Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol.
The endoplasmic reticulum enzyme 3-hydroxy-3-methylglutaryl-CoA reductase produces mevalonate, which is converted to sterols and to other products, including geranylgeraniol groups attached to proteins. The enzyme is known to be ubiquitinated and rapidly degraded when sterols and nonsterol end products of mevalonate metabolism accumulate in cells. Here, we use RNA interference to show that sterol-accelerated ubiquitination of reductase requires Insig-1 and Insig-2, membrane-bound proteins of the endoplasmic reticulum that were shown previously to accelerate degradation of reductase when overexpressed by transfection. Alanine substitution experiments reveal that binding of reductase to Insigs and subsequent ubiquitination require the tetrapeptide sequence YIYF in the second membrane-spanning helix of reductase. The YIYF peptide is also found in the sterol-sensing domain of SCAP, another protein that binds to Insigs in a sterol-stimulated fashion. When lysine 248 of reductase is substituted with arginine, Insig binding persists, but the reductase is no longer ubiquitinated and degradation is markedly slowed. Lysine 248 is predicted to lie immediately adjacent to a membrane-spanning helix, suggesting that a membrane-bound ubiquitin transferase is responsible. Finally, we show that Insig-dependent, sterol-stimulated degradation of reductase is further accelerated when cells are also supplied with the 20-carbon isoprenoid geranylgeraniol, but not the 15-carbon farnesol, raising the possibility that the nonsterol potentiator of reductase regulation is a geranylgeranylated protein.
Sever
Navdar
N
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046, USA.
Song
Bao-Liang
BL
Yabe
Daisuke
D
Goldstein
Joseph L
JL
Brown
Michael S
MS
DeBose-Boyd
Russell A
RA
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004224:Diterpenes / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005204:Farnesol / Q000737:chemistry; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D008239:Lysine / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism; D008798:Mevalonic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013261:Sterols / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*
D002352:Carrier Proteins; D004224:Diterpenes; C108626:INSIG1 protein, human; C467727:INSIG2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; D011506:Proteins; D013261:Sterols; D025801:Ubiquitin; D005204:Farnesol; C017338:geranylgeraniol; D006903:Hydroxymethylglutaryl CoA Reductases; D008239:Lysine; D000409:Alanine; D008798:Mevalonic Acid