A C-terminal amphipathic helix is necessary for the in vivo tubule-shaping function of a plant reticulon.
Reticulons (RTNs) are a class of endoplasmic reticulum (ER) membrane proteins that are capable of maintaining high membrane curvature, thus helping shape the ER membrane into tubules. The mechanism of action of RTNs is hypothesized to be a combination of wedging, resulting from the transmembrane topology of their conserved reticulon homology domain, and scaffolding, arising from the ability of RTNs to form low-mobility homo-oligomers within the membrane. We studied the plant RTN isoform RTN13, which has previously been shown to locate to ER tubules and the edges of ER cisternae and to induce constrictions in ER tubules when overexpressed, and identified a region in the C terminus containing a putative amphipathic helix (APH). Here we show that deletion of this region or disruption of the hydrophobic face of the predicted helix abolishes the ability of RTN13 to induce constrictions of ER tubules in vivo. These mutants, however, still retain their ability to interact and form low-mobility oligomers in the ER membrane. Hence, our evidence indicates that the conserved APH is a key structural feature for RTN13 function in vivo, and we propose that RTN, like other membrane morphogens, rely on APHs for their function.
School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom;
Emily
E
Breeze
School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom;
Natasha
N
Dzimitrowicz
Department of Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom;
Verena
V
Kriechbaumer
Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom;
Rhiannon
R
Brooks
Central Laser Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot OX11 0QX, United Kingdom;
Stanley W
SW
Botchway
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;
Jacob P
JP
Brady
Department of Biological and Medical Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom;
Chris
C
Hawes
Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom;
Ann M
AM
Dixon
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;
Jason R
JR
Schnell
Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom.
Mark D
MD
Fricker
School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom; l.frigerio@warwick.ac.uk.
Lorenzo
L
Frigerio
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism*; D017124:Conserved Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D057927:Hydrophobic and Hydrophilic Interactions; D007425:Intracellular Membranes / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D019441:Plant Epidermis / Q000166:cytology; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014026:Nicotiana / Q000166:cytology
D029681:Arabidopsis Proteins
amphipathic helix; endoplasmic reticulum; membrane curvature; plant; reticulon