Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Jun, Yong-Woo, Lee, Jin-A, Jang, Deok-Jin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2019
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
_version_ 1783426985039495168
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
work_keys_str_mv AT junyongwoo novelgfpfusedproteinprobesfordetectingphosphatidylinositol4phosphateintheplasmamembrane
AT leejina novelgfpfusedproteinprobesfordetectingphosphatidylinositol4phosphateintheplasmamembrane
AT jangdeokjin novelgfpfusedproteinprobesfordetectingphosphatidylinositol4phosphateintheplasmamembrane