2ND-SITE CLEAVAGE IN STEROL REGULATORY ELEMENT-BINDING PROTEIN OCCURSAT TRANSMEMBRANE JUNCTION AS DETERMINED BY CYSTEINE PANNING

In response to sterol deprivation, two sequential proteolytic cleavage s release the NH2-terminal fragments of sterol regulatory element-bind ing proteins (SREBPs) from cell membranes, The fragments translocate t o the nucleus where they activate genes involved in cholesterol and fa tty acid metabolism. The SREBPs are bound to membranes in a hairpin fa shion. 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-termi nal fragment is then released by cleavage at Site-2, which is believed to lie within the first transmembrane segment. Here, we use a novel c ysteine panning method to identify the second cleavage site (Site-2) i n human SREBP-2 as the Leu(484) Cys(485) bond that lies at the junctio n between the cytoplasmic NH2-terminal fragment and the first transmem brane segment. We transfected cells with cDNAs encoding fusion protein s with single cysteine residues at positions to the NH2-terminal and C OOH-terminal sides of cysteine 485. The NH2-terminal fragments were te sted for susceptibility to modification with N-alpha-(3-maleimidylprop ionyl)biocytin, which attaches a biotin group to cysteine sulfhydryls, Cysteines to the NH2-terminal side of cysteine 485 were retained on t he NH2-terminal fragment, but cysteines to the COOH-terminal side of l eucine 484 were lost. Leucine 484 is three residues to the COOH-termin al side of the tetrapeptide Asp-Arg-Ser-Arg, which immediately precede s the first transmembrane segment and is required for Site-2 cleavage.