TY - JOUR
T1 - Systematic Analysis of Phosphatidylinositol-5-phosphate-Interacting Proteins Using Yeast Proteome Microarrays
AU - Herianto, Samuel
AU - Rathod, Jagat
AU - Shah, Pramod
AU - Chen, You Zuo
AU - Wu, Wei Sheng
AU - Liang, Biqing
AU - Chen, Chien Sheng
N1 - Funding Information:
This research was supported by Taiwan’s Ministry of Science and Technology (MOST) under grant MOST 108–2320-B-006-038-MY3 to C.S.C., 109–2811-M-006-524 to B.Q., and 108–2628-E-006-004-MY3 to W.S.W. C.S.C. also acknowledges funding support in part from the Center of Allergy and Mucosal Immunity and the Headquarters of University Advancement at National Cheng Kung University, which is sponsored by the Ministry of Education in Taiwan.
Publisher Copyright:
© 2020 American Chemical Society.
PY - 2021/1/19
Y1 - 2021/1/19
N2 - We used yeast proteome microarrays (∼5800 purified proteins) to conduct a high-throughput and systematic screening of PI5P-interacting proteins with PI5P-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 PI5P-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 PI5P-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 PI5P-containing liposomes but not to PI5P-deprived liposomes or PI-containing liposomes. Additionally, the glycine (G6) and lysine (K7) residues of the Walker A motif (-GPAGTG6K7S-) were found to be critical to the PI5P-binding ability. This study not only identified an additional set of PI5P-interacting proteins but also revealed the strong PI5P-binding affinity (Kd = 1.81 × 10-7 M) of the Walker A motif beyond the motif's nucleotide-binding characteristic.
AB - We used yeast proteome microarrays (∼5800 purified proteins) to conduct a high-throughput and systematic screening of PI5P-interacting proteins with PI5P-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 PI5P-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 PI5P-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 PI5P-containing liposomes but not to PI5P-deprived liposomes or PI-containing liposomes. Additionally, the glycine (G6) and lysine (K7) residues of the Walker A motif (-GPAGTG6K7S-) were found to be critical to the PI5P-binding ability. This study not only identified an additional set of PI5P-interacting proteins but also revealed the strong PI5P-binding affinity (Kd = 1.81 × 10-7 M) of the Walker A motif beyond the motif's nucleotide-binding characteristic.
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U2 - 10.1021/acs.analchem.0c03463
DO - 10.1021/acs.analchem.0c03463
M3 - Article
AN - SCOPUS:85098963591
SN - 0003-2700
VL - 93
SP - 868
EP - 877
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 2
ER -