Abstract
The pivotal event for sterol-induced degradation of the cholesterol biosynthetic enzyme HMG-CoA reductase is binding of its membrane domain to Insig proteins in the endoplasmic reticulum. Insigs are carriers for gp78, an E3 ubiquitin ligase that marks reductase for proteasomal degradation. We report here the isolation of mutant Chinese hamster ovary cell lines, designated SRD-16, -17, and -18, in which sterol-induced ubiquitination and degradation of reductase are severely impaired. These cells were produced by chemical mutagenesis and selection with SR-12813, a compound that mimics sterols in stimulating ubiquitination and degradation of reductase. Each SRD cell line was found to contain a point mutation in one reductase allele, resulting in substitutions of aspartate for serine-60 (SRD-16), arginine for glycine-87 (SRD-17), and proline for alanine-333 (SRD-18). Sterols failed to promote ubiquitination and degradation of these reductase mutants, owing to their decreased affinity for Insigs. Thus, three different point mutations in reductase, all of which localize to the membrane domain, disrupt Insig binding and abolish sterol-accelerated degradation of the enzyme.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 318-327 |
| Number of pages | 10 |
| Journal | Journal of Lipid Research |
| Volume | 48 |
| Issue number | 2 |
| DOIs | |
| State | Published - Feb 2007 |
Fingerprint
Keywords
- 3-hydroxy-3-methylglutaryl coenzyme A reductase
- Cholesterol homeostasis
- Endoplasmic reticulum-associated degradation
- Membrane attachment region
- Mutagenesis
- Somatic cell genetics
- Ubiquitination
ASJC Scopus subject areas
- Endocrinology
Cite this
Mutations within the membrane domain of HMG-CoA reductase confer resistance to sterol-accelerated degradation. / Lee, Peter C W; Nguyen, Andrew D.; DeBose-Boyd, Russell A.
In: Journal of Lipid Research, Vol. 48, No. 2, 02.2007, p. 318-327.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Mutations within the membrane domain of HMG-CoA reductase confer resistance to sterol-accelerated degradation
AU - Lee, Peter C W
AU - Nguyen, Andrew D.
AU - DeBose-Boyd, Russell A.
PY - 2007/2
Y1 - 2007/2
N2 - The pivotal event for sterol-induced degradation of the cholesterol biosynthetic enzyme HMG-CoA reductase is binding of its membrane domain to Insig proteins in the endoplasmic reticulum. Insigs are carriers for gp78, an E3 ubiquitin ligase that marks reductase for proteasomal degradation. We report here the isolation of mutant Chinese hamster ovary cell lines, designated SRD-16, -17, and -18, in which sterol-induced ubiquitination and degradation of reductase are severely impaired. These cells were produced by chemical mutagenesis and selection with SR-12813, a compound that mimics sterols in stimulating ubiquitination and degradation of reductase. Each SRD cell line was found to contain a point mutation in one reductase allele, resulting in substitutions of aspartate for serine-60 (SRD-16), arginine for glycine-87 (SRD-17), and proline for alanine-333 (SRD-18). Sterols failed to promote ubiquitination and degradation of these reductase mutants, owing to their decreased affinity for Insigs. Thus, three different point mutations in reductase, all of which localize to the membrane domain, disrupt Insig binding and abolish sterol-accelerated degradation of the enzyme.
AB - The pivotal event for sterol-induced degradation of the cholesterol biosynthetic enzyme HMG-CoA reductase is binding of its membrane domain to Insig proteins in the endoplasmic reticulum. Insigs are carriers for gp78, an E3 ubiquitin ligase that marks reductase for proteasomal degradation. We report here the isolation of mutant Chinese hamster ovary cell lines, designated SRD-16, -17, and -18, in which sterol-induced ubiquitination and degradation of reductase are severely impaired. These cells were produced by chemical mutagenesis and selection with SR-12813, a compound that mimics sterols in stimulating ubiquitination and degradation of reductase. Each SRD cell line was found to contain a point mutation in one reductase allele, resulting in substitutions of aspartate for serine-60 (SRD-16), arginine for glycine-87 (SRD-17), and proline for alanine-333 (SRD-18). Sterols failed to promote ubiquitination and degradation of these reductase mutants, owing to their decreased affinity for Insigs. Thus, three different point mutations in reductase, all of which localize to the membrane domain, disrupt Insig binding and abolish sterol-accelerated degradation of the enzyme.
KW - 3-hydroxy-3-methylglutaryl coenzyme A reductase
KW - Cholesterol homeostasis
KW - Endoplasmic reticulum-associated degradation
KW - Membrane attachment region
KW - Mutagenesis
KW - Somatic cell genetics
KW - Ubiquitination
UR - http://www.scopus.com/inward/record.url?scp=33846882148&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33846882148&partnerID=8YFLogxK
U2 - 10.1194/jlr.M600476-JLR200
DO - 10.1194/jlr.M600476-JLR200
M3 - Article
C2 - 17090658
AN - SCOPUS:33846882148
VL - 48
SP - 318
EP - 327
JO - Journal of Lipid Research
JF - Journal of Lipid Research
SN - 0022-2275
IS - 2
ER -