SUT1-promoted sterol uptake involves the ABC transporter Aus1 and the mannoprotein Dan1 whose synergistic action is sufficient for this process

Abstract

Efficient sterol influx in the yeast Saccharomyces cerevisiae is restricted to anaerobiosis or to haem deficiency resulting from mutations. Constitutive expression of SUT1, an hypoxic gene encoding a transcriptional regulator, induces sterol uptake in aerobiosis. A genome-wide approach using DNA microarray was used to identify the mediators of SUT1 effects on aerobic sterol uptake. A total of 121 ORFs (open reading frames) were significantly and differentially expressed after SUT1 overexpression, 61 down-regulated and 60 up-regulated. Among these genes, the role of the putative ABC transporter (ATP-binding-cassette transporter) Aus1, and of the cell-wall mannoprotein Dan1, was characterized better. These two genes play an essential role in aerobic sterol uptake, since their deletion compromised the SUT1 effects, but individual overexpression of either of these genes in a wild-type background was not sufficient for this process. However, constitutive co-expression of AUS1 and DAN1 in a wild-type background resulted in sterol influx in aerobiosis. These results suggest that the corresponding proteins may act synergistically in vivo to promote sterol uptake.

Figure 1. UPC2 inactivation does not compromise the SUT1 effects on sterol uptake

Serial 10-fold dilutions of cell suspensions of YPA1-1 (upc2Δ) and YPA3 (WT, wild-type), transformed with plasmid pNF1 harbouring the SUT1 gene (resp. upc2Δ[SUT1] and WT[SUT1]) or with the empty vector pNEV-N (resp. upc2Δ[0] and WT[0]), were propagated on selective medium and subsequently spotted on to complete medium (YPD), YPD containing 80 μg/ml ergosterol in Tergitol/ethanol (1:1) with 1 μg/ml fenpropimorph (FEN) or without the inhibitor (TER) and YPD containing 2% (v/v) Tergitol/ethanol (1:1) and 1 μg/ml fenpropimorph (TEF and ergosterol omitted).

Figure 2. SUT1 overexpression impairs respiration

(A) Serial 10-fold dilutions of cell suspensions of YPA3, transformed with pNEV-N (WT[0]) or pNF1 (WT[SUT1]), were spotted on to selective medium containing glucose (20 g/l) as a carbon source (YNB) or a mixture of glycerol (20 g/l) and 1% ethanol (YNBGE). The plates were incubated at 30 °C for 2 days (YNB) or 1 week (YNBGE). (B) Oxygen consumption of the same cells. Results are expressed as the means±S.D. for three independent experiments. Statistical significance of differences was examined by the Mann–Whitney test (P <0.05, n=3).

Figure 3. Effects of AUS1 and DAN1 on sterol uptake induced by SUT1

Serial 10-fold dilutions of a suspension of Y11787 (aus1Δ) and YPA4 (dan1Δ) transformed with Yep13-SUT1 were propagated on selective medium and subsequently spotted on to complete medium (YPD) and complete medium containing 1 μg/ml fenpropimorph, 80 μg/ml ergosterol in Tergitol/ethanol (1:1, v/v) (FEN). The same strains co-transformed with Yep13-SUT1 and pNEV-AUS1 or pNEV-DAN1 were used to ensure that the effects could be complemented by AUS1 or DAN1 respectively. The YPA3 strain (WT) transformed with Yep13-SUT1 was used as a positive control.

Figure 4. Effect of AUS1 constitutive expression in a rox1Δ strain

Serial 10-fold dilutions of cell suspensions of YPA5 (rox1Δ) untransformed or transformed with either pNEV-AUS1 or pNF1 harbouring the SUT1 gene, were propagated on selective medium and subsequently spotted on to either complete medium (YPD) or complete medium containing 1 μg/ml fenpropimorph, 80 μg/ml ergosterol in Tergitol/ethanol (1:1) (FEN). The strain YPA6 (rox1Δdan1Δ) transformed with pNF1 was also spotted on to the same media (lower panels). The largest isolated colonies growing in the presence of fenpropimorph (with the exception of the rox1Δ[SUT1] strain) correspond to spontaneous resistant mutants.