Final Report Summary - BIOCHEMIRT (Biochemical characterization of Arabidopsis Fe uptake transporter IRT1)
Iron is an essential nutrient for life. Plants require it as a key component for photosynthesis, oxidative respiration and free radical control among several other processes. However, iron low solubility in soils hampers plant growth and crop production. In order to ensure iron uptake, plants have developed two strategies. Strategy I plants solubilise iron by acidifying the soil with a proton pump, subsequently reduces the Fe (III) to Fe (II) with a reductase and the Fe (II) is subsequently incorporated into the plant by a ZIP transporter. In contrast, Strategy II plants directly remobilise the iron from soils with iron chelators, and the complex chelate-iron enters the plant through YSL transporters.
Given that increased iron uptake would result in higher crop production, we intended to study, from the structure-function point of view, the ZIP transporter responsible for iron uptake in Strategy I plants. As a model we opted for the Arabidopsis thaliana IRT1 transporter. The project included IRT1 purification, determination of its transport kinetics, stoichiometry and its oligomeric state. An essential prerequisite for these analyses resides in the purification of the transporter with a high yield, since some of these assays require relatively big amounts of purified protein. Therefore we dedicated the first months of this proposal to obtain pure iRT1 protein.
Initially, we explored the possibility of expressing IRT1 in E. coli. However, this gene was toxic for all the tested strains. This is not unsual, and it was considered in Decision point 1. 1 of the research proposal. According to this point, a yeast expression system was used as an alternative. To do so, we expressed irt1 cDNA under a galactose inducible promoter that adds a N-terminal (His) 6 tag. This construct was directly assembled in a commercial diploid yeast strain developed for protein expression, INVSc1, by homologous recombination, avoiding the transformation of E. coli cells that might mutate the gene. Cells were grown in 2 % glucose SC medium overnight and subsequently transferred to fresh medium containing 2 % galactose and 1 % raffinose as the sole sugars. After 12 hours of growth, we did observe accumulation of IRT1 protein by Western blot.
In order to purify a membrane protein, the microsomal fraction has to be solubilised with detergents. To find the optimal solubilisation for IRT1 containing microsomes, a series of detergents were used. Our studies indicated that INVSc1 microsomes were solublised the most by phoscholine-12 (90 % of protein solubilised). The solubilised membranes were ultracentrifuged to remove lipids and non-solublised proteins, and the supernatant was incubated with nickel-agarose beads to bind His-tagged IRT1. After the elution from the nickel-agarose column, the protein was concentrated, quantified and its purity assessed by SDS-PAGE . However, this protein was not pure enough for our purposes and most importantly the yield were extremely low to do any structure-function studies (< 100 ug/liter of culture). Several attemps were carried out to improve this yield (other inducers, other strains and other growth conditions), but to no avail.
Currently, other expression systems are being explored: Lactococcus lactis and Pichia pastoris. Both organisms have been used to successfully express membrane proteins in the past. However, since the fellow is taking a tenure-track position in his home country, these assays will be carried out outside the scope of this fellowship in collaboration with French teams that regularly use these methods.
Given that increased iron uptake would result in higher crop production, we intended to study, from the structure-function point of view, the ZIP transporter responsible for iron uptake in Strategy I plants. As a model we opted for the Arabidopsis thaliana IRT1 transporter. The project included IRT1 purification, determination of its transport kinetics, stoichiometry and its oligomeric state. An essential prerequisite for these analyses resides in the purification of the transporter with a high yield, since some of these assays require relatively big amounts of purified protein. Therefore we dedicated the first months of this proposal to obtain pure iRT1 protein.
Initially, we explored the possibility of expressing IRT1 in E. coli. However, this gene was toxic for all the tested strains. This is not unsual, and it was considered in Decision point 1. 1 of the research proposal. According to this point, a yeast expression system was used as an alternative. To do so, we expressed irt1 cDNA under a galactose inducible promoter that adds a N-terminal (His) 6 tag. This construct was directly assembled in a commercial diploid yeast strain developed for protein expression, INVSc1, by homologous recombination, avoiding the transformation of E. coli cells that might mutate the gene. Cells were grown in 2 % glucose SC medium overnight and subsequently transferred to fresh medium containing 2 % galactose and 1 % raffinose as the sole sugars. After 12 hours of growth, we did observe accumulation of IRT1 protein by Western blot.
In order to purify a membrane protein, the microsomal fraction has to be solubilised with detergents. To find the optimal solubilisation for IRT1 containing microsomes, a series of detergents were used. Our studies indicated that INVSc1 microsomes were solublised the most by phoscholine-12 (90 % of protein solubilised). The solubilised membranes were ultracentrifuged to remove lipids and non-solublised proteins, and the supernatant was incubated with nickel-agarose beads to bind His-tagged IRT1. After the elution from the nickel-agarose column, the protein was concentrated, quantified and its purity assessed by SDS-PAGE . However, this protein was not pure enough for our purposes and most importantly the yield were extremely low to do any structure-function studies (< 100 ug/liter of culture). Several attemps were carried out to improve this yield (other inducers, other strains and other growth conditions), but to no avail.
Currently, other expression systems are being explored: Lactococcus lactis and Pichia pastoris. Both organisms have been used to successfully express membrane proteins in the past. However, since the fellow is taking a tenure-track position in his home country, these assays will be carried out outside the scope of this fellowship in collaboration with French teams that regularly use these methods.
