Seminario De Metabolismo De Lipoproteinas

Cell, Vol. 99, 703–712, December 23, 1999, Copyright ©1999 by Cell Press

Transport-Dependent Proteolysis of SREBP: Relocation of Site-1 Protease from Golgi to ER Obviates the Need for SREBP Transport to Golgi
Russell A. DeBose-Boyd, Michael S. Brown,* Wei-Ping Li, Axel Nohturfft, Joseph L. Goldstein,* and Peter J. Espenshade Department of Molecular Genetics University of Texas SouthwesternMedical Center Dallas, Texas 75390-9046 complex is formed by an interaction between the cytosolic COOH-terminal regulatory domain of the SREBPs and the cytosolic WD40 domain of SCAP. SCAP targets the SREBPs to S1P, a membrane-bound serine protease whose active site faces the lumen of the ER and postER organelles (Sakai et al., 1998b). S1P cleaves the luminal loop of the SREBPs following thetetrapeptide sequence Arg-Xaa-Xaa-Leu, thereby separating SREBP into two halves (Duncan et al., 1997). This event allows a second protease, designated Site-2 protease (S2P), to cleave the NH2-terminal transmembrane domain, thereby liberating the NH2-terminal segments of SREBPs from the membrane and allowing them to enter the nucleus (Sakai et al., 1996; Rawson et al., 1997). This complex proteolyticactivation mechanism allows lipid synthesis to be controlled by the lipid content of cell membranes (Brown and Goldstein, 1997). When cells are depleted of sterols, the cleavage reactions are rapid, and the NH2-terminal segments of SREBPs readily enter the nucleus. When cells are overloaded with sterols, the Site-1 cleavage reaction is blocked. The SREBPs remain bound to cell membranes, and the targetgenes are no longer activated. Cholesterol synthesis stops as a result of decreased transcription of genes encoding 3-hydroxy3-methylglutaryl coenzyme A (HMG CoA) synthase, HMG CoA reductase, and other enzymes of the cholesterol biosynthetic pathway. Cholesterol uptake is blocked by a downregulation of the low-density lipoprotein (LDL) receptor, and the synthesis of unsaturated fatty acids ispartially decreased as a result of reduced transcription of genes encoding acetyl CoA carboxylase, fatty acid synthase, and stearoyl CoA desaturase (Brown and Goldstein, 1997; Edwards and Ericsson, 1999). Biochemical and genetic data show that SCAP is the key to the regulation of Site-1 cleavage (Hua et al., 1996a; Nohturfft et al., 1996, 1999; Rawson et al., 1999). Mutant Chinese hamster ovary (CHO)cells that lack SCAP fail to cleave SREBPs at Site-1, and they are therefore auxotrophic for cholesterol and unsaturated fatty acids (Rawson et al., 1999). Sterols block Site-1 cleavage by reducing the activity of SCAP. This inhibition does not occur when SCAP bears substitution mutations at either of two positions in its membrane attachment domain (Tyr298Cys or Asp443Asn) (Hua et al., 1996a;Nohturfft et al., 1996, 1998b). This portion of the membrane attachment domain has been designated the sterol-sensing region. Mutant CHO cells that produce these altered forms of SCAP overproduce cholesterol and fail to slow this synthesis when cholesterol overaccumulates in the cell. Evidence indicates that SCAP acts by escorting SREBPs to a post-ER compartment where the active S1P resides and thatsterols may prevent cleavage by blocking this transport (Nohturfft et al., 1998b, 1999). This hypothesis emerged from studies of the N-linked carbohydrates attached to a luminal loop in the polytopic membrane domain of SCAP. When cells are depleted of sterols, these carbohydrates are largely resistant to cleavage by endoglycosidase H (endo H),

Summary Cholesterol homeostasis in animal cells isachieved by regulated cleavage of membrane-bound transcription factors, designated SREBPs. Proteolytic release of the active domains of SREBPs from membranes requires a sterol-sensing protein, SCAP, which forms a complex with SREBPs. In sterol-depleted cells, SCAP escorts SREBPs from ER to Golgi, where SREBPs are cleaved by Site-1 protease (S1P). Sterols block this transport and abolish cleavage....