To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents.

SummaryThe giant non-fimbrial adhesin SiiE is essential to establish intimate contact between Salmonella enterica and the apical surface of polarized epithelial cells. SiiE is secreted by a type I secretion system (T1SS) encoded by Salmonella Pathogenicity Island 4 (SPI4). We identified SiiA and SiiB as two regulatory proteins encoded by SPI4. Mutant strains in siiA or siiB still secrete SiiE, but are highly reduced in adhesion to, and invasion of polarized cells. SiiA and SiiB are inner membrane proteins with one and three transmembrane (TM) helices respectively. TM2 and TM3 of SiiB are similar to members of the ExbB/TolQ family, while the TM of SiiA is similar to MotB and a conserved aspartate residue in this TM is essential for SPI4-encoded T1SS function. Co-immunoprecipitation, bacterial two-hybrid and FRET demonstrate homoand heterotypic protein interactions for SiiA and SiiB. SiiB, but not SiiA also interacts with the SPI4-T1SS ATPase SiiF. The integrity of the Walker A box in SiiF was required for SiiB-SiiF interaction and SiiF dimer formation. Based on these data, we describe SiiA and SiiB as new, exclusively virulence-associated members of the Mot/Exb/Tol family of membrane proteins. Both proteins are involved in a novel mechanism of controlling SPI4-T1SS-dependent adhesion, most likely by formation of a proton-conducting channel.

Inside prokaryotic cells, passive translational diffusion typically limits the rates with which cytoplasmic proteins can reach their locations. Diffusion is thus fundamental to most cellular processes, but the understanding of protein mobility in the highly crowded and non-homogeneous environment of a bacterial cell is still limited. Here we investigated the mobility of a large set of proteins in the cytoplasm of Escherichia coli, by employing fluorescence correlation spectroscopy (FCS) combined with simulations and theoretical modeling. We conclude that cytoplasmic protein mobility could be well described by Brownian diffusion in the confined geometry of the bacterial cell and at the high viscosity imposed by macromolecular crowding. We observed similar size dependence of protein diffusion for the majority of tested proteins, whether native or foreign to E. coli. For the faster-diffusing proteins, this size dependence is well consistent with the Stokes-Einstein relation once taking into account the specific dumbbell shape of protein fusions. Pronounced subdiffusion and hindered mobility are only observed for proteins with extensive interactions within the cytoplasm. Finally, while protein diffusion becomes markedly faster in actively growing cells, at high temperature, or upon treatment with rifampicin, and slower at high osmolarity, all of these perturbations affect proteins of different sizes in the same proportions, which could thus be described as changes of a well-defined cytoplasmic viscosity.