Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 May 21;27(10):1491-501.
doi: 10.1038/emboj.2008.83. Epub 2008 Apr 17.

Oxygen-regulated Degradation of Fission Yeast SREBP by Ofd1, a Prolyl Hydroxylase Family Member

Affiliations
Free PMC article

Oxygen-regulated Degradation of Fission Yeast SREBP by Ofd1, a Prolyl Hydroxylase Family Member

Bridget T Hughes et al. EMBO J. .
Free PMC article

Abstract

Sre1, the fission yeast sterol regulatory element binding protein, is an endoplasmic reticulum membrane-bound transcription factor that responds to changes in oxygen-dependent sterol synthesis as an indirect measure of oxygen availability. Under low oxygen, Sre1 is proteolytically cleaved and the released N-terminal transcription factor (Sre1N) activates gene expression essential for hypoxic growth. Here, we describe an oxygen-dependent mechanism for regulation of Sre1 that is independent of sterol-regulated proteolysis. Using yeast expressing only Sre1N, we show that Sre1N turnover is regulated by oxygen. Ofd1, an uncharacterized prolyl 4-hydroxylase-like 2-oxoglutarate-Fe(II) dioxygenase, accelerates Sre1N degradation in the presence of oxygen. However, unlike the prolyl 4-hydroxylases that regulate mammalian hypoxia-inducible factor, Ofd1 uses multiple domains to regulate Sre1N degradation by oxygen; the Ofd1 N-terminal dioxygenase domain is required for oxygen sensing and the Ofd1 C-terminal domain accelerates Sre1N degradation. Our data support a model whereby the Ofd1 N-terminal dioxygenase domain is an oxygen sensor that regulates the activity of the C-terminal degradation domain.

