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.
Mesh Terms:
Animals, Binding, Competitive, Carrier Proteins, Catalysis, Cell Line, Cytosol, Digitonin, Endoplasmic Reticulum, Fibroblasts, Humans, Hydroxymethylglutaryl CoA Reductases, Immunoblotting, Intracellular Signaling Peptides and Proteins, Ligases, Liver, Male, Membrane Proteins, Models, Chemical, Precipitin Tests, RNA Interference, Rats, Rats, Sprague-Dawley, Sterols, Ubiquitin, Ubiquitin-Protein Ligases
J. Biol. Chem.
Date: Jul. 02, 2004
109196
Switch View:
  • PTM Genes 1