Direct Demonstration of Rapid Degradation of Nuclear Sterol Regulatory Element-binding Proteins by the Ubiquitin-Proteasome Pathway

Abstract

Sterol regulatory element-binding proteins (SREBPs) are synthesized as membrane-bound precursors and processed to generate transcriptionally active forms. The active SREBPs translocate to the nucleus, induce the expression of responsive genes, and are degraded very rapidly. Treatment with proteasome inhibitors elevates the amount of the endogenous nuclear SREBPs, but not the precursors, in HeLa cells. Nuclear forms of human SREBP-1a (amino acids 1-487) and SREBP-2 (amino acids 1-481), which are transiently expressed in stable Chinese hamster ovary cell lines (CHO-487 and -481), are also stabilized by proteasome inhibitors, suggesting that the nuclear SREBPs are likely to be substrates for the proteasome-dependent proteolysis. The stabilized nuclear SREBPs actively induce the expression of responsive genes including hydroxymethylglutaryl (HMG)-CoA synthase, fatty acid synthase, and the low density lipoprotein receptor. The rapid turnover of nuclear SREBP-1a is not affected by the intracellular sterol levels, and the half-life is estimated to be approximately 3 h. The nuclear SREBPs are found conjugated with a polyubiquitin chain. When this conjugation is inhibited by overexpression of mutant ubiquitin that is defective in polyubiquitination, the nuclear SREBPs are partly stabilized and induce the expression of the responsive gene, suggesting that the ubiquitin-conjugated SREBPs are substrates for the proteasome. Taken together, these results demonstrate that the ubiquitin-prot…