Manipulating Endoplasmic Reticulum-Plasma Membrane Tethering in Plants Through Fluorescent Protein Complementation
The bimolecular fluorescence complementation (BiFC) assay has been widely used to examine interactions between integral and peripheral proteins within putative plasma membrane (PM) microdomains. In the course of using BiFC assays to examine the co-localization of plasma membrane (PM) targeted recept...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-05-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fpls.2019.00635/full |
| _version_ | 1819071350250143744 |
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| author | Kai Tao Kai Tao Justin R. Waletich Felipe Arredondo Brett M. Tyler Brett M. Tyler Brett M. Tyler |
| author_facet | Kai Tao Kai Tao Justin R. Waletich Felipe Arredondo Brett M. Tyler Brett M. Tyler Brett M. Tyler |
| author_sort | Kai Tao |
| collection | DOAJ |
| description | The bimolecular fluorescence complementation (BiFC) assay has been widely used to examine interactions between integral and peripheral proteins within putative plasma membrane (PM) microdomains. In the course of using BiFC assays to examine the co-localization of plasma membrane (PM) targeted receptor-like kinases (RLKs), such as FLS2, with PM micro-domain proteins such as remorins, we unexpectedly observed heterogeneous distribution patterns of fluorescence on the PM of Nicotiana benthamiana leaf cortical cells. These patterns appeared to co-localize with the endoplasmic reticulum (ER) and with ER-PM contact sites, and closely resembled patterns caused by over-expression of the ER-PM tether protein Synaptotagmin1 (SYT1). Using domain swap experiments with SYT1, we inferred that non-specific dimerization between FLS2-VenusN and VenusC-StRem1.3 could create artificial ER-PM tether proteins analogous to SYT1. The same patterns of ER-PM tethering were produced when a representative set of integral membrane proteins were partnered in BiFC complexes with PM-targeted peripheral membrane proteins, including PtdIns(4)P-binding proteins. We inferred that spontaneous formation of mature fluorescent proteins caused the BiFC complexes to trap the integral membrane proteins in the ER during delivery to the PM, producing a PM-ER tether. This phenomenon could be a useful tool to deliberately manipulate ER-PM tethering or to test protein membrane localization. However, this study also highlights the risk of using the BiFC assay to study membrane protein interactions in plants, due to the possibility of alterations in cellular structures and membrane organization, or misinterpretation of protein-protein interactions. A number of published studies using this approach may therefore need to be revisited. |
| first_indexed | 2024-12-21T17:20:26Z |
| format | Article |
| id | doaj.art-93fbfdb50c9948289bac4b775956e55d |
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| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-21T17:20:26Z |
| publishDate | 2019-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-93fbfdb50c9948289bac4b775956e55d2022-12-21T18:56:10ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2019-05-011010.3389/fpls.2019.00635441756Manipulating Endoplasmic Reticulum-Plasma Membrane Tethering in Plants Through Fluorescent Protein ComplementationKai Tao0Kai Tao1Justin R. Waletich2Felipe Arredondo3Brett M. Tyler4Brett M. Tyler5Brett M. Tyler6Molecular and Cellular Biology Program, Oregon State University, Corvallis, OR, United StatesDepartment of Botany and Plant Pathology, Oregon State University, Corvallis, OR, United StatesDepartment of Botany and Plant Pathology, Oregon State University, Corvallis, OR, United StatesDepartment of Botany and Plant Pathology, Oregon State University, Corvallis, OR, United StatesMolecular and Cellular Biology Program, Oregon State University, Corvallis, OR, United StatesDepartment of Botany and Plant Pathology, Oregon State University, Corvallis, OR, United StatesCenter for Genome Research and Biocomputing, Oregon State University, Corvallis, OR, United StatesThe bimolecular fluorescence complementation (BiFC) assay has been widely used to examine interactions between integral and peripheral proteins within putative plasma membrane (PM) microdomains. In the course of using BiFC assays to examine the co-localization of plasma membrane (PM) targeted receptor-like kinases (RLKs), such as FLS2, with PM micro-domain proteins such as remorins, we unexpectedly observed heterogeneous distribution patterns of fluorescence on the PM of Nicotiana benthamiana leaf cortical cells. These patterns appeared to co-localize with the endoplasmic reticulum (ER) and with ER-PM contact sites, and closely resembled patterns caused by over-expression of the ER-PM tether protein Synaptotagmin1 (SYT1). Using domain swap experiments with SYT1, we inferred that non-specific dimerization between FLS2-VenusN and VenusC-StRem1.3 could create artificial ER-PM tether proteins analogous to SYT1. The same patterns of ER-PM tethering were produced when a representative set of integral membrane proteins were partnered in BiFC complexes with PM-targeted peripheral membrane proteins, including PtdIns(4)P-binding proteins. We inferred that spontaneous formation of mature fluorescent proteins caused the BiFC complexes to trap the integral membrane proteins in the ER during delivery to the PM, producing a PM-ER tether. This phenomenon could be a useful tool to deliberately manipulate ER-PM tethering or to test protein membrane localization. However, this study also highlights the risk of using the BiFC assay to study membrane protein interactions in plants, due to the possibility of alterations in cellular structures and membrane organization, or misinterpretation of protein-protein interactions. A number of published studies using this approach may therefore need to be revisited.https://www.frontiersin.org/article/10.3389/fpls.2019.00635/fulltetheringplasma membraneendoplasmic recticulumperipheral membrane proteinintegral membrane protein |
| spellingShingle | Kai Tao Kai Tao Justin R. Waletich Felipe Arredondo Brett M. Tyler Brett M. Tyler Brett M. Tyler Manipulating Endoplasmic Reticulum-Plasma Membrane Tethering in Plants Through Fluorescent Protein Complementation Frontiers in Plant Science tethering plasma membrane endoplasmic recticulum peripheral membrane protein integral membrane protein |
| title | Manipulating Endoplasmic Reticulum-Plasma Membrane Tethering in Plants Through Fluorescent Protein Complementation |
| title_full | Manipulating Endoplasmic Reticulum-Plasma Membrane Tethering in Plants Through Fluorescent Protein Complementation |
| title_fullStr | Manipulating Endoplasmic Reticulum-Plasma Membrane Tethering in Plants Through Fluorescent Protein Complementation |
| title_full_unstemmed | Manipulating Endoplasmic Reticulum-Plasma Membrane Tethering in Plants Through Fluorescent Protein Complementation |
| title_short | Manipulating Endoplasmic Reticulum-Plasma Membrane Tethering in Plants Through Fluorescent Protein Complementation |
| title_sort | manipulating endoplasmic reticulum plasma membrane tethering in plants through fluorescent protein complementation |
| topic | tethering plasma membrane endoplasmic recticulum peripheral membrane protein integral membrane protein |
| url | https://www.frontiersin.org/article/10.3389/fpls.2019.00635/full |
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