Insig required for sterol-mediated inhibition of Scap/SREBP binding to COPII proteins in vitro.
When added to living cells, sterols such as cholesterol and 25-hydroxycholesterol block the lateral movement of sterol regulatory element-binding proteins (SREBPs) into COPII-coated vesicles on endoplasmic reticulum (ER) membranes and thereby prevent the SREBPs from reaching the Golgi complex for processing to the mature forms that activate cholesterol synthesis. Sorting of SREBPs into COPII vesicles is mediated by Sar1 and the coat proteins Sec23 and Sec24. Here, we explore the mechanism of sterol inhibition in vitro through use of protein pull-down assays. We show that addition of cholesterol or 25-hydroxycholesterol to microsomal membranes in vitro blocks Sar1-dependent binding of the Sec23/24 complex to Scap, the SREBP escort protein. This in vitro inhibition is dependent on the presence of Insig-1, an ER resident protein that is necessary for sterol-mediated inhibition of Scap/SREBP transport in intact cells. Sec23/24 binding to Scap requires the hexapeptide sequence MELADL located in a cytoplasmic loop of Scap. This hexapeptide acts as a sterol-regulated ER sorting signal. These studies define the biochemical parameters responsible for regulated sorting of an ER membrane protein into COPII-coated vesicles.
Sun
Li-Ping
LP
Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9046, USA.
Li
Lu
L
Goldstein
Joseph L
JL
Brown
Michael S
MS
D000595:Amino Acid Sequence; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D016466:CHO Cells; D022181:COP-Coated Vesicles / Q000737:chemistry*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003470:Culture Media / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D007313:Insecta; D050356:Lipid Metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; D003470:Culture Media; D004268:DNA-Binding Proteins; D010455:Peptides; D011994:Recombinant Proteins; C512739:SEC23A protein, human; C497387:SREBF1 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols; D014157:Transcription Factors; D033921:Vesicular Transport Proteins; D005982:Glutathione Transferase