Mapping bilayer thickness in the ER membrane

Abstract

In the plasma membrane and in synthetic membranes, resident lipids may laterally unmix to form domains of distinct biophysical properties. Whether lipids also drive the lateral organization of intracellular membranes is largely unknown. Here, we describe genetically encoded fluorescent reporters visualizing local variations in bilayer thickness. Using them, we demonstrate that long-chained ceramides promote the formation of discrete domains of increased bilayer thickness in the yeast ER, particularly in the future plane of cleavage and at ER–trans-Golgi contact sites. Thickening of the ER membrane in the cleavage plane contributed to the formation of lateral diffusion barriers, which restricted the passage of short, but not long, protein transmembrane domains between the mother and bud ER compartments. Together, our data establish that the ER membrane is laterally organized and that ceramides drive this process, and provide insights into the physical nature and biophysical mechanisms of the lateral diffusion barriers that compartmentalize the ER.

Investigating the structural properties of hydrophobic solvent-rich lipid bilayers

“…To put this difference into some biological context, in the case of hexadecane this corresponds to roughly one entire a-helical turn (3.6 residues, 0.54 nm), a very common secondary structure conformation of transmembrane (TM) proteins. Interestingly, this difference is within the range of the observed difference between Golgi and Plasma Membrane proteins for vertebrates 39 or in the range of what is sufficient to alter the localization of synthetic TM peptides in the yeast ER 40 .…”

The role of sigma-1 receptor in organization of endoplasmic reticulum signaling microdomains

“…The amino acid sequences of TM17 and TM27 were designed based on synthetic peptides previously used in GUV studies of membrane thickness (Kaiser et al, 2011). Similar WALP-GFP fusion proteins were successfully used for measurements of ER lipid heterogeneity in yeast cells (Prasad, Sliwa-Gonzalez, & Barral, 2020). The hydrophobic length of MBP-TM17, calculated using 1.5 Å rise per residue (Hildebrand, Preissner, & Frommel, 2004), matches well with the hydrophobic thickness of a DOPC bilayer (25.5 Å, and 26.8 Å, respectively) (Kucerka et al, 2005).…”

The role of sigma 1 receptor in organization of endoplasmic reticulum signaling microdomains

“…The amino acid sequences of TM17 and TM27 were designed based on the synthetic peptides previously used in GUV studies of membrane thickness ( Kaiser et al, 2011 ). Similar WALP-GFP fusion proteins were successfully used for measurements of the ER lipid heterogeneity in yeast cells ( Prasad et al, 2020 ). The hydrophobic length of MBP-TM17, calculated using 1.5 Å rise per residue ( Hildebrand et al, 2004 ), matches well with the hydrophobic thickness of the DOPC bilayer (25.5 Å and 26.8 Å, respectively) ( Kučerka et al, 2006 ).…”