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Novel GFP-fused protein probes for detecting phosphatidylinositol-4-phosphate in the plasma membrane
Phosphatidylinositol-4-phosphate (PI4P) plays a crucial role in cellular functions, including protein trafficking, and is mainly located in the cytoplasmic surface of intracellular membranes, which include the trans-Golgi network (TGN) and the plasma membrane. However, many PI4P-binding domains of m...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Taylor & Francis
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567041/ https://www.ncbi.nlm.nih.gov/pubmed/31231579 http://dx.doi.org/10.1080/19768354.2019.1599424 |
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author | Jun, Yong-Woo Lee, Jin-A Jang, Deok-Jin |
author_facet | Jun, Yong-Woo Lee, Jin-A Jang, Deok-Jin |
author_sort | Jun, Yong-Woo |
collection | PubMed |
description | Phosphatidylinositol-4-phosphate (PI4P) plays a crucial role in cellular functions, including protein trafficking, and is mainly located in the cytoplasmic surface of intracellular membranes, which include the trans-Golgi network (TGN) and the plasma membrane. However, many PI4P-binding domains of membrane-associated proteins are localized only to the TGN because of the requirement of a second binding protein such as ADP-ribosylation factor 1 (ARF1) in order to be stably localized to the specific membrane. In this study, we developed new probes that were capable of detecting PI4P at the plasma membrane using the known TGN-targeting PI4P-binding domains. The PI4P-specific binding pleckstrin homology (PH) domain of various proteins including CERT, OSBP, OSH1, and FAPP1 was combined with the N-terminal moderately hydrophobic domain of the short-form of Aplysia phosphodiesterase 4 (S(N30)), which aids in plasma membrane association but cannot alone facilitate this association. As a result, we found that the addition of S(N30) to the N-terminus of the GFP-fused PH domain of OSBP (S(N30)-GFP-OSBP-PH), OSH1 (S(N30)-GFP-OSH1-PH), or FAPP1 (S(N30)-GFP-FAPP1-PH) could induce plasma membrane localization, as well as retain TGN localization. The plasma membrane localization of S(N30)-GFP-FAPP1-PH is mediated by PI4P binding only, whereas those of S(N30)-GFP-OSBP-PH and S(N30)-GFP-OSH1-PH are mediated by either PI4P or PI(4,5)P(2) binding. Taken together, we developed new probes that detect PI4P at the plasma membrane using a combination of a moderately hydrophobic domain with the known TGN-targeting PI4P-specific binding PH domain. |
format | Online Article Text |
id | pubmed-6567041 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Taylor & Francis |
record_format | MEDLINE/PubMed |
spelling | pubmed-65670412019-06-21 Novel GFP-fused protein probes for detecting phosphatidylinositol-4-phosphate in the plasma membrane Jun, Yong-Woo Lee, Jin-A Jang, Deok-Jin Anim Cells Syst (Seoul) Molecular & Cellular Biology Phosphatidylinositol-4-phosphate (PI4P) plays a crucial role in cellular functions, including protein trafficking, and is mainly located in the cytoplasmic surface of intracellular membranes, which include the trans-Golgi network (TGN) and the plasma membrane. However, many PI4P-binding domains of membrane-associated proteins are localized only to the TGN because of the requirement of a second binding protein such as ADP-ribosylation factor 1 (ARF1) in order to be stably localized to the specific membrane. In this study, we developed new probes that were capable of detecting PI4P at the plasma membrane using the known TGN-targeting PI4P-binding domains. The PI4P-specific binding pleckstrin homology (PH) domain of various proteins including CERT, OSBP, OSH1, and FAPP1 was combined with the N-terminal moderately hydrophobic domain of the short-form of Aplysia phosphodiesterase 4 (S(N30)), which aids in plasma membrane association but cannot alone facilitate this association. As a result, we found that the addition of S(N30) to the N-terminus of the GFP-fused PH domain of OSBP (S(N30)-GFP-OSBP-PH), OSH1 (S(N30)-GFP-OSH1-PH), or FAPP1 (S(N30)-GFP-FAPP1-PH) could induce plasma membrane localization, as well as retain TGN localization. The plasma membrane localization of S(N30)-GFP-FAPP1-PH is mediated by PI4P binding only, whereas those of S(N30)-GFP-OSBP-PH and S(N30)-GFP-OSH1-PH are mediated by either PI4P or PI(4,5)P(2) binding. Taken together, we developed new probes that detect PI4P at the plasma membrane using a combination of a moderately hydrophobic domain with the known TGN-targeting PI4P-specific binding PH domain. Taylor & Francis 2019-04-11 /pmc/articles/PMC6567041/ /pubmed/31231579 http://dx.doi.org/10.1080/19768354.2019.1599424 Text en © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Molecular & Cellular Biology Jun, Yong-Woo Lee, Jin-A Jang, Deok-Jin Novel GFP-fused protein probes for detecting phosphatidylinositol-4-phosphate in the plasma membrane |
title | Novel GFP-fused protein probes for detecting phosphatidylinositol-4-phosphate in the plasma membrane |
title_full | Novel GFP-fused protein probes for detecting phosphatidylinositol-4-phosphate in the plasma membrane |
title_fullStr | Novel GFP-fused protein probes for detecting phosphatidylinositol-4-phosphate in the plasma membrane |
title_full_unstemmed | Novel GFP-fused protein probes for detecting phosphatidylinositol-4-phosphate in the plasma membrane |
title_short | Novel GFP-fused protein probes for detecting phosphatidylinositol-4-phosphate in the plasma membrane |
title_sort | novel gfp-fused protein probes for detecting phosphatidylinositol-4-phosphate in the plasma membrane |
topic | Molecular & Cellular Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567041/ https://www.ncbi.nlm.nih.gov/pubmed/31231579 http://dx.doi.org/10.1080/19768354.2019.1599424 |
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