Abstract
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.
Highlights
A variety of internal organelles and structures are closely associated with the plasma membrane (PM), especially in plant cells where the large central vacuole compresses the cytoplasm into a thin layer against the PM
When StRem1.3 was replaced by a mutant, StRem1.3∗ that could not bind the PM, the bimolecular fluorescence complementation (BiFC) complexes displayed the localization expected for FLS2 alone (Supplementary Figures S1B,C)
Though the exclusion of membrane proteins by endoplasmic reticulum (ER)-PM junctions has not been reported on plants, when we examined the distribution of membrane-associated protein AtFlotillin1 (Supplementary Figure S3E), reduced distribution in regions of the membrane displaying either SYT1-YFP or especially the FLS2-StRem1.3 complexes was apparent, StRem1.3 and Other Peripheral Membrane Proteins Can Replace the C-Terminus of SYT1 in ER-PM Tethering
Summary
The bimolecular fluorescence complementation (BiFC) assay has been widely used to examine interactions between integral and peripheral proteins within putative plasma membrane (PM) microdomains. Using domain swap experiments involving the endoplasmic reticulum-PM tether protein SYT1, we have obtained evidence that BiFC complexes involving one integral membrane protein and one peripheral membrane protein can act as synthetic EM-PM tethers, producing images that resemble microdomain co-localization, but are artifacts; a number of published studies should be revisited
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