Figures

Figure 1
Figure 1
Sre1N accumulates under low oxygen. (A) Diagram of Sre1. A stop codon was inserted after aa 440 directly before the first predicted transmembrane domain to generate strain sre1N. bHLH denotes the N-terminal transcription factor domain of Sre1. (B) Wild-type and sre1N cells were grown in the presence or absence of oxygen for 6 h. P and N denote the precursor and nuclear forms, respectively. (C) sre1N cells were grown for increasing times in the absence of oxygen. (D) sre1N cells were grown at the indicated oxygen concentration for 6 h. Whole-cell lysates were subjected to western blot analysis with an antibody directed against the N-terminus of Sre1 (anti-Sre1 IgG). For all figures, molecular mass markers (kDa) are shown.
Figure 2
Figure 2
Sre1N is stabilized under low oxygen. (A) Yeast cells expressing Sre1N from either the wild-type (sre1N) or SRE2+3 mutated sre1N promoter (sre1N-MP) were grown in the presence or absence of oxygen for 6 h. (B) sre1N or sre1N-MP cells were grown for increasing times in the absence of oxygen. (C) sre1N-MP cells were grown in the absence of oxygen for 6 h. At t=0, cells were shifted to the presence of oxygen and samples were collected at the indicated times. (D) Wild-type cells were grown for 6 h in the absence of oxygen. At t=0, cultures were shifted to the presence of oxygen and samples were collected at the indicated times. Whole-cell extracts were subjected to western blot analysis using anti-Sre1 IgG and total RNA (5 μg) was subjected to northern analysis with the indicated 32P-labelled probes. α-Tubulin (tub1+) mRNA served as a loading control.
Figure 3
Figure 3
Sre1N is degraded by the proteasome. (A) sre1N cells were grown ±2% DMSO (D) or 200 μM proteasome inhibitor II (P) for 4 h. (B) Wild-type and mts3-1 cells were grown in the absence of oxygen at 30°C for 4 h. The hypoxic workstation was shifted to 35.5°C for an additional 2 h. At t=0, cycloheximide (100 μg/ml) was added and cells were shifted to the presence of oxygen at 35.5°C. Samples were collected at the indicated times. The percent of Sre1N remaining at each time point relative to t=0 is quantified to the right. Whole-cell lysates were subjected to western blot analysis using anti-Sre1 IgG. P denotes the precursor form of Sre1 and N denotes the nuclear form.
Figure 4
Figure 4
Ofd1 is a 2-OG-Fe(II) dioxygenase that negatively regulates Sre1N. (A) sre1N cells were grown for 6 h ±0.5% DMSO, 10 mM DMOG, or 200 μM DFX, or for 6 h in the absence of oxygen. (B) sre1N and sre1N ofd1Δ cells were grown in the presence or absence of oxygen for 6 h. (C) Wild-type and ofd1Δ cells were analysed by indirect immunofluorescence using affinity-purified anti-Ofd1 antibody and 4′,6-diamidino-2-phenylindole (DAPI) to stain DNA. (D) sre1N and sre1N ofd1Δ cells were grown for 6 h in the presence or absence of 0.5% DMSO (V), 10 mM DMOG (D), or 200 μM DFX (X). (E) sre1N-MP and sre1N-MP ofd1Δ cells were grown in the presence of 0.5% DMSO or 10 mM DMOG for 6 h. (F) sre1N-MP and sre1N-MP ofd1Δ cells were grown in the presence or absence of oxygen for 6 h. Whole-cell extracts were subjected to western blot analysis using anti-Sre1 IgG or anti-Ofd1 IgG as indicated. (B, D) Both long and short exposures to film are shown. Total RNA (5 μg) was subjected to northern analysis with the indicated 32P-labelled probes. α-Tubulin (tub1+) mRNA served as a loading control.
Figure 5
Figure 5
Ofd1 accelerates Sre1N turnover in the presence of oxygen. (A) sre1N and sre1N ofd1Δ cells were grown in the absence of oxygen for 6 h. At t=0, cycloheximide (100 μg/ml) was added and cells were shifted to the presence of oxygen. Samples were collected at the indicated times. (B) Wild-type and ofd1Δ cells were grown in the absence of oxygen for 6 h. At t=0, cycloheximide (100 μg/ml) was added and cells were shifted to the presence of oxygen. Samples were collected at the indicated times. P denotes the precursor form of Sre1 and N denotes the nuclear form. (C) Wild-type and ofd1Δ cells expressing Cut8-HA were grown in the absence of oxygen for 6 h. At t=0, cycloheximide (100 μg/ml) was added and cells were shifted to the presence of oxygen. Samples were collected at the indicated times. (A–C) Whole-cell extracts were subjected to western blot analysis using anti-Sre1 IgG or anti-HA IgG as indicated. The percent of Sre1N or Cut8-HA remaining at each time point relative to t=0 is quantified to the right; ofd1+ (squares) and ofd1Δ (triangles).
Figure 6
Figure 6
Prolyl 4-hydroxylase domain of Ofd1 is required for oxygen sensing, but not Sre1N degradation. (A) Sequence alignment of the prolyl 4-hydroxylase domains of S. pombe Ofd1 (aa 23–229) and human HIF PHD3 (NP_071356) (aa 11–213). Identical residues are shaded in black and similar residues are shaded in grey. Residues predicted to be required for iron coordination are indicated by an asterisk. (B) sre1N, sre1N ofd1Δ, sre1N ofd1-H142A, sre1N ofd1-H210A, or sre1N ofd1-H142A;D144A cells were grown in 0.5% DMSO or 10 mM DMOG for 6 h. (C) sre1N, sre1N ofd1Δ, sre1N ofd1-H142A, sre1N ofd1-H210A, or sre1N ofd1-H142A;D144A cells were grown in the presence or absence of oxygen for 3 h. (D) sre1N, sre1N ofd1-H142A, or sre1N ofd1Δ cells were grown in the absence of oxygen for 6 h. At t=0, cycloheximide (100 μg/ml) was added and cells were shifted to the presence of oxygen. Samples were collected at the indicated times. P denotes the precursor form of Sre1 and N denotes the nuclear form. The percent of Sre1N remaining at each time point relative to t=0 is quantified to the right. Whole-cell extracts were subjected to western blot analysis using anti-Sre1 IgG or anti-Ofd1 IgG as indicated.
Figure 7
Figure 7
The C-terminus of Ofd1 is required for accelerating Sre1N degradation. (A) Domain structure of Ofd1 homologues from S. pombe, S. cerevisiae Tpa1p, D. melanogaster CG31120-PA, X. tropicalis LOC549076, and H. sapiens OGFOD1. The black box denotes the prolyl-4-hydroxylase (P4Hc) domain. The percent identity (similarity) of the P4Hc domain of each homologue to the P4Hc domain of S. pombe Ofd1 is shown in the black box. The percent identity (similarity) to S. pombe Ofd1 across the entire length of each homologue is shown to the right. (B) The indicated amino acids of Ofd1 were expressed from a plasmid using the thiamine-repressible Nmt* promoter in sre1N (lane 1) or sre1N ofd1Δ cells (lanes 2–8). Cells were grown in the presence of oxygen in minimal medium lacking thiamine to induce expression of Ofd1. (C) ofd1Δ cells containing an Nmt* plasmid expressing full-length Ofd1, the C-terminus of Ofd1 (Ofd1-C, aa 242–515), or empty vector were grown overnight in minimal medium lacking thiamine to induce expression of Ofd1. Cells were shifted to rich medium and grown in the absence of oxygen for 6 h. At t=0, cycloheximide (100 μg/ml) was added and cells were shifted to the presence of oxygen. Samples were collected at the indicated times. The percent of Sre1N remaining at each time point relative to t=0 is quantified below. Whole-cell lysates were subjected to western blot analysis using anti-Sre1 IgG or anti-Ofd1 IgG.
Figure 8
Figure 8
Oxygen regulates Sre1 by two distinct mechanisms. (A) Model for regulation of Sre1 by oxygen. Sre1 proteolysis is controlled by sterol synthesis, which requires oxygen (1). Under low oxygen, sterol synthesis is inhibited and Sre1 proteolysis is upregulated, leading to increased Sre1N. In addition, degradation of Sre1N is regulated by oxygen (2). In the absence of oxygen, the Ofd1 N-Reg inhibits the Ofd1 CTDD, leading to increased Sre1N stability. In the presence of oxygen, N-Reg inhibition is blocked and Ofd1 CTDD accelerates Sre1N degradation. (B) Wild-type or ofd1Δ cells were grown in the absence of oxygen or treated with 200 μM compactin (CPN) for the indicated times. Whole-cell lysates were subjected to western blot analysis using anti-Sre1 IgG.

Similar articles

See all similar articles

Cited by 61 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

Feedback