Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4
- Center for Genomic Regulation (CRG), Spain
- Universitat Pompeu Fabra, Spain
- University of Southern Denmark, Denmark
Figures
Figure 1 with 1 supplement
Erg1 is a substrate of the Doa10 complex.
(A) Schematic representation of the mevalonate pathway and its different end products. The steps catalyzed by HMG-CoA reductase and the squalene monooxygenase Erg1 are indicated. Adapted from Goldste…
Figure 1—figure supplement 1
Abundance of Erg1 but not of other components of the Erg pathway is altered in Doa10 complex mutants.
(A) Abundance of the indicated Erg pathway components in endoplasmic reticulum-associated protein degradation (ERAD) mutants relative to wt cells, as detected by mass spectrometry upon SILAC …
Figure 2 with 1 supplement
Doa10-dependent degradation of Erg1 depends on a single lysine residue.
(A) Expression of ERG1 or ERG1-derivatives with the indicated lysine mutations from a plasmid rescues the growth of yeast cells upon repression of the endogenous ERG1. A yeast strain expressing …
Figure 2—figure supplement 1
Doa10-dependent degradation of Erg1 depends on the lysine residue at position 311.
(A) The degradation of Erg1 or the indicated Erg1 lysine mutants expressed from the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was followed after inhibition of protein …
Figure 3 with 1 supplement
Flux through the sterol pathway regulates Erg1 degradation.
(A) Schematic representation of the ergosterol biosynthetic pathway highlighting the enzymatic steps affected by the small inhibitor zaragozic acid, Ro48-807, and fluconazole. The step catalyzed by …
Figure 3—figure supplement 1
Sterol depletion affects Doa10-dependent degradation of Erg1 but not of other Doa10 substrates.
(A) The degradation of the Doa10 substrates Vma12-Ndc10902–956-HA and Erg1 was followed after inhibition of protein synthesis by cycloheximide in wt control cells (DMSO) or in wt cells treated for 2 …
Figure 4
Doa10-dependent degradation of Erg1 affects sterol homeostasis and, together with sterol esterification, is essential to prevent buildup of sterol intermediates.
(A) Relative amounts of ergosterol, lanosterol, and ergostadienol in cells with the indicated genotype. Cells were grown in synthetic complete (SC) media until early stationary phase, and lipids …
Figure 5 with 1 supplement
Doa10 homologue Teb4 promotes degradation of human squalene monooxygenase.
(A) The degradation of endogenous squalene monooxygenase (SM) was followed after inhibition of protein synthesis by cycloheximide in sterol-deprived HEK293 cells treated with control siRNA or siRNA …
Figure 5—figure supplement 1
Overexpression of dominant negative Teb4(C9A), but not of wt Teb4, strongly delays degradation of human squalene monooxygenase.
The degradation of endogenous squalene monooxygenase (SM) was followed after inhibition of protein synthesis by cycloheximide in sterol-deprived HEK293 cells transfected with Teb4-myc or …
Figure 6
A central role of endoplasmic reticulum-associated protein degradation in sterol homeostasis.
(A) Schematic representation of the feedback inhibition systems required for sterol homeostasis in yeast (left) and mammals (right) previously characterized (dotted lines) and described here (solid …
Additional files
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Supplementary file 1
(A) Yeast strains used in this study. (B) Plasmids used in this study. (C) Primers used in this study.
- https://doi.org/10.7554/eLife.00953.013
