Ubiquitination of 3-hydroxy-3-methylglutaryl-CoA reductase in permeabilized cells mediated by cytosolic E1 and a putative membrane-bound ubiquitin ligase.
The endoplasmic reticulum (ER) enzyme, 3-hydroxy-3-methylglutaryl-CoA reductase, catalyzes the production of mevalonate, a rate-controlling step in cholesterol biosynthesis. Excess sterols promote ubiquitination and subsequent degradation of reductase as part of a negative feedback regulatory mechanism. To characterize the process in more detail, we here report the development of a permeabilized cell system that supports reductase ubiquitination stimulated by the addition of sterols in vitro. Sterol-dependent ubiquitination of reductase in permeabilized cells is dependent upon exogenous cytosol, ATP, and either Insig-1 or Insig-2, two membrane-bound ER proteins shown previously to mediate sterol regulation of reductase degradation in intact cells. Oxysterols, but not cholesterol, promote reductase ubiquitination under our conditions. Finally, we show that ubiquitin-activating enzyme (E1) can efficiently replace cytosol to ubiquitinate reductase in response to sterol treatment, suggesting that other molecules required for ubiquitination of reductase, such as the ubiquitin-conjugating and -ligating enzymes (E2 and E3), are localized to ER membranes.
Song
Bao-Liang
BL
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046, USA.
DeBose-Boyd
Russell A
RA
D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism*; D004072:Digitonin / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D008025:Ligases / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008956:Models, Chemical; D011233:Precipitin Tests; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D013261:Sterols / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
D002352:Carrier Proteins; C108626:INSIG1 protein, human; C467727:INSIG2 protein, human; C491574:Insig1 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D013261:Sterols; D025801:Ubiquitin; D006903:Hydroxymethylglutaryl CoA Reductases; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D004072:Digitonin