Identification of the biotin transporter in Escherichia coli, biotinylation of histones in Saccharomyces cerevisiae and analysis of biotin sensing in Saccharomyces cerevisiae

摘要: Biotin transport in E. coli was already observed 1972. Results this work demonstrate yigM is the gene encoding biotin transporter. The protein has 10 predicited transmembrane domains and shows homology to members of subfamiliy carboxylate/amino acid/amine transporters. Deletion led complete loss, overexpression with different expression systems a dose dependent increase uptake. function YigM shown whole cells reconstituted membrane vesicles. Weak inhibition uptake by protonophores, respiratory chain blocker NaN3, as well not energized vesicles suggest facilitated diffusion mechanism. E. overexpressing displayed saturable kinetics (KM 74 ± 14 nM). This value similar previously reported measurements wt cells, indicating that only transporter coli. set plasmamembrane transporters extended yigM, for mammals, eucaryotic unicellular organisms, gram-positive bacteria archea have been described before. regulation yigM-gene experiments luciferase-reporter construct. In S. cerevisiae evidence role pyruvate carboxylases, especially carboxylase 2 sensing provided. Single-knockouts two PYC genes reduce response low concentrations, GFP-reporter-assays western-blot. A double-knockout mutant both even suppresses signals completely. Pyc2p demonstrated be independent changes expression, biotinylation enzymatic activity. However fusing an epitope tag truncation at C-terminus revealed end seems influence sensing. Additionally vivo histones H2B, H3 H4 demonstrated. possible biotinylated discussed.