Oligomerization state influences the degradation rate of 3-hydroxy-3-methylglutaryl-CoA reductase.
The steady-state level of the resident endoplasmic reticulum protein, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), is regulated, in part, by accelerated degradation in response to excess sterols or mevalonate. Previous studies of a chimeric protein (HM-Gal) composed of the membrane domain of HMGR fused to Escherichia coli beta-galactosidase, as a replacement of the normal HMGR cytosolic domain, have shown that the regulated degradation of this chimeric protein, HM-Gal, is identical to that of HMGR (Chun, K. T., Bar-Nun, S., and Simoni, R. D. (1990) J. Biol. Chem. 265, 22004-22010; Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). Since the cytosolic domain can be replaced with beta-galactosidase without effect on regulated degradation, it has been assumed that the cytosolic domain was not important to this process and also that the membrane domain of HMGR was both necessary and sufficient for regulated degradation. In contrast to our previous results with HM-Gal, we observed in this study that replacement of the cytosolic domain of HMGR with various heterologous proteins can have an effect on the regulated degradation, and the effect correlates with the oligomeric state of the replacement cytosolic protein. Chimeric proteins that are oligomeric in structure are relatively stable, and those that are monomeric are unstable. To test the hypothesis that the oligomeric state of the cytosolic domain of HMGR influences degradation, we use an "inducible" system for altering the oligomeric state of a protein in vivo. Using a chimeric protein that contains the membrane domain of HMGR fused to three copies of FK506-binding protein 12, we were able to induce oligomerization by addition of a "double-headed" FK506-like "dimerizer" drug (AP1510) and to monitor the degradation rate of both the monomeric form and the drug-induced oligomeric form of the protein. We show that this chimeric protein, HM-3FKBP, is unstable in the monomeric state and is stabilized by AP1510-induced oligomerization. We also examined the degradation rate of HMGR as a function of concentrations within the cell. HMGR is a functional dimer; therefore, its oligomeric state and, we predict, its degradation rate should be concentration-dependent. We observed that it is degraded more rapidly at lower concentrations.
Cheng
H H
HH
Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA.
Xu
L
L
Kumagai
H
H
Simoni
R D
RD
D019281:Dimerization; D004795:Enzyme Stability / Q000187:drug effects; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020104:Immunophilins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013261:Sterols / Q000378:metabolism; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins
C119717:AP 1510; D008565:Membrane Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013261:Sterols; D006903:Hydroxymethylglutaryl CoA Reductases; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; D016559:Tacrolimus