In Vitro Reconstitution of the Endoplasmic Reticulum.

Reconstitution of cellular organelles in vitro offers the possibility to perform quantitative and qualitative experiments in a controlled environment that cannot be done with the same accuracy in living cells. Following a previous report, the subsequent list of protocols describes how to reconstitute and quantify a tubular ER network in vitro based on purified microsomes from culture cells and cytosol from Xenopus laevis egg extracts. Biological material preparation and reconstitution assays require mostly basic laboratory instrumentation and chemicals, and can be executed without any specific training, making them appealing to a wide range of laboratories. Moreover, to promote conditions that are markedly more reflective of in vivo environments, this method describes for the first time in the literature, the purification of microsomes from HeLa cells in some detail. Basic Protocol 1 in this article describes the reconstitution process on different substrates including the associated fluorescence imaging process. Purification of ER microsomes and cytosol, both of which are needed for this approach, are described in detail in Support Protocols 1 and 2, respectively. Coating of surfaces with polyacrylamide gels is described in Support Protocol 3. Basic Protocol 2 outlines how to segment and skeletonize fluorescence images of ER networks, and how to quantify segment lengths between the network's branching points. The described quantitative evaluation provides a meaningful approach to analyze the topology and geometry of organelle structures. © 2017 by John Wiley & Sons, Inc.

Oscillating Electric Field Measures the Rotation Rate in a Native Rotary Enzyme.

Rotary enzymes are complex, highly challenging biomolecular machines whose biochemical working mechanism involves intersubunit rotation. The true intrinsic rate of rotation of any rotary enzyme is not known in a native, unmodified state. Here we use the effect of an oscillating electric (AC) field on the biochemical activity of a rotary enzyme, the vacuolar proton-ATPase (V-ATPase), to directly measure its mean rate of rotation in its native membrane environment, without any genetic, chemical or mechanical modification of the enzyme, for the first time. The results suggest that a transmembrane AC field is able to synchronise the steps of ion-pumping in individual enzymes via a hold-and-release mechanism, which opens up the possibility of biotechnological exploitation. Our approach is likely to work for other transmembrane ion-transporting assemblies, not only rotary enzymes, to determine intrinsic in situ rates of ion pumping.

Shaping the endoplasmic reticulum in vitro.

Organelles in eukaryotic cells often have complex shapes that deviate significantly from simple spheres. A prime example is the endoplasmic reticulum (ER) that forms an extensive network of membrane tubules in many mammalian cell types and in reconstitution assays in vitro. Despite the successful hunt for molecular determinants of ER shape we are still far from having a comprehensive understanding of ER network morphogenesis. Here, we have studied the hitherto neglected influence of the host substrate when reconstituting ER networks in vitro as compared to ER networks in vivo. In culture cells we observed cytoplasm-spanning ER networks with tubules being connected almost exclusively by three-way junctions and segment lengths being narrowly distributed around a mean length of about 1µm. In contrast, networks reconstituted from purified ER microsomes on flat glass or gel substrates of varying stiffness showed significantly broader length distributions with an up to fourfold larger mean length. Self-assembly of ER microsomes on small oil droplets, however, yielded networks that resembled more closely the native ER network of mammalian cells. We conclude from these observations that the ER microsomes' inherent self-assembly capacity is sufficient to support network formation with a native geometry if the influence of the host substrate's surface chemistry becomes negligible. We hypothesize that under these conditions the networks' preference for three-way junctions follows from creating 'starfish-shaped' vesicles when ER microsomes with a protein-induced spontaneous curvature undergo fusion.

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes.

The rate of rotation of the rotor in the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or steady parts of the enzyme, is estimated in native vacuolar membrane vesicles from Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are formed spontaneously after exposing purified yeast vacuoles to osmotic shock. The fraction of total ATPase activity originating from the V-ATPase is determined by using the potent and specific inhibitor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during ten min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit of the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data are incompatible with models assuming multiple binding sites), to the inhibitor titration curve determines the concentration of the enzyme. Combining this with the known ATP/rotation stoichiometry of the V-ATPase and the assayed concentration of inorganic phosphate liberated by the V-ATPase, leads to an average rate of ~10 Hz for full 360° rotation (and a range of 6-32 Hz, considering the ± standard deviation of the enzyme concentration), which, from the time-dependence of the activity, extrapolates to ~14 Hz (8-48 Hz) at the beginning of the reaction. These are lower-limit estimates. To our knowledge, this is the first report of the rotation rate in a V-ATPase that is not subjected to genetic or chemical modification and is not fixed to a solid support; instead it is functioning in its native membrane environment.

Stabilization of Na,K-ATPase by ionic interactions.

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 degrees C, as does the kidney enzyme at 42 degrees C (but not at 20 degrees C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (Tm approximately 45 degrees C) than does the kidney enzyme (Tm approximately 55 degrees C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 degrees C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.