Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata, Endoplasmic Reticulum, and the Plasma Membrane.
The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modeling, may play important roles in linking the cortical ER to the plasma membrane.
Plant Cell Biology, Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom (V.K., C.H.);Central Laser Facility, Science and Technology Facilities Council (STFC) Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot OX11 0QX, United Kingdom (S.W.B.);Warwickshire Private Hospital (WPH) Proteomics Facility Research Technology Platform (S.E.S.) and School of Life Sciences (S.E.S., L.F.), University of Warwick, Coventry CV4 7AL, United Kingdom; andInstitute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom (K.K., K.O.) vkriechbaumer@brookes.ac.uk.
Verena
http://orcid.org/0000-0003-3782-5834
V
Kriechbaumer
Plant Cell Biology, Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom (V.K., C.H.);Central Laser Facility, Science and Technology Facilities Council (STFC) Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot OX11 0QX, United Kingdom (S.W.B.);Warwickshire Private Hospital (WPH) Proteomics Facility Research Technology Platform (S.E.S.) and School of Life Sciences (S.E.S., L.F.), University of Warwick, Coventry CV4 7AL, United Kingdom; andInstitute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom (K.K., K.O.).
Stanley W
http://orcid.org/0000-0002-3268-9303
SW
Botchway
Plant Cell Biology, Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom (V.K., C.H.);Central Laser Facility, Science and Technology Facilities Council (STFC) Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot OX11 0QX, United Kingdom (S.W.B.);Warwickshire Private Hospital (WPH) Proteomics Facility Research Technology Platform (S.E.S.) and School of Life Sciences (S.E.S., L.F.), University of Warwick, Coventry CV4 7AL, United Kingdom; andInstitute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom (K.K., K.O.).
Susan E
http://orcid.org/0000-0002-0538-1477
SE
Slade
Plant Cell Biology, Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom (V.K., C.H.);Central Laser Facility, Science and Technology Facilities Council (STFC) Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot OX11 0QX, United Kingdom (S.W.B.);Warwickshire Private Hospital (WPH) Proteomics Facility Research Technology Platform (S.E.S.) and School of Life Sciences (S.E.S., L.F.), University of Warwick, Coventry CV4 7AL, United Kingdom; andInstitute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom (K.K., K.O.).
Kirsten
K
Knox
Plant Cell Biology, Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom (V.K., C.H.);Central Laser Facility, Science and Technology Facilities Council (STFC) Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot OX11 0QX, United Kingdom (S.W.B.);Warwickshire Private Hospital (WPH) Proteomics Facility Research Technology Platform (S.E.S.) and School of Life Sciences (S.E.S., L.F.), University of Warwick, Coventry CV4 7AL, United Kingdom; andInstitute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom (K.K., K.O.).
Lorenzo
http://orcid.org/0000-0003-4100-6022
L
Frigerio
Plant Cell Biology, Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom (V.K., C.H.);Central Laser Facility, Science and Technology Facilities Council (STFC) Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot OX11 0QX, United Kingdom (S.W.B.);Warwickshire Private Hospital (WPH) Proteomics Facility Research Technology Platform (S.E.S.) and School of Life Sciences (S.E.S., L.F.), University of Warwick, Coventry CV4 7AL, United Kingdom; andInstitute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom (K.K., K.O.).
Karl
K
Oparka
Plant Cell Biology, Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom (V.K., C.H.);Central Laser Facility, Science and Technology Facilities Council (STFC) Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot OX11 0QX, United Kingdom (S.W.B.);Warwickshire Private Hospital (WPH) Proteomics Facility Research Technology Platform (S.E.S.) and School of Life Sciences (S.E.S., L.F.), University of Warwick, Coventry CV4 7AL, United Kingdom; andInstitute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom (K.K., K.O.).
Chris
http://orcid.org/0000-0003-4856-7690
C
Hawes
D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D031425:Plasmodesmata / Q000378:metabolism* / Q000648:ultrastructure; D025941:Protein Interaction Mapping; D040901:Proteomics; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
D029681:Arabidopsis Proteins; D008565:Membrane Proteins; C000607430:RTNLB3 protein, Arabidopsis; C000607431:reticulon-like protein B6, Arabidopsis; D049452:Green Fluorescent Proteins