Overexpression of Membrane Domain of SCAP Prevents Sterols from Inhibiting SCAP·SREBP Exit from Endoplasmic Reticulum*

SCAP (SREBP cleavage-activating protein) forms a complex with sterol regulatory element-binding proteins (SREBPs) and escorts them from the endoplasmic reticulum (ER) to the Golgi complex where proteases release transcriptionally active segments of SREBPs, which enter the nucleus to activate lipid synthesis. The NH2-terminal segment of SCAP contains eight transmembrane helices, five of which (TM2–6) comprise the sterol-sensing domain. This domain responds to sterols by causing the SCAP·SREBP complex to be retained in the ER, preventing proteolytic release and reducing transcription of lipogenic genes. Here, we use transfection techniques to overexpress a segment of SCAP containing transmembrane helices 1–6 in hamster and human cells. This segment does not interfere with SCAP·SREBP movement to the Golgi in the absence of sterols, but it prevents sterols from suppressing this movement. This block is abolished when SCAP(TM1–6) contains a point mutation (Y298C) that is known to abolish the activity of the sterol-sensing domain. We interpret these findings to indicate that sterols cause the SCAP·SREBP complex to bind to an ER retention protein through an interaction that involves the sterol-sensing domain. The SCAP(TM1–6) segment competes with the SCAP·SREBP complex for binding to this putative retention protein, thereby liberating the SCAP·SREBP complex so that it can move to the Golgi despite the presence of sterols. These studies provide a potential mechanistic explanation for the ability of sterols to block SCAP·SREBP movement from the ER and thereby to control lipid synthesis in animal cells.

[1]  J. Goldstein,et al.  Cleavage of Sterol Regulatory Element-binding Proteins (SREBPs) at Site-1 Requires Interaction with SREBP Cleavage-activating Protein , 1998, The Journal of Biological Chemistry.

[2]  J. Goldstein,et al.  Membrane-bound domain of HMG CoA reductase is required for sterol-enhanced degradation of the enzyme , 1985, Cell.

[3]  P. Espenshade,et al.  Transport-Dependent Proteolysis of SREBP Relocation of Site-1 Protease from Golgi to ER Obviates the Need for SREBP Transport to Golgi , 1999, Cell.

[4]  R. D. Simoni,et al.  Molecular Dissection of the Role of the Membrane Domain in the Regulated Degradation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase (*) , 1995, The Journal of Biological Chemistry.

[5]  J. Goldstein,et al.  The SREBP Pathway: Regulation of Cholesterol Metabolism by Proteolysis of a Membrane-Bound Transcription Factor , 1997, Cell.

[6]  M. Brown,et al.  Failure to Cleave Sterol Regulatory Element-binding Proteins (SREBPs) Causes Cholesterol Auxotrophy in Chinese Hamster Ovary Cells with Genetic Absence of SREBP Cleavage-activating Protein* , 1999, The Journal of Biological Chemistry.

[7]  J. Goldstein,et al.  Topology of SREBP Cleavage-activating Protein, a Polytopic Membrane Protein with a Sterol-sensing Domain* , 1998, The Journal of Biological Chemistry.

[8]  Raman Nambudripad,et al.  The ancient regulatory-protein family of WD-repeat proteins , 1994, Nature.

[9]  X. Hua,et al.  Sterol Resistance in CHO Cells Traced to Point Mutation in SREBP Cleavage–Activating Protein , 1996, Cell.

[10]  M. Brown,et al.  Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. , 1983, Methods in enzymology.

[11]  M. Brown,et al.  Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[12]  X. Hua,et al.  Regulated Cleavage of Sterol Regulatory Element Binding Proteins Requires Sequences on Both Sides of the Endoplasmic Reticulum Membrane (*) , 1996, The Journal of Biological Chemistry.

[13]  M. S. Brown,et al.  Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[14]  X. Hua,et al.  Hairpin Orientation of Sterol Regulatory Element-binding Protein-2 in Cell Membranes as Determined by Protease Protection * , 1995, The Journal of Biological Chemistry.

[15]  M. Brown,et al.  Second-site Cleavage in Sterol Regulatory Element-binding Protein Occurs at Transmembrane Junction as Determined by Cysteine Panning* , 1998, The Journal of Biological Chemistry.

[16]  M. Brown,et al.  A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[17]  X. Hua,et al.  Sterol-Regulated Release of SREBP-2 from Cell Membranes Requires Two Sequential Cleavages, One Within a Transmembrane Segment , 1996, Cell.

[18]  P. Espenshade,et al.  Molecular identification of the sterol-regulated luminal protease that cleaves SREBPs and controls lipid composition of animal cells. , 1998, Molecular cell.

[19]  J. Goldstein,et al.  Identification of Complexes between the COOH-terminal Domains of Sterol Regulatory Element-binding Proteins (SREBPs) and SREBP Cleavage-Activating Protein* , 1997, The Journal of Biological Chemistry.