Mga2p Processing by Hypoxia and Unsaturated Fatty Acids in Saccharomyces cerevisiae : Impact on LORE-Dependent Gene Expression

Abstract

In Saccharomyces cerevisiae, OLE1 encodes a ⌬9 fatty acid desaturase, an enzyme that plays a critical role in maintaining the correct ratio of saturated to monounsaturated fatty acids in the cell membrane. Previous studies have demonstrated that (i) OLE1 expression is repressed by unsaturated fatty acids (UFAs) and induced by low oxygen tension, (ii) a component of this regulation is mediated through the same low oxygen response element (LORE) in the OLE1 promoter, and (iii) Mga2p is involved in LORE-dependent hypoxic induction of OLE1. We now report that LORE-CYC1 basal promoter-lacZ fusion reporter assays demonstrate that UFAs repress the reporter expression under hypoxic conditions in a dose-dependent manner via LORE. Electrophoretic mobility shift assays show that UFAs repress the hypoxia-induced complex formation with LORE. Studies with a construct encoding a truncated form of Mga2p support the hypothesis that both hypoxia and UFA signals affect the processing of Mga2p and the UFA repression of OLE1 hypoxic induction is mediated through Mga2p. Data from Western blot assays provide evidence that under normoxic conditions, Mga2p processing produces approximately equimolar levels of the membrane-bound and processed forms and is unaffected by UFAs. Hypoxic induction of OLE1, however, is associated with increased processing of the protein, resulting in an approximately fivefold increase in the soluble active form that is counteracted by exposure of the cells to unsaturated fatty acids. Data from this study suggest that the Mga2p-LORE interaction plays an important role in OLE1 expression under both normoxic and hypoxic conditions. Saccharomyces cerevisiae is a facultative aerobe that can utilize oxygen for energy production and therefore has developed a number of mechanisms by which to sense and respond to changes in oxygen availability. The regulatory pathways that are involved with the response to hypoxia (low oxygen) have been studied for decades, and there has been significant progress in this field (3,23,43).In mammalian cells, the best-studied hypoxia signal transduction pathway is mediated by hypoxia-inducible factor 1 (HIF-1) (3, 32). HIF-1 is a heterodimer comprised of HIF-1␣ and ARNT, two basic helix-loop-helix proteins in the PAS family (39). ARNT mRNA and protein levels are not significantly affected by ambient oxygen tension. HIF-1␣ mRNA levels are also not appreciably affected by oxygen tension, but HIF-1␣ protein is only minimally present in normoxia and is rapidly degraded by the ubiquitin-proteasome pathway under this condition (15,16,21,30). This process is dependent on the von Hippel-Lindau tumor suppressor protein (pVHL) (24), which serves as the recognition component of a ubiquitin ligase that promotes ubiquitin-dependent proteolysis of HIF-1␣ and directly interacts with a highly conserved region within the HIF-1␣ oxygen-dependent degradation domain (7,22,28,35).Recent studies have indicated that a proline hydroxylation within that conserved region is responsible for targeting HIF-1␣ for pVH...

A dual signalling pathway for the hypoxic expression of lipid genes, dependent on the glucose sensor Rag4, is revealed by the analysis of the KlMGA2 gene in Kluyveromyces lactis

“…Similar to OLE1 of S. cerevisiae (Jiang et al, 2002), KlOLE1 transcription is induced by hypoxia and repressed by UFAs. Oxygen shortage promptly induces KlOLE1 transcription overcoming UFA repression, while a preliminary incubation with UFAs blocks hypoxic induction.…”

Deletion of a subgroup of ribosome-related genes minimizes hypoxia-induced changes and confers hypoxia tolerance

“…The precise level of oxygen was also detected by using a CHEMetrics rhodazine oxygen detection kit (K-7511) with the minimum detection limit at 1 ppb and a range of 0 -20 ppb (56). The hypoxic state was further confirmed by measuring oxygen-controlled promoter activities, including UAS1/CYC1, ANB1, and OLE1 (45,50,67). Sustained cell growth under hypoxic conditions was supplemented with Tween 80 and ergosterol, as described previously (13,45).…”

Role of the non‐respiratory pathways in the utilization of molecular oxygen by Saccharomyces cerevisiae

“…Hence, oxygen and UFA signal transduction pathways likely interact at the OLE1 gene level. 140,199 The Mga2p-LORE interaction seems to play an important role in OLE1 expression under aerobic, oxygen-limited and anaerobic conditions, while Rox1p is likely not involved in its aerobic repression 86,140 (see also section on Oxygen sensing). Free or esterified (Tween 80) oleate is required for anaerobic growth.…”