Protchenko Olga, Shakoury-Elizeh Minoo, Keane Patricia, Storey Joshua, Androphy Rachel, Philpott Caroline C
Genetics and Metabolism Section, Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bldg. 10, Rm. 9B-16, Bethesda, MD 20892, USA.
Eukaryot Cell. 2008 May;7(5):859-71. doi: 10.1128/EC.00414-07. Epub 2008 Mar 7.
Unlike pathogenic fungi, the budding yeast Saccharomyces cerevisiae is not efficient at using heme as a nutritional source of iron. Here we report that for this yeast, heme uptake is induced under conditions of heme starvation. Heme synthesis requires oxygen, and yeast grown anaerobically exhibited an increased uptake of hemin. Similarly, a strain lacking aminolevulinate synthase exhibited a sixfold increase in hemin uptake when grown without 2-aminolevulinic acid. We used microarray analysis of cells grown under reduced oxygen tension or reduced intracellular heme conditions to identify candidate genes involved in heme uptake. Surprisingly, overexpression of PUG1 (protoporphyrin uptake gene 1) resulted in reduced utilization of exogenous heme by a heme-deficient strain and, conversely, increased the utilization of protoporphyrin IX. Pug1p was localized to the plasma membrane by indirect immunofluorescence and subcellular fractionation. Strains overexpressing PUG1 exhibited decreased accumulation of [(55)Fe]hemin but increased accumulation of protoporphyrin IX compared to the wild-type strain. To measure the effect of PUG1 overexpression on intracellular heme pools, we used a CYC1-lacZ reporter, which is activated in the presence of heme, and we monitored the activity of a heme-containing metalloreductase, Fre1p, expressed from a constitutive promoter. The data from these experiments were consistent with a role for Pug1p in inducible protoporphyrin IX influx and heme efflux.
与致病真菌不同，出芽酵母酿酒酵母在利用血红素作为铁的营养来源方面效率不高。在此我们报告，对于这种酵母，血红素摄取在血红素饥饿条件下会被诱导。血红素合成需要氧气，厌氧培养的酵母对血红素的摄取增加。同样，缺乏氨基乙酰丙酸合酶的菌株在无2-氨基乙酰丙酸的情况下生长时，血红素摄取增加了六倍。我们对在低氧张力或细胞内血红素水平降低条件下生长的细胞进行微阵列分析，以鉴定参与血红素摄取的候选基因。令人惊讶的是，PUG1（原卟啉摄取基因1）的过表达导致血红素缺陷菌株对外源血红素的利用减少，相反，增加了原卟啉IX的利用。通过间接免疫荧光和亚细胞分级分离，Pug1p定位于质膜。与野生型菌株相比，过表达PUG1的菌株[(55)Fe]血红素的积累减少，但原卟啉IX的积累增加。为了测量PUG1过表达对细胞内血红素池的影响，我们使用了CYC1-lacZ报告基因，其在血红素存在时被激活，并且我们监测了由组成型启动子表达的含血红素的金属还原酶Fre1p的活性。这些实验的数据与Pug1p在诱导性原卟啉IX内流和血红素外流中的作用一致。
