Molecular identification of the sterol-regulated luminal protease that cleaves SREBPs and controls lipid composition of animal cells.
The lipid composition of animal cells is controlled by SREBPs, transcription factors released from membranes by sterol-regulated proteolysis. Release is initiated by Site-1 protease (S1P), which cleaves SREBPs in the ER luminal loop between two membrane-spanning regions. To clone S1P, we prepared pCMV-PLAP-BP2, which encodes a fusion protein that contains placental alkaline phosphatase (PLAP) in the ER lumen flanked by cleavage sites for signal peptidase and S1P. In sterol-deprived cells, cleavage by both proteases leads to PLAP secretion. PLAP is not secreted by SRD-12B cells, cholesterol auxotrophs that lack S1P. We transfected SRD-12B cells with pCMV-PLAP-BP2 plus pools of CHO cDNAs and identified a cDNA that restores Site-1 cleavage and PLAP secretion. The cDNA encodes S1P, an intraluminal 1052-amino-acid membrane-bound subtilisin-like protease. We propose that S1P is the sterol-regulated protease that controls lipid metabolism in animal cells.
Sakai
J
J
Department of Molecular Genetics, University of Texas, Southwestern Medical Center, Dallas 75235, USA.
Rawson
R B
RB
Espenshade
P J
PJ
Cheng
D
D
Seegmiller
A C
AC
Goldstein
J L
JL
Brown
M S
MS
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics; D010920:Placenta / Q000201:enzymology; D043484:Proprotein Convertases; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols / Q000378:metabolism*; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C497387:SREBF1 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D013261:Sterols; D014157:Transcription Factors; D000469:Alkaline Phosphatase; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; C115575:membrane-bound transcription factor peptidase, site 1