Proteins with sequence homology to SREBP, Scap, and INSIG, called Sre1, Scp1, and Ins1, respectively, have been identified in the fission yeast S. pombe
). Sre1 contains two predicted transmembrane segments and a bHLH DNA-binding domain with the unique tyrosine residue found in all SREBP orthologs. Initial studies of these proteins revealed many similarities to the mammalian SREBP system (). First, Sre1 is synthesized as a membrane-bound precursor and is cleaved in response to a decrease in ergosterol, the fungal equivalent of cholesterol. This has been demonstrated using specific pharmacological inhibitors of sterol biosynthesis, including statin and azole drugs (18
). Second, Sre1 and Scp1 physically interact, and Scp1 is required for Sre1 cleavage and activation (19
). Third, in vitro
and in vivo
experiments determined that Sre1 binds directly to conserved SRE sequences in target gene promoters (45
). Finally, Sre1 activates genes required for ergosterol biosynthesis, and Sre1 mutants are defective for maintaining proper sterol levels (18
). On the basis of these findings, it is clear that the SREBP pathway is conserved in fungi as an important regulator of sterol homeostasis.
Conserved SREBP pathway components in fungi
Fig. 2. SREBP pathway in fungi. (A) S. pombe. In the presence of oxygen, sterols inhibit proteolysis of Sre1 and Ofd1 accelerates Sre1N degradation. In the absence of oxygen, both Sre1 proteolysis and stability increase, leading to upregulation of genes required (more ...)
Despite these similarities, the S. pombe
Sre1 pathway displays interesting differences from the mammalian SREBP pathway. Most importantly S. pombe
Sre1 is a major regulator of the fission yeast hypoxic response (19
). Sre1 activity is dramatically upregulated in the absence of oxygen. One reason for this is that, unlike mammals, fission yeast is unable to transport sterols from the extracellular medium and cells rely solely on the biosynthesis of ergosterol, which is heavily oxygen-requiring. Consistent with this, under low-oxygen conditions, ergosterol synthesis decreases and Sre1 cleavage is induced. In this way, environmental oxygen concentrations directly affect cellular sterol levels and indirectly regulate Sre1 activity. In addition to regulating sterol biosynthetic enzymes, Sre1 activates genes coding for enzymes in other oxygen-dependent metabolic pathways, including heme, sphingolipid, and ubiquinone biosynthesis. Thus, Sre1 regulates the transcriptional responses under both low-sterol and low-oxygen conditions (45
). Sre1 does not regulate pathways involved in respiration or fermentation, suggesting that other transcription factors exist to regulate changes in energy metabolism under low-oxygen conditions. Transcriptional profiling experiments revealed that Sre1 is a central regulator of hypoxic gene expression and that consequently, Sre1 is essential for growth under anaerobic conditions.
Further investigation of Sre1 in the hypoxic transcriptional response revealed a sterol-independent mechanism for the regulation of Sre1 activity. The cleaved soluble N terminus of Sre1 (Sre1N) undergoes oxygen-mediated regulation by the 2-o
ioxygenase domain-containing protein Ofd1 (20
). Ofd1 regulates Sre1N through a mechanism reminiscent of the mammalian hypoxia-inducible factor (HIF) transcription factor pathway (22
). In that system, the alpha subunit of HIF is negatively regulated under normoxic conditions by the members of the 2-oxoglutarate Fe(II) dioxygenase prolyl hydroxylase (PHD) family of enzymes. PHDs bind directly to oxygen and catalyze the hydroxylation of HIF alpha, resulting in its ubiquitinylation and proteasomal degradation. When oxygen levels are low, PHDs can no longer catalyze this reaction and HIF is stabilized and activates the transcriptional response under low-oxygen conditions. Similarly, S. pombe
Ofd1 negatively regulates Sre1N stability in an oxygen-dependent manner () (20
). Under normoxia, Ofd1 accelerates the proteasomal degradation of Sre1N. Under anaerobic conditions, Ofd1 is bound to the nuclear protein Nro1 (n
egulator of O
fd1), which prevents Sre1N degradation, leading to increased activation of Sre1 target gene expression (25
). In this model, oxygen disrupts the binding of Ofd1 to Nro1, thereby allowing Ofd1 to promote Sre1N degradation. Important questions remain regarding the mechanism by which Ofd1 regulates Sre1N stability and whether Ofd1 acts as a prolyl hydroxylase similar to the mammalian PHD enzymes. The dual regulation by oxygen of Sre1 cleavage and protein turnover confers robust amplification of Sre1N levels under low-oxygen conditions. Using these two modules, cells are able to fine-tune levels of Sre1 activity in response to the oxygen and lipid environment.
While S. pombe
has an INSIG homolog called Ins1, it does not function directly in the Sre1 pathway (6
). Mammalian INSIG regulates sterol homeostasis by two mechanisms: (i) INSIG regulates the sterol-dependent retention of Scap-SREBP on the ER membrane (), and (ii) INSIG promotes the degradation of the sterol biosynthetic enzyme HMG-CoA reductase (HMGR) under high-sterol conditions (13
). S. pombe
Ins1 functions to regulate sterol homeostasis similarly to this second mechanism (6
). However, instead of regulating HMGR protein stability, Ins1 is a negative regulator of HMGR enzymatic activity.
In addition to Sre1, fission yeast carries another SREBP-like protein, called Sre2 (19
). Sre2 contains the conserved tyrosine in its DNA-binding domain and has two predicted transmembrane segments. However, unlike Sre1 or SREBP, Sre2 has a short C-terminal cytoplasmic tail (23 amino acids) that does not bind Scp1. Therefore, decreased sterol levels do not activate Sre2, and Sre2 is cleaved constitutively. This sterol-independent cleavage raises questions as to why Sre2 is associated with the membrane when its release from the membrane does not appear to be regulated by sterol levels. Further studies will be required for identification of the transcriptional targets of Sre2 and the factors that regulate its activity.
Another difference between the fission yeast and mammalian SREBP systems is the lack of site 1 and site 2 protease homologs in the fission yeast genome, necessitating an alternate mechanism for Sre1 and Sre2 cleavage. Further studies of the mechanism of Sre1 and Sre2 proteolysis in S. pombe may reveal interesting insights into the evolution of SREBPs as well as potentially novel factors involved in mammalian SREBP processing.