Abstract
Artificial giant vesicles have proven highly useful as membrane models in a large variety of biophysical and biochemical studies. They feature accessibility for manipulation and detection, but lack the compositional complexity needed to reconstitute complicated cellular processes. For the plasma membrane (PM), this gap was bridged by the establishment of giant PM vesicles (GPMVs). These native membranes have facilitated studies of protein and lipid diffusion, protein interactions, electrophysiology, fluorescence analysis of lateral domain formation and protein and lipid partitioning as well as mechanical membrane properties and remodeling. The endoplasmic reticulum (ER) is key to a plethora of biological processes in any eukaryotic cell. However, its intracellular location and dynamic and intricate tubular morphology makes it experimentally even less accessible than the PM. A model membrane, which will allow the afore-mentioned types of studies on GPMVs to be performed on ER membranes outside the cell, is therefore genuinely needed. Here, we introduce the formation of giant ER vesicles, termed GERVs, as a new tool for biochemistry and biophysics. To obtain GERVs, we have isolated ER membranes from Saccharomyces cerevisiae and fused them by exploiting the atlastin-like fusion protein Sey1p. We demonstrate the production of GERVs and their utility for further studies.
Highlights
Artificial giant vesicles have proven highly useful as membrane models in a large variety of biophysical and biochemical studies
Since the endoplasmic reticulum (ER) has a plethora of functions in the cell, it is highly desirable to devise a method for generating giant vesicles from ER membranes while preserving protein function
GERVs were formed when purified ER membranes were genetically enriched with Sey1p and a further ER-membrane protein (Bet1p-mCherry, Fig. 1d)
Summary
Artificial giant vesicles have proven highly useful as membrane models in a large variety of biophysical and biochemical studies They feature accessibility for manipulation and detection, but lack the compositional complexity needed to reconstitute complicated cellular processes. On the other hand, when membrane-related processes are reconstituted outside the cell using artificial model membranes to facilitate experimental access, massive simplifications ensue and critical choices need to be made. Since the ER has a plethora of functions in the cell, it is highly desirable to devise a method for generating giant vesicles from ER membranes while preserving protein function These GERVs are likely to facilitate and advance studies on co-translational membrane protein insertion, lipid synthesis, protein modifications, ER contact sites, unfolded protein response, ER-associated protein degradation, ion channels and calcium signaling as well as vesicular transport of newly synthesized proteins and lipids
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