No abstract

Multidrug transporters provide a survival strategy for living organisms. As expected given their central role in survival, these transporters are ubiquitous, and in many genomes, several genes coding for putative transporters have been identified. However, in an organism such as Escherichia coli mutations in genes coding for transporters other than the major AcrAB-TolC multidrug efflux transporter have only a marginal effect on phenotype. Thus, whether the physiological role of the transporters identified is indeed drug export has been questioned. We show here that the minor effect of single mutations is due to the overlapping functionality of several transporters. This was revealed by generating multiple chromosomal deletion mutations in genes coding for transporters that share the same substrate and testing their effect on the resistance phenotype. In addition, complementation studies imply that AcrAB-TolC confers robust resistance provided that single-component transporters in the plasma membrane are functional. This finding supports the contention that hydrophobic drugs are removed in a 2-stage process: AcrAB-TolC removes substrates from the periplasmic space, while single-component transporters remove them from the cell. The overlapping specificities of the transporters ensure coverage of a wide range of xenobiotics and provide robustness in the response to environmental stress. This strategy also confers evolvability to the organism by reducing constraints on change and allowing the accumulation of nonlethal variation.AcrAB ͉ drug resistance ͉ EmrE ͉ MdfA ͉ multidrug transporters

We introduce a novel optical flow estimation process based on a spatio-temporal model with varying coefficients multiplying a set of basis functions at each pixel. Previous optical flow estimation methodologies did not use such an over parameterized representation of the flow field as the problem is ill-posed even without introducing any additional parameters: Neighborhood based methods like LucasKanade determine the flow in each pixel by constraining the flow to to be constant in a small area. Modern variational methods represent the optic flow directly via its x and y components at each pixel. The benefit of over-parametrization becomes evident in the smoothness term, which instead of directly penalizing for changes in the optic flow, integrates a cost on the deviation from the assumed optic flow model. Previous variational optical flow techniques are special cases of the proposed method, used in conjunction with a constant flow basis function. Experimental results with the novel flow estimation process yielded significant improvements with respect to the best results published so far.

In all kingdoms of life, ATP binding cassette (ABC) transporters are essential to many cellular functions. In this large superfamily of proteins, two catalytic sites hydrolyze ATP to power uphill substrate translocation. A central question in the field concerns the relationship between the two ATPase catalytic sites: Are the sites independent of one another? Are both needed for function? Do they function cooperatively? These issues have been resolved for type I ABC transporters but never for a type II ABC transporter. The many mechanistic differences between type I and type II ABC transporters raise the question whether in respect to ATP hydrolysis the two subtypes are similar or different. We have addressed this question by studying the Escherichia coli vitamin B 12 type II ABC transporter BtuCD. We have constructed and purified a series of BtuCD variants where both, one, or none of the ATPase sites were rendered inactive by mutation. We find that, in a membrane environment, the ATPase sites of BtuCD are highly cooperative with a Hill coefficient of 2. We also find that, when one of the ATPase sites is inactive, ATP hydrolysis and vitamin B 12 transport by BtuCD is reduced by 95%. These exact features are also shared by the archetypical type I maltose ABC transporter. Remarkably, mutants that have lost 95% of their ATPase and transport capabilities still retain the ability to fully use vitamin B 12 in vivo. The results demonstrate that, despite the many differences between type I and type II ABC transporters, the fundamental mechanism of ATP hydrolysis remains conserved.cooperativity | membrane permeation | membrane proteins A TP binding cassette (ABC) transporters comprise one of the largest membrane protein superfamilies of any proteome (1-3). From bacteria to humans, they participate in processes such as cancer and bacterial multidrug resistance, antigen presentation, signal transduction, DNA repair, translation, cell division, homeostasis maintenance, detoxification, nutrient import, and antiviral defense (4-13). Hydrolyzing ATP to drive transport, ABC transporters shuttle cargo molecules to and fro the various cellular compartments, through the impermeable barriers of cell membranes. Notable mammalian examples include the multidrug exporters (4) and the transporter associated with antigen presentation (14). In prokaryotes, ABC transporters often function as importers and depend on a high-affinity substrate binding protein (SBP) that delivers the substrate to the cognate transporter. Structural and functional information derived from studies of prokaryotic systems largely shaped our mechanistic view of this superfamily of proteins (15)(16)(17)(18)(19)(20)(21)(22). An "alternating access" mechanistic model has been formulated over the years, according to which ATP hydrolysis power a sequence of conformational changes that shift the transporter between intracellular and extracellular accessible conformations (23)(24)(25)(26). This model has been most extensively demonstrated for the maltose transporter that i...

An over-parametrization model based total varia-The constant a is the relative weight of the regularization. tion signal and image denoising method is proposed and analyzed For a -0 we obtain the trivial solution of f (x) = fNoiy. in this paper. In cases where some structural information is For a -oc we obtain the solution f"(x) = 0 from the provided on the signals or images of interest, this method may Euler-Lagrange equation, which results in a linear solution lead to substantial improvements in denoising performance. for f(x). However, Equation (1) shows that only the constant