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.
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...
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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