Analysis of Phosphoinositide-Binding Properties and Subcellular Localization of GFP-Fusion Proteins |
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Authors: | Yong-Woo Jun Sangyeol Kim Kun-Hyung Kim Jin-A Lee Chae-Seok Lim Iksoo Chang Byung-Chang Suh Bong-Kiun Kaang Deok-Jin Jang |
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Affiliation: | 1. , Department of Ecological Science, College of Ecology and Environment, Kyungpook National University, Sang‐ju, Korea;2. Center for Proteome Biophysics, Daegu, Korea;3. , Department of Physics, Pusan National University, Pusan, Korea;4. , Department of Biotechnology, College of Life Science and Nano Technology, Hannam University, Daejeon, Korea;5. , Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea;6. , Department of Brain Science, Daegu Gyeongbuk Institute of Science and Technology, Daegu, Korea |
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Abstract: | Specific protein‐phosphoinositide (PI) interactions are known to play a key role in the targeting of proteins to specific cellular membranes. Investigation of these interactions would be greatly facilitated if GFP‐fusion proteins expressed in mammalian cells and used for their subcellular localization could also be employed for in vitro lipid binding. In this study, we found that lysates of cells overexpressing GFP‐fusion proteins could be used for in vitro protein‐PI binding assays. We applied this approach to examine the PI‐binding properties of Aplysia Sec7 protein (ApSec7) and its isoform ApSec7(VPKIS), in which a VPKIS sequence is inserted into the PH domain of ApSec7. EGFP‐ApSec7 but not EGFP‐ApSec7(VPKIS) did specifically bind to PI(3,4,5)P3 in an in vitro lipid‐coated bead assay. Overexpression of EGFP‐ApSec7 but not EGFP‐ApSec7(VPKIS) did induce neurite outgrowth in Aplysia sensory neurons. Structure modeling analysis revealed that the inserted VPKIS caused misfolding around the PI(3,4,5)P3‐binding pocket of ApSec7 and disturbed the binding of PI(3,4,5)P3 to the pleckstrin homology (PH) domain. Our data indicate that plasma membrane localization of EGFP‐ApSec7 via the interaction between its PH domain and PI(3,4,5)P3 might play a key role in neurite outgrowth in Aplysia. |
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Keywords: | GFP‐fusion protein Phosphoinositide PI(3 4 5)P3 Aplysia Sec7 In vitro protein‐phosphoinositide binding Neurite outgrowth |
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