Iron is an essential metal cofactor that is required for many biological processes. assimilation, which may also serve as a source of iron for cell growth. (9), unless exogenous ALA is provided, allowing heme biosynthesis from step 2 to proceed. An additional way to maintain is any amino acid residue). In (growth in the presence of hemin as a sole source of iron. In contrast, growth defect due to the absence of Rbt5 could be restored CB 300919 by adding increasing concentrations of hemin (or hemoglobin), suggesting the existence of additional cellular components or mechanism(s) for acquisition of heme (14). Genome sequence of has revealed other genes encoding CFEM-related proteins, including Pga7, Csa1, Csa2, and Ssr1. In the case of Pga7, its inactivation causes a greater growth defect phenotype than an in a mouse model of systemic infection (14). Besides the cell-surface glycosylphosphatidylinositol (GPI)-anchored proteins Rbt5 and Pga7, additional proteins are involved in exogenous heme acquisition. These proteins include heme oxygenase Hmx1 (15, 16), vacuolar ATPase Vma11, and proteins of the ESCRT (endosomal sorting complex required for transport) system that may be involved in heme trafficking to the vacuole for processing and its utilization as a source of iron (17). However, the mechanism responsible CB 300919 for heme internalization by heme-responsive GPI-anchored proteins remains unclear because of their obvious lack of a cytoplasmic domain. Furthermore, their connection with the ESCRT CB 300919 system and an endocytic pathway to transport cargo to the vacuole remains unclear. Other yeasts such as and use Rbt5-like proteins to acquire heme (18, 19). In the case of because Vps23, a component of the ESCRT system, is also required for heme acquisition (21). Two pathways of iron acquisition have so far been identified in (22). The first pathway consists of a ferrireductase and a ferroxidase-permease complex for high affinity elemental iron uptake (23). The ferrireductase Frp1 reduces Fe3+ to Fe2+ ions prior to uptake through transport by the Fio1-Fip1 heteromeric complex. The second pathway consists of the production and uptake of siderophores. The siderophore synthetase Sib1 and l-ornithine liquid cultures were seeded to an strain genotypes DNA Constructs To generate the pKS-5UTR-fep1+-loxP-kanMX6-loxP-fep13UTR plasmid, a 3,123-bp NotI-EcoRV PCR-amplified DNA segment containing the and YEp357R(27) to CB 300919 generate pSP1and pSP1was used to introduce mutations in all three GATA boxes (positions ?122 to ?127, ?131 to 136, and ?136 to ?141 relative to the A of the ATG codon of fusion plasmids, four high performance liquid chromatography-purified complementary oligonucleotides were annealed pairwise (wild-type strands 1 + 2 and mutated strands 3 + 4) to form double-stranded DNAs. The resulting double-stranded DNAs containing either three consensus GATA-binding sites or three mutated sites were then amplified by PCR. Because of the fact that the primers contained NotI and SpeI restriction sites, the two purified PCR-amplified fragments were digested with these enzymes and inserted immediately upstream of the minimal in pSP1(30). PCR amplification of the mutant allele containing site-specific mutations was created CB 300919 using a similar approach, except that the plasmid pBP-1317and pSKand (5-CAATCTAGAATCAATTAGTGAGGGATAGTCTG-3), (5-GCCATCTTATATAGTACTGGAAATTCAATGAATTAAG-3), (5-CCCACTTCTTCCAGGCATCTG-3), and (5-GTCGGAGTTGGTGTCCACTTTG-3). Two primers derived from an 18 S ribosomal DNA coding region were used as internal background controls: 18 S-a (5-CAGCTTGCGTTGAATACGTCCC-3) and 18 S-b (5-AGCCAATCCAGAGGCCTCACTA-3). Each qPCR experiment was performed in triplicate, and all ChIP experiments were repeated at least three times using independent chromatin preparations. Direct and Indirect Immunofluorescence Microscopy Mid-logarithmic for 30 min at 4 C. The supernatant containing soluble proteins was set aside, whereas the pellet fraction was resuspended in a buffer consisting of 25 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm EDTA, 1 mm dithiothreitol, 1% Triton X-100, and the mixture of protease inhibitors. Once resuspended, the pellet Notch1 fraction was incubated on ice for 30 min and then recentrifuged at 100,000 for 30 min at 4 C. The supernatant fraction that contained dissolved membrane proteins was used for Western blot analysis or hemin-agarose pulldown assays. In the case of pulldown assays with hemin-agarose, proteins (50 g) were incubated with 500 l of hemin-agarose beads, and the suspensions were mixed end-over-end for 20 min at 25 C. The beads.