ISOLATION OF CHOLESTEROL-REQUIRING MUTANT CHINESE-HAMSTER OVARY CELLSWITH DEFECTS IN CLEAVAGE OF STEROL REGULATORY ELEMENT-BINDING PROTEINS AT SITE-1

The synthesis and uptake of cholesterol requires transcription factors designated sterol regulatory element-binding proteins (SREBPs). SREBP s are bound to membranes in a hairpin orientation with their transcrip tionally active NH2-terminal segments facing the cytosol, The NH2-term inal segments are released from membranes by two-step proteolysis init iated by site 1 protease (S1P), which cleaves in the luminal loop betw een two membrane-spanning segments, Next, site 2 protease (S2P) releas es the NH2-terminal fragment of SREBP. The S2P gene was recently isola ted by complementation cloning using Chinese hamster ovary cells that require cholesterol for growth, due to a mutation in the S2P gene. A s imilar approach cannot be used for S1P because all previous cholestero l auxotrophs manifest defects in S2P, which is encoded by a single cop y gene. To circumvent this problem, in the current studies we transfec ted Chinese hamster ovary cells with the S2P cDNA, assuring multiple c opies. We mutagenized the cells, selected for cholesterol auxotrophy, and identified two mutant cell lines (SRD-12A and -12B) that fail to c leave SREBPs at site 1, Complementation analysis demonstrated that the defects in both cell lines are recessive and noncomplementing, indica ting a mutation in the same gene. These cells should now be useful for expression cloning of the sterol-regulated SIP gene.