Abstract
Insig-1 and Insig-2 are membrane proteins of the endoplasmic reticulum that regulate lipid metabolism by the following two actions: 1) sterol-induced binding to 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an action that leads to ubiquitination and degradation of the enzyme; and 2) sterol-induced binding to SREBP cleavage-activating protein, an action that blocks the proteolytic processing of sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors that enhance the synthesis of cholesterol and fatty acids. Here we report the isolation of a new mutant line of Chinese hamster ovary cells, designated SRD-14, in which Insig-1 mRNA and protein are not produced due to a partial deletion of the INSIG-1 gene. The SRD-14 cells were produced by gamma-irradiation, followed by selection with the 1,1-bisphosphonate ester SR-12813, which mimics sterols in accelerating reductase degradation but does not block SREBP processing. SRD-14 cells fail to respond to sterols by promoting reductase ubiquitination and degradation. The rate at which sterols suppress SREBP processing is significantly slower in SRD-14 cells than wild type CHO-7 cells. Sterol regulation of reductase degradation and SREBP processing is restored when SRD-14 cells are transfected with expression plasmids encoding either Insig-1 or Insig-2. These results provide formal genetic proof for the essential role of Insig-1 in feedback control of lipid synthesis in cultured cells.
Highlights
Insig-1 and Insig-2, a pair of closely related endoplasmic reticulum (ER)1-localized membrane proteins, play a substantial role in the feedback control of lipid synthesis in animal cells
Insig-1 and Insig-2 are membrane proteins of the endoplasmic reticulum that regulate lipid metabolism by the following two actions: 1) sterol-induced binding to 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an action that leads to ubiquitination and degradation of the enzyme; and 2) sterol-induced binding to sterol regulatory element-binding protein (SREBP) cleavage-activating protein, an action that blocks the proteolytic processing of sterol regulatory elementbinding proteins (SREBPs), membrane-bound transcription factors that enhance the synthesis of cholesterol and fatty acids
1) SR-12813 replaced sterols in promoting Insig-dependent degradation and ubiquitination of reductase but not ER retention of the SCAP1⁄7SREBP-2 complex (Fig. 1). 2) Chinese hamster ovary (CHO)-7 cells failed to grow in the presence of SR-12813, and this was overcome by the addition of exogenous cholesterol to the growth medium (Fig. 2A). 3) SR-12813 did not influence the stability of degradation-resistant mutant forms of reductase (Fig. 1D), and the drug failed to kill cells that expressed the mutant but not the wild type form of full-length reductase (Fig. 2B)
Summary
Materials—We obtained MG-132 from Calbiochem; horseradish peroxidase-conjugated, donkey anti-mouse, and anti-rabbit IgGs were from Jackson ImmunoResearch; SR-12813 was from Dr Timothy M. Aliquots of nuclear extract and membrane fractions were subjected to 8% SDS-PAGE, and immunoblot analysis was carried out as described [3]. Mutagenesis and Isolation of SR-12813-resistant Cells Deficient in Insig-1—On day 0, 2.5 ϫ 107 CHO-7 cells were subjected to ␥-irradiation as described previously [44]. Individual colonies were isolated with cloning cylinders, and Insig-1 or Insig-2 expression was assessed by immunoblot analysis with anti-Myc. Cells from a single colony were cloned by limiting dilution and maintained in medium B containing 5% LPDS and 500 g/ml G418 at 37 °C, 8 –9% CO2. Hamster Insig-1 cDNA probes for Northern and Southern blot analysis were prepared by PCR amplification of reverse-transcribed total RNA isolated from wild type CHO-7 cells using the following forward and reverse primers: 5Ј-CCAAGGATCCATGCCCAGGCTGCACGACCAC-3Ј and 5Ј-CCAAGCGGCCGCTCAGTCACTGTGAGGCTTTTCCGG3Ј. Filters were exposed to film with intensifying screens for the indicated time at Ϫ80 °C
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
