Crystalline Patches Research Articles

Enlarged Crystalline Patches on the Plasma Membrane of Yeast Protoplasts

The crystalline patches of intramembraneous particles that form in the yeast plasma membrane, under stationary state physiological conditions, represent a potentially interesting specimen for high resolution electron microscopy. Isolation of these crystalline membrane patches first requires removal of the cell wall and the formation of osmotically fragile yeast protoplasts. In developing a procedure for the isolation of these crystalline membrane patches, we have found that the intramembraneous particles form much larger crystalline patches in protoplasts than in intact yeast cells. We have performed deep etch experiments and have found that the crystalline array of particles is not expressed on the extracellular surface of the plasma membrane.

Binding of polylysine to charged bilayer membranes. Molecular organization of a lipid · peptide complex

The interaction between a positively charged peptide (poly- l-lysine) and model membranes containing charged lipids has been investigated. Conformational changes of the polypeptide as well as changes in the membrane lipid distribution were observed upon lipid-protein agglutination: 1. 1. The strong binding of polylysine is shown directly by the use of spin-labelled polypeptide. Upon binding to phosphatidic acid a shift in the hyperfine coupling constant from 16.5 to 14.6 Oe is observed. The spectrum of the lipid-bound peptide is superimposed on the spectrum of polylysine in solution. Half of the lysine groups are bound to the charged membranes. A change in the conformation of polylysine from a random coil to a partially ordered configuration is suggested. 2. 2. Spin labelling of the lipid component gives evidence concerning the molecular organization of a lipid mixture containing charged phosphatidic acid. Addition of polylysine induces the formation of crystalline patches of bound phosphatidic acid. 3. 3. Excimer forming pyrene decanoic acid has been employed. Addition of positively charged polylysine (pH 9.0) to phosphatidic acid membranes increases the transition temperature of the lipid from T t = 50 to T t = 62° C . Thus, a lipid segregation of lipid into regions of phosphatidic acid bound to the peptide which differ in their microviscosity from the surrounding membrane is induced. One lysine group binds one phosphatidic acid molecule, but only half of the phosphatidic acid is bound. 4. 4. Direct evidence for charge-induced domain formation in lipid mixtures containing phosphatidic acid is given by electron microscopy. Addition of polylysine leads to a change in the surface curvature of the bound charged lipid. The domain size is estimated from the electron micrographs. The number of domains present is dependent on both the ratio of charged to uncharged lipids as well as on the amount of polylysine added to the vesicles. The size of the domains is not dependent on membrane composition. However, the size seems to increase in a stepwise manner that is correlated with a multiple of the area covered by one polylysine molecule.

A spin label study of the interaction of acth with Y-1 adrenal cell membranes

By the use of hydrophilic and lipophilic spin labels the effects of α 1–24-ACTH binding to its receptor upon both protein conformation and lipid bulk fluidity and distribution were studied. The results are consistent with a model in which conformational changes induced by hormone binding cause increased lateral mobility of the resulting hormone-receptor complexes. By aggregation of several of these hormone-receptor complexes, crystalline patches of phospholipid on the outer monolayer of the plasma membrane are formed. It is suggested that the formation of these crystalline patches acts as the transducer to activate the adenyl cyclase system which is bound to the inner surface of the plasma membrane. The model predicts the presence of a temperature-dependent time-lag for the stimulation of adenyl cyclase activity.

Chemically induced lipid phase separation in model membranes containing charged lipids: A spin label study

The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca 2+ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, W ex. By measuring W ex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithinphosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers. In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8·10 −8 cm 2/s at 59°C. Addition of Ca 2+ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca 2+-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of W ex on the Ca 2+ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca 2+ concentration clusters containing about 30 mol% lecithin are formed. At high lecithin or Ca 2+ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn 2+ using ESR spectroscopy. Polylysine leads to the same strong increase in the lecithin segregation as Ca 2+. The transition of the phosphatidic acid bound by the polypeptide is shifted from T t = 47.5° to T t = 62°C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.