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Saccharomyces cerevisiae as a viable model for the overexpression and purification of proteins involved in iron homeostasis
Author(s) -
Hati Debolina
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.631.20
Subject(s) - saccharomyces cerevisiae , mutant , microbiology and biotechnology , yeast , biology , transcription factor , biochemistry , chemistry , gene
Iron is indispensable for the eukaryotic cell but excess of iron is toxic. Disruptions in iron metabolism leads to numerous human diseases, hence the study of iron regulation and metabolism is of high importance. Saccharomyces cerevisiae is used as a model system in our lab to study iron trafficking and homeostasis pathways to better understand iron regulation at the cellular and molecular level. In yeast, two paralogous transcriptional activators, Aft1 and Aft2, play a central role in iron regulation by activating the transcription of target genes in response to iron deprivation. Monothiol glutaredoxins Grx3 and Grx4 that utilize glutathione to bind [2Fe‐2S] clusters interact specifically with Aft1 and Aft2, transferring an Fe‐S cluster to Aft1/2 that inhibits its DNA binding activity. Aft1 is considered the primary regulator of iron homeostasis because aft1 Δ mutants exhibit a stronger iron deficiency phenotype than aft2 Δ mutants. Aft1 and Aft2 share 39% sequence homology in their N‐terminal DNA binding domains. The only available structure is of a truncated version of Aft2 that contains this homologous portion and includes the domains responsible for DNA binding and iron sensing. Aft1/2 have been shown to be very unstable and difficult to overexpress and purify in Escherichia coli . Hence we optimized the growth conditions to overexpress Aft1 in its native organism, Saccharomyces cerevisiae , and further try to purify Aft1 from the same. Further studies will be carried out in order to investigate the in‐vitro interaction between full length Aft1/2 and Grx3/4‐Fra2 to better understand the mechanism of Aft1/2 inhibition at the molecular level. Support or Funding Information Supported by Dr. Caryn E. Outten's Research Funding This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .