title: Reconstitution of sterol-regulated endoplasmic reticulum-to-Golgi transport of SREBP-2 in insect cells by co-expression of mammalian SCAP and Insigs.
abstract: In mammalian cells, membrane-bound sterol regulatory element-binding proteins (SREBPs) are transported from ER to Golgi where they are processed proteolytically to generate soluble transcription factors that activate lipid synthesis. ER-to-Golgi transport requires SCAP, a sterol-regulated escort protein. In sterol-treated cells, the SCAP/SREBP complex binds to Insig-1 or Insig-2, which retains the complex in the ER, blocking SREBP processing and decreasing lipid synthesis. In Drosophila cells, the endogenous SCAP/SREBP complex is transported to Golgi, but transport is blocked by phosphatidylethanolamine instead of sterols. Here, we show that mammalian SREBP-2 is not transported to Golgi when expressed in Drosophila cells. Transport requires co-expression of mammalian SCAP. Sterols block transport of the mammalian SCAP/SREBP-2 complex, but only when mammalian Insig-1 or -2 is co-expressed. These reconstitution studies define SCAP and Insig as the minimal requirements for sterol-regulated transport of SREBPs from ER to Golgi. They indicate that insect cells can respond to sterols when proper regulatory proteins are expressed.
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046.
authors.forename: Irina Y
authors.initials: IY
authors.lastname: Dobrosotskaya
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Goldstein
authors.forename: Michael S
authors.initials: MS
authors.lastname: Brown
authors.forename: Robert B
authors.initials: RB
authors.lastname: Rawson
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D019856:Ethanolamine / Q000494:pharmacology; D005822:Genetic Vectors; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D047908:Intracellular Signaling Peptides and Proteins; D008322:Mammals; D008565:Membrane Proteins / Q000235:genetics*; D019308:Palmitic Acid / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011506:Proteins / Q000235:genetics*; D051782:Sterol Regulatory Element Binding Protein 2; D013261:Sterols / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C108626:INSIG1 protein, human; C467727:INSIG2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011506:Proteins; C102196:SREBP cleavage-activating protein; D051782:Sterol Regulatory Element Binding Protein 2; D013261:Sterols; D014157:Transcription Factors; D019308:Palmitic Acid; D019856:Ethanolamine