SUMMARY: A fundamental feature of cellular plasma membranes (PM) is asymmetric lipid distribution between the bilayer leaflets. However, neither the detailed, comprehensive compositions of individual PM leaflets, nor how these contribute to structural membrane asymmetries have been defined. We report the distinct lipidomes and biophysical properties of both monolayers in living mammalian PMs. Phospholipid unsaturation is dramatically asymmetric, with the cytoplasmic leaflet being ~2-fold more unsaturated than the exoplasmic. Atomistic simulations and spectroscopy of leaflet-selective fluorescent probes reveal that the outer PM leaflet is more packed and less diffusive than the inner leaflet, with this biophysical asymmetry maintained in the endocytic system. The structural asymmetry of the PM is reflected in asymmetric structures of protein transmembrane domains (TMD). These structural asymmetries are conserved throughout Eukaryota, suggesting fundamental cellular design principles.

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Protozoan microtubules are sensitive to disruption by dinitroanilines, compounds that kill intracellular Toxoplasma gondii parasites without affecting microtubules in vertebrate host cells. We previously isolated a number of resistant Toxoplasma lines that harbor mutations to the ␣1-tubulin gene. Some of the mutations are localized in or near the M and N loops, domains that coordinate lateral interactions between protofilaments. Other resistance mutations map to a computationally identified binding site beneath the N loop. Allelic replacement of wild-type ␣1-tubulin with the individual mutations is sufficient to confer dinitroaniline resistance. Some mutations seem to increase microtubule length, suggesting that they increase subunit affinity. All mutations are associated with replication defects that decrease parasite viability. When parasites bearing the N loop mutation Phe52Tyr are grown without dinitroaniline selection, they spontaneously acquired secondary mutations in the M loop (Ala273Val) or in an ␣-tubulin-specific insert that stabilizes the M loop (Asp367Val). Parasites with the double mutations have both reduced resistance and diminished incidence of replication defects, suggesting that the secondary mutations decrease protofilament affinity to increase parasite fitness. INTRODUCTIONMicrotubules are essential components of diverse structures in eukaryotic cells. These structures include spindles used for chromosome segregation, flagellar/ciliary axonemes used for motility, and cytoplasmic microtubule arrays used as tracks for vesicle transport (Nogales, 2000). Microtubules are built by the polymerization of ␣-␤-tubulin heterodimers, and they typically contain 13 protofilaments, a substructure formed by the longitudinal head-to-tail association of the heterodimers. The lateral association of protofilaments forms a microtubule, which consists of a 24-nm-wide cylinder with a hollow lumen (Downing and Nogales, 1998a,b;Li et al., 2002).Because ␣-and ␤-tubulins are ancestrally related proteins, they contain many domains that have similar structure and function, such as the M and N loops, which coordinate contacts between adjacent protofilaments (Downing, 2000;Lowe et al., 2001;Li et al., 2002). However, ␣-and ␤-tubulins also have important differences. For example, although both ␣-and ␤-tubulins bind to guanosine triphosphate (GTP), only the ␤-tubulin GTP is hydrolyzed (Davis et al., 1994;Sage et al., 1995;Anders and Botstein, 2001;Dougherty et al., 2001). Polymerization-dependent ␤-tubulin GTP hydrolysis is stimulated by a GTPase-activating domain of ␣-tubulin from the adjacent dimer within the protofilament. When GTP is hydrolyzed, dimers undergo a conformation change that promotes microtubule disassembly (Nicholson et al., 1999;Nogales et al., 2003; Nogales and Wang, 2006a,b). Appropriate microtubule disassembly is as essential as assembly to the appropriate function of tubulin. Expression of mutant ␣-tubulin genes that lack a functional GTPase activation domain causes a lethal phenotype in budding yeast...

