title: Second-site cleavage in sterol regulatory element-binding protein occurs at transmembrane junction as determined by cysteine panning.
abstract: In response to sterol deprivation, two sequential proteolytic cleavages release the NH2-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 NH2-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 NH2-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 Leu484-Cys485 bond that lies at the junction between the cytoplasmic NH2-terminal fragment and the first transmembrane segment. We transfected cells with cDNAs encoding fusion proteins with single cysteine residues at positions to the NH2-terminal and COOH-terminal sides of cysteine 485. The NH2-terminal fragments were tested for susceptibility to modification with Nalpha-(3-maleimidylpropionyl)biocytin, which attaches a biotin group to cysteine sulfhydryls. Cysteines to the NH2-terminal side of cysteine 485 were retained on the NH2-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 Asp-Arg-Ser-Arg, which immediately precedes the first transmembrane segment and is required for Site-2 cleavage.
authors.affiliation: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
authors.forename: E A
authors.initials: EA
authors.lastname: Duncan
authors.forename: U P
authors.initials: UP
authors.lastname: Davé
authors.forename: J
authors.initials: J
authors.lastname: Sakai
authors.forename: J L
authors.initials: JL
authors.lastname: Goldstein
authors.forename: M S
authors.initials: MS
authors.lastname: Brown
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C497390:SREBF2 protein, human; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors; D003545:Cysteine