Inositol phosphate and phosphoinositides are metabolites that have several regulatory functions in eukaryotes, including the control of gene expression, protein trafficking, signal transduction, and cell development. They perform these regulatory function by binding to proteins, thereby changing protein conformation, catalytic activity, or protein interactions. The identification of proteins that bind to inositol phosphate or phosphoinositides is essential to understand how those metabolites perform their regulatory function.
This protocol has a simple workflow that's sensitive, non-radioactive, lyposome free and uses reagents that are commercially available. In this method, a finished chromatography with biotinylated inositol phosphate or phosphoinositides, is used to isolate interacting proteins, they are then identified by western blot, or mass spectrometry. For blood stream forms, grow T.brucei cells to mid log phase in HMI-9 media, supplemented with 10%FBS, at 37 degree Celsius, with 5%Carbon Dioxide.
Keep the cell density between 800, 000 and 1.6 million cells per milliliter. When ready, centrifuge the cells at 1600 times G, and at room temperature for 10 minutes. Discard the supernatant, and gently resuspend the pellet in 10 milliletres of PBS-G pre-heated at 37 degrees Celsius to wash the cells.
Centrifuge the cells at 1600 times G and at room temperature for five minutes to complete the wash. Repeat this washing procedure twice more. Then resuspend the pellet in one milliliter of PBS-G.
Transfer this suspension to a one point five milliliter tube, then centrifuge at 1600 times G for five minutes. Discard the supernatant, and resuspend the pellet in zero point five milliliters of lysis buffer, supplemented with one point five X protease inhibitor cocktail, and one X phosphatase inhibitor cocktail that has been pre-chilled on ice to lyse the cells. Centrifuge the lysate at 1400 times G and at four degrees Celsius for 10 minutes.
After this collect the supernatant, which contains the extracted parasite proteins, into a new one point five milliliter tube for the binding assays. Set aside 5%of the total lysate for western blot analysis. Collect 50 microliters of inositol phosphates or phosphoinositides conjugated to agarose beads, and add them 500 milliliters of binding buffer.
Centrifuge at 1000 times G for one minute. Discard the supernatant, and resuspend in 50 microliters of binding buffer to equilibrate the beads. Use non-conjugated beads as a control, and use beads with different phosphate configurations including non-phosphorylated forms, to control for unspecific interactions.
Next, add 50 microliters of IP or PI beads to the cell lysate or the purified proteins. Keep the volume of beads within 10%of the total lysate. If necessary, use binding buffer to adjust the volume of the binding reaction.
Incubate the reaction for either one hour or overnight at four degrees Celsius, while rotating at 50rpm. After this, centrifuge the mix at 1000 times G, and at four degrees Celsius for one minute. Remove the supernatant, and keep the pellet, making sure to keep 5%of the supernatant for western blot analysis.
Then add one milliliter of washing buffer and tap or swirl the tube to resuspend the resin. Centrifuge the reaction at 1000 times G, and at four degrees Celsius for one minute, and discard the supernatant. Repeat this process for a total of five washes.
After this, add 50 microliters of 2X Laemmli buffer supplemented with 710 millimolar 2-mercaptoethanol to the beads. Tap or vortex to mix, and dilute the proteins. Next, heat at 95 degrees Celsius for five minutes, and centrifuge at 10, 000 times G for one minute.
Collect the supernatant, and either freeze the eluate at minus 80 degrees Celsius, or proceed to western blot analysis. The method presented here is used to analyze the binding of PIs by repressor-activator protein 1 from T.brucei lysate, or by recombinant T.brucei repressor-activator protein 1 protein. As RAP1 lacks canonical PI binding domains, the binding assays are performed with PIs that are non-phosphorylated, or that are phosphorylated at different positions of the inositol ring, and with non-conjugated agarose beads.
Western analysis shows that RAP1 binds preferentially to PI(3, 4, 5)P3 beads, but that it also binds to lesser extent to PI(4, 5)P2 beads. However, it did not bind to any other PIs or agarose beads. To test whether RAP1 binds directly to PIs, a C terminally tagged 6XHis recombinant RAP1 protein is expressed and purified to homogeneity from E.Coli.
Western blotting shows that increasing the concentration of PI(3, 4, 5)P3, but not PI(4, 5)P2, inhibits the interaction of recombinant RAP1 with PI(3, 4, 5)P3 beads. The addition of T.brucei purified PIP5Pase enzyme to the reaction restored PI(3, 4, 5)P3 binding by recombinant RAP1. This is due to PIP5Pase dephosphorylation of free PI(3, 4, 5)P3, and thus indicates that the phosphorylation pattern of this metabolite is essential for our RAP1 binding.
T.brucei proteins that bind to Ins(1, 4, 5)P3 are then identified by affinity chromatography followed by mass spectrometry. SDS-PAGE analysis shows enrichment in proteins eluted from Ins(1, 4, 5)P3 beads, compared to proteins eluted from the control agarose beads. Mass spectrometry analysis of the eluted proteins identified over 250 proteins of which 84 were enriched with Ins(1, 4, 5)P3 beads, compared to control beads.
The enrichment of proteins bound to Ins(1, 4, 5)P3, compared to control beads, correlates with the protein signal detected by SDS-PAGE. A critical step in this protocol is the use of appropriate controls, to discriminate specific from non-specific interactions. We recommend using non-conjugated agarose beads, and beads conjugated with inositol phosphate or phosphoinositides, using various phosphate configurations, including non-phosphorylated forms.
This method can be applied to other single-celled protozoan parasites, such as Trypanosoma cruzi, leishmania, or plasmodium. It can also be easily adapted to other organisms, including yeast or mammalian cells. This method can be coupled to quantitative mass spectrometry, such as SILAC, to identify dynamic interactions of proteins with inositol phosphate or phosphoinostides.
In can also be combined with subcellular fractionation, to identify organelle specific proteins that bind to these metabolites. This approach has helped to identify proteins that bind to inositol phosphate and phosphoinositides in T.brucei, mammalian cells, and yeast. It has helped identify new protein domains that bind to phosphoinositides.
It has paved the way to understand the regulatory function of these metabolites, their role in gene expression, cell development, and signal transduction in eukaryotes. Some of the reagents in this protocol are toxic, or flammable. Use personal protective equipment, and where necessary, work in a fume hood.