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Dinitroanilines (oryzalin, trifluralin, ethafluralin) disrupt microtubules in protozoa but not in vertebrate cells, causing selective death of intracellular Toxoplasma gondii parasites without affecting host cells. Parasites containing a1-tubulin point mutations are dinitroaniline resistant but show increased rates of aberrant replication relative to wild-type parasites. T. gondii parasites bearing the F52Y mutation were previously demonstrated to spontaneously acquire two intragenic mutations that decrease both resistance levels and replication defects. Parasites bearing the G142S mutation are largely dependent on oryzalin for viable growth in culture. We isolated 46 T. gondii lines that have suppressed microtubule defects associated with the G142S or the F52Y mutations by acquiring secondary mutations. These compensatory mutations were a1-tubulin pseudorevertants or extragenic suppressors (the majority alter the b1-tubulin gene). Many secondary mutations were located in tubulin domains that suggest that they function by destabilizing microtubules. Most strikingly, we identified seven novel mutations that localize to an eight-amino-acid insert that stabilizes the a1-tubulin M loop, including one (P364R) that acts as a compensatory mutation in both F52Y and G142S lines. These lines have reduced dinitroaniline resistance but most perform better than parental lines in competition assays, indicating that there is a trade-off between resistance and replication fitness.

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Palmitoylation is a widespread, reversible lipid modification that has been implicated in regulating a variety of cellular processes. Approximately one thousand proteins are annotated as being palmitoylated, and for some of these, including several oncogenes of the Ras and Src families, palmitoylation is indispensable for protein function. Despite this wealth of disease-relevant targets, there are currently few effective pharmacological tools to interfere with protein palmitoylation. One reason for this lack of development is the dearth of assays to efficiently screen for small molecular inhibitors of palmitoylation. To address this shortcoming, we have developed a robust, high-throughput compatible, click chemistry-based approach to identify small molecules that interfere with the palmitoylation of Ras, a high value therapeutic target that is mutated in up to a third of human cancers. This assay design shows excellent performance in 384-well format and is sensitive to known, non-specific palmitoylation inhibitors. Further, we demonstrate an ideal counter-screening strategy, which relies on a target peptide from an unrelated protein, the Src-family kinase Fyn. The screening approach described here provides an integrated platform to identify specific modulators of palmitoylated proteins, demonstrated here for Ras and Fyn, but potentially applicable to pharmaceutical targets involved in a variety of human diseases.

A fundamental feature of cellular plasma membranes (PM) is the asymmetric distribution of lipids between the bilayer leaflets. This asymmetry is central to cellular physiology, regulating signaling, apoptosis, coagulation, and cell-cell fusion. While the broad transbilayer distributions of some lipid types are well established, the detailed lipidomic asymmetry of the PM has not been characterized and thus the compositions of the individual leaflets remain poorly understood. Further, how these compositional differences contribute to structural asymmetries between PM leaflets in living cells is not defined. Here, we report the distinct lipidomes and biophysical properties of both monolayers in living mammalian PMs. Using mass spectrometry coupled to enzymatic digestion, we report the detailed compositions of PM leaflets of human erythrocytes. We find a dramatic asymmetry in phospholipid unsaturation, with the cytoplasmic leaflet being ~2-fold more unsaturated than the exoplasmic. Atomistic simulations of lipid mixtures compiled from the lipidomic observations suggested significant asymmetries in lipid order, packing, and dynamics between the two PM leaflets. These were probed directly in the PM of living cells by Fluorescence Lifetime Imaging Microscopy (FLIM) of leaflet-selective, environment-sensitive probes. The outer PM leaflet is highly packed and ordered, resembling a liquid ordered phase, whereas the inner leaflet is significantly more disordered. This biophysical asymmetry is maintained in the endocytic system. These observations reveal the detailed compositional and biophysical asymmetry of mammalian plasma membranes, elucidating fundamental design principles of living membranes. SIGNIFICANCE STATEMENT:The asymmetric distribution of lipids between the two bilayer leaflets is a fundamental feature of mammalian plasma membranes. Here, we quantify the comprehensive lipidomes of the two PM leaflets in living mammalian cells and examine their consequences on leaflet physical properties. Using computer simulations, we predict distinct biophysical features for the two leaflets and confirm these with a variety of spectroscopic and microscopic techniques. We find that the outer leaflet of the membrane contains many more fully saturated lipids, which endows it with higher lipid packing and lower diffusivity.