurs at Site-1 in the lumenal loop. The NH 2 -terminal fragment is then released by cleavage at Site-2, which is believed to lie within the first transmembrane segment. Here, we use a novel cysteine panning method to identify the second cleavage site (Site-2) in human SREBP-2 as the Leu 484 -Cys 485 bond that lies at the junction between the cytoplasmic NH 2 -terminal fragment and the first transmembrane segment. We transfected cells with cDNAs encoding fusion proteins with single cysteine residues at positions to the NH 2 -terminal and COOH-terminal sides of cysteine 485. The NH 2 -terminal fragments were tested for susceptibility to modification with N ␣ -(3-maleimidylpropionyl)biocytin, which attaches a biotin group to cysteine sulfhydryls. Cysteines to the NH 2 -terminal side of cysteine 485 were retained on the NH 2 -terminal fragment, but cysteines to the COOH-terminal side of leucine 484 were lost. Leucine 484 is three residues to the COOH-terminal side of the tetrapeptide AspArg-Ser-Arg, which immediately precedes the first transmembrane segment and is required for Site-2 cleavage.Cells maintain cholesterol homeostasis through a feedback pathway that depends upon the regulated proteolytic processing of a pair of membrane-bound transcription factors, sterol regulatory element-binding proteins-1 and -2 (SREBP-1 and SREBP-2) 1 (reviewed in Ref. 1). Cells deprived of cholesterol process the SREBPs proteolytically to release the NH 2 -terminal domains which travel to the nucleus to activate transcription of genes that regulate several metabolic pathways: uptake of cholesterol and fatty acids (low density lipoprotein receptor and lipoprotein lipase) (1, 2), synthesis of cholesterol (3-hydroxy-3-methylglutaryl-CoA reductase, 3-hydroxy-3-methylglutaryl-CoA synthase, farnesyl diphosphate synthase, squalene synthase) (1, 3-5), synthesis of fatty acids (acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase) (1, 2, 6, 7), and synthesis of triglycerides (glycerol-3-phosphate acyltransferase) (8). When cells accumulate sterols, proteolysis of the SREBPs is suppressed, nuclear SREBPs decline, and the transcription of the target genes is reduced. This regulation assures a steady supply of cholesterol and fatty acids while preventing their overaccumulation.SREBPs are unique among transcription factors because they are synthesized as membrane-bound precursors with three domains. The precursors are bound to the endoplasmic reticulum and nuclear envelope in a hairpin orientation (9). The cytoplasmic NH 2...

We conclude that MCH nonspecifically increases ingestive behavior. Furthermore, MCH had no apparent effect on anxiety. The ability of MCH to increase alcohol and/or sucrose intake may be explained by the effect of MCH on energy balance and/or reward processes.

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