Characterization of two Chinese hamster ovary cell lines expressing the COOH-terminal domains of sterol regulatory element-binding protein (SREBP)-1.
Sterol regulatory element-binding proteins (SREBPs) regulate transcription of genes encoding enzymes in the cholesterol biosynthetic pathway and the LDL receptor. These proteins are synthesized as membrane-bound precursors and processed to generate the NH2-terminal domains, mature transcription factors. We established two Chinese hamster ovary (CHO) cell lines, CHO-421 expressing the truncated hamster SREBP-1 (amino acids 421-1133) with two transmenbrane domains and CHO-557 expressing another truncated SREBP-1 (amino acids 557-1133) without any transmembrane domains, to investigate the fate of the COOH terminus after cleavage of the NH2-terminal mature SREBP. The cell fractionation experiments revealed that the two proteins, regardless of the absence of transmembrane domains in the SREBP (557-1133), similarly localized in the nuclear envelope and the microsomal membrane fractions, suggesting that these proteins appear to be tightly bound to a membrane protein(s) localizing on the nuclear and endoplasmic reticulum (ER) membranes. Although we predicted that overexpression of the COOH-terminal domains, which were thought to be involved in the regulation of SREBP processing, would result in disruption of the SREBP-dependent transcriptional regulation of several genes, the mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase in these two cell lines were regulated in a sterol-dependent manner. Pulse-chase experiments revealed that the SREBP (421-1133) was relatively rapidly degraded (t1/2 = 4-6 hr) and that the intracellular cholesterol level did not affect the half-life time. The degradation of the SREBP (421-1133) was not suppressed by the treatment of a calpain inhibitor, N-acetyl-leucyl-leucyl-norleucynal (ALLN), which blocks the proteolysis of some proteins within or near the ER. In CHO-557 cells the SREBP (557-1133) was much more rapidly degraded (t1/2 = 1-2 hr), suggesting that the cytosolic COOH-terminal domain is accessible to the enzymatic attacks from the cytoplasm. Taken together, overexpression of the COOH-terminal domains does not affect the regulation of SREBP processing and the domains are rapidly turned over by the cytosolic proteolytic process distinct from the ALLN-sensitive ER degradative pathway.
Kawabe
Y
Y
Department of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan.
Imanaka
T
T
Kodama
T
T
Takano
T
T
Sato
R
R
D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017353:Gene Deletion; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000235:genetics / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D007976:Leupeptins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; C063261:acetylleucyl-leucyl-norleucinal; D006904:Hydroxymethylglutaryl-CoA Synthase; D002154:Calpain