Characterization of micron-scale protein-depleted plasma membrane domains in phosphatidylserine-deficient yeast cells

ABSTRACT Membrane phase separation to form micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large-scale phase separation has not been reported in the plasma membrane of the living cells. We show here that a stable micron-scale protein-depleted region is generated in the plasma membrane of yeast mutants lacking phosphatidylserine at high temperatures. We named this region the ‘void zone’. Transmembrane proteins and certain peripheral membrane proteins and phospholipids are excluded from the void zone. The void zone is rich in ergosterol, and requires ergosterol and sphingolipids for its formation. Such properties are also found in the cholesterol-enriched domains of phase-separated artificial membranes, but the void zone is a novel membrane domain that requires energy and various cellular functions for its formation. The formation of the void zone indicates that the plasma membrane in living cells has the potential to undergo phase separation with certain lipid compositions. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.

New Applications for the Kinetic Exclusion Assay (KinExA)

This dissertation examines the fundamental principles and applicability of the kinetic exclusion assay (KinExA), developed and marketed by Sapidyne Instruments of Boise, Idaho, since 1995. Chapter One reviews and consolidates the manufacturer’s guidance and many early papers that delineate the practical and theoretical aspects of the technology. In brief, KinExA is a two stage analytical system. In stage one, a number of solutions are prepared, whereby one of the partners is kept constant (the constant binding partner, or CBP), while the other (the titrant) is varied, usually in serial dilution. As the titrant is increased, the free CBP decreases, and is analyzed by a sophisticated and precise microfluidic fluorometric device (stage two). The resulting signal can be related mathematically to the affinity (KD) of the two molecules for each other, and to the kinetic parameters of binding (kon) and dissociation (koff). A comparison of KinExA with other current technology available for quantification of interactions is provided. In Chapter Two, I investigate the use of KinExA technology with DNA aptamers. DNA aptamers are short nucleotide oligomers selected to bind a target ligand with affinity and specificity rivaling that of antibodies. These remarkable features make them promising alternatives for analytical and therapeutic applications that traditionally use antibodies as biorecognition elements. Numerous traditional and emerging analytical techniques have been proposed and successfully implemented to utilize aptamers for sensing purposes. In this work, I exploited the analytical capabilities offered by the KinExA technology to measure the affinity of fluorescent aptamers for their target molecule thrombin, and quantify the concentration of analyte in solution. Standard binding curves constructed by using equilibrated mixtures of aptamers titrated with thrombin were fitted with a 1:1 binding model and provided an effective KD of the binding in the sub-nanomolar range. However, the experimental results suggest that this simple model does not satisfactorily describe the binding process; therefore, the possibility that the aptamer is composed of a mixture of two or more distinct KD populations is discussed. The same standard curves, together with a four-parameter logistic equation, were used to determine “unknown” concentrations of thrombin in mock samples. The ability to identify and characterize complex binding stoichiometry, together with the determination of target analyte concentrations in the pM–nM range, supports the adoption of this technology for kinetics, equilibrium, and analytical purposes by employing aptamers as biorecognition elements. In Chapter Three, I explore complex capture agents in the KinExA system. Liposomes made from purified reagents, or from natural cellular membranes, are attached to the beads used in the KinExA process to capture the analyte. Model molecules representing lipophilic dyes, antibodies, and bacterial toxins were successfully captured by the beads for measurement. Residual free ligand captured after pre-equilibration with membrane components, presented as either liposomes or whole cells, could be quantified, and kinetic parameters determined. By this process the “bi-molecular” interaction of the B subunit of cholera toxin for the ganglioside GM1 incorporated into artificial membranes could be quantified, and shown to be dependent upon the presence of the ganglioside in the membrane. The diffusion into artificial membranes of the lipophilic dye DID could be quantified and shown to be dependent upon the amount of lipid available in the equilibration step. In addition, the bulk affinity of a commercial polyclonal antibody for the surface antigens of their target red blood cells could be evaluated. This membrane immobilization process appears to be generally applicable to any membrane system. Thus, it promises to be valuable for the study of signaling molecules for which purified soluble target cellular components may result in misleading information, or for which soluble fragments are unavailable. Likewise, this process should aid in the search for drugs which mimic or antagonize these signaling ligands.

Soft materials as biological and artificial membranes

This review focuses on soft materials involved in biological and artificial membranes. The illustration is a conceptual scheme of artificial membranes synthesized by human-made hydrophilic and hydrophobic soft materials.

Phosphatidylserine prevents the generation of a protein-free giant plasma membrane domain in yeast

Membrane phase separation accompanied with micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large phase separation has not been reported in the plasma membrane of the living cells. We demonstrate here that a stable micron-scale protein-free region is generated in the plasma membrane of the yeast mutants lacking phosphatidylserine. We named this region the “void zone”. Transmembrane proteins, peripheral membrane proteins, and certain phospholipids are excluded from the void zone. The void zone is rich in ergosterol and requires ergosterol and sphingolipids for its formation. These characteristics of the void zone are similar to the properties of the cholesterol-enriched domain in phase-separated artificial membranes. We propose that phosphatidylserine prevents the formation of the void zone by preferentially interacting with ergosterol. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.Summary statementYeast cells lacking phosphatidylserine generate protein-free plasma membrane domains, and vacuoles contact with this domain. This is the first report of micron-scale plasma membrane domains in living cells.