Systematic Analysis Of Phosphatidylinositol-5-Phosphate-Interacting Proteins Using Yeast Proteome Microarrays

We used yeast proteome microarrays (similar to 5800 purified proteins) to conduct a high-throughput and systematic screening of PISP-interacting proteins with PISP-tagged fluorescent liposomal nanovesicles. Lissamine rhodamine B-dipalmitoyl phosphatidylethanol was incorporated into the liposome bilayer to provide the nanovesicles with fluorescence without any encapsulants, which not only made the liposome fabrication much easier without the need for purification but also improved the chip-probing quality. A special chip assay was washed very gently without the traditional spin-dry step. Forty-five PISP-interacting proteins were identified in triplicate with this special chip assay. Subsequently, we used flow cytometry to validate these interactions, and a total of 41 PISP-interacting proteins were confirmed. Enrichment analysis revealed that these proteins have significant functions associated with ribosome biogenesis, rRNA processing, ribosome binding, GTP binding, and hydrolase activity. Their component enrichment is located in the nucleolus. The InterPro domain analysis indicated that PI5P-interacting proteins are enriched in the P-loop containing nucleoside triphosphate hydrolases domain (P-loop). Additionally, using the MEME program, we identified a consensus motif (IVGPAGTGKSTLF) that contains the Walker A sequence, a well-known nucleotide-binding motif. Furthermore, using a quartz crystal microbalance, both the consensus motif and Walker A motif showed strong affinities to PISP-containing liposomes but not to PISP-deprived liposomes or PI-containing liposomes. Additionally, the glycine (G6) and lysine (K7) residues of the Walker A motif (-GPAGTG(6)K(7)S-) were found to be critical to the PISP-binding ability. This study not only identified an additional set of PISP-interacting proteins but also revealed the strong PI5P-binding affinity (K-d = 1.81 X 10(-7) M) of the Walker A motif beyond the motif's nucleotide-binding characteristic.