Gap junctions have traditionally been characterized as nonspecific pores between cells passing molecules up to 1 kDa in molecular mass. Nonetheless, it has become increasingly evident that different members of the connexin (Cx) family mediate quite distinct physiological processes and are often not interchangeable. Consistent with this observation, differences in permeability to natural metabolites have been reported for different connexins, although the physical basis for selectivity has not been established. Comparative studies of different members of the connexin family have provided evidence for ionic charge selectivity, but surprisingly little is known about how connexin composition affects the size of the pore. We have employed a series of Alexa dyes, which share similar structural characteristics but range in size from molecular weight 350 to 760, to probe the permeabilities and size limits of different connexin channels expressed in Xenopus oocytes. Correlated dye transfer and electrical measurements on each cell pair, in conjunction with a three-dimensional mathematical model of dye diffusion in the oocyte system, allowed us to obtain single channel permeabilities for all three dyes in six homotypic and four heterotypic channels. Cx43 and Cx32 channels passed all three dyes with similar efficiency, whereas Cx26, Cx40, and Cx45 channels showed a significant drop-off in permeability with the largest dye. Cx37 channels only showed significant permeability for the smaller two dyes, but at two- to sixfold lower levels than other connexins tested. In the heterotypic cases studied (Cx26/Cx32 and Cx43/Cx37), permeability characteristics were found to resemble the more restrictive parental homotypic channel. The most surprising finding of the study was that the absolute permeabilities calculated for all gap junctional channels in this study are, with one exception, at least 2 orders of magnitude greater than predicted purely on the basis of hindered pore diffusion. Consequently, affinity between the probes and the pore creating an energetically favorable in-pore environment, which would elevate permeant concentration within the pore and hence the flux, is strongly implicated.

In Gram-negative bacterial pathogens, specialized chaperones bind to secreted effector proteins and maintain them in a partially unfolded form competent for translocation by type III secretion systems/injectisomes. How diverse sets of effector-chaperone complexes are recognized by injectisomes is unclear. Here we describe a new mechanism of effector-chaperone recognition by the Chlamydia injectisome, a unique and ancestral line of these evolutionarily conserved secretion systems. By yeast two-hybrid analysis we identified networks of Chlamydia-specific proteins that interacted with the basal structure of the injectisome, including two hubs of protein-protein interactions that linked known secreted effector proteins to CdsQ, the putative cytoplasmic C-ring component of the secretion apparatus. One of these protein-interaction hubs is defined by Ct260/Mcsc (Multiple cargo secretion chaperone). Mcsc binds to and stabilizes at least two secreted hydrophobic proteins, Cap1 and Ct618, that localize to the membrane of the pathogenic vacuole (“inclusion”). The resulting complexes bind to CdsQ, suggesting that in Chlamydia, the C-ring of the injectisome mediates the recognition of a subset of inclusion membrane proteins in complex with their chaperone. The selective recognition of inclusion membrane proteins by chaperones may provide a mechanism to co-ordinate the translocation of subsets of inclusion membrane proteins at different stages in infection.

SummaryOur understanding of how obligate intracellular pathogens co-opt eukaryotic cellular functions has been limited by their intractability to genetic manipulation and by the abundance of pathogen-specific genes with no known functional homologues. In this report we describe a gene expression system to characterize proteins of unknown function from the obligate intracellular bacterial pathogen Chlamydia trachomatis . We have devised a homologous recombination-based cloning strategy to construct an ordered array of Saccharomyces cerevisiae strains expressing all Chlamydia -specific genes. These strains were screened to identify chlamydial proteins that impaired various yeast cellular functions or that displayed tropism towards eukaryotic organelles. In addition, to identify bacterial factors that are secreted into the host cell, recombinant chlamydial proteins were screened for reactivity towards antisera raised against vacuolar membranes purified from infected mammalian cells. We report the identification of 34 C. trachomatis proteins that impact yeast cellular functions or are tropic for a range of eukaryotic organelles including mitochondria, nucleus and cytoplasmic lipid droplets, and a new family of Chlamydia -specific proteins that are exported from the parasitopherous vacuole. The versatility of molecular manipulations and protein expression in yeast allows for the rapid construction of comprehensive protein expression arrays to explore the function of pathogen-specific gene products from microorganisms that are difficult to genetically manipulate, grow in culture or too dangerous for routine analysis in the laboratory.

In Gram-negative bacterial pathogens, specialized chaperones bind to secreted effector proteins and maintain them in a partially unfolded form competent for translocation by type III secretion systems/injectisomes. How diverse sets of effectorchaperone complexes are recognized by injectisomes is unclear. Here we describe a new mechanism of effector-chaperone recognition by the Chlamydia injectisome, a unique and ancestral line of these evolutionarily conserved secretion systems. By yeast two-hybrid analysis we identified networks of Chlamydia-specific proteins that interacted with the basal structure of the injectisome, including two hubs of protein-protein interactions that linked known secreted effector proteins to CdsQ, the putative cytoplasmic C-ring component of the secretion apparatus. One of these protein-interaction hubs is defined by Ct260/ Mcsc (Multiple cargo secretion chaperone). Mcsc binds to and stabilizes at least two secreted hydrophobic proteins, Cap1 and Ct618, that localize to the membrane of the pathogenic vacuole (''inclusion''). The resulting complexes bind to CdsQ, suggesting that in Chlamydia, the C-ring of the injectisome mediates the recognition of a subset of inclusion membrane proteins in complex with their chaperone. The selective recognition of inclusion membrane proteins by chaperones may provide a mechanism to co-ordinate the translocation of subsets of inclusion membrane proteins at different stages in infection.