Projection structure of P-glycoprotein by electron microscopy. Evidence for a closed conformation of the nucleotide binding domains

The structure of P-glycoprotein (Pgp) from mouse has been studied by electron microscopy and image analysis. Two-dimensional crystals of Pgp in a lipid bilayer were generated by reconstituting pure, detergent-solubilized protein containing a C-terminal six-histidine tag using the lipid monolayer technique. The crystals belong to plane group P1 with a = b = 104 +/- 2 A and gamma = 90 +/- 4 degrees. The projection structure of Pgp calculated at a resolution of 22 A shows two closely interacting protein domains that can be interpreted as the N- and C-terminal halves of the protein. The projection structure of Pgp is consistent with the recently published x-ray structure of MsbA, a lipid A flippase from Escherichia coli with high sequence homology to Pgp but only when the two MsbA subunits are rotated to bring their nucleotide binding domains together.     

Real-time RT-PCR quantitation of mCLCA1 and mCLCA2 reveals differentially regulated expression in pre- and postnatal murine tissues

Members of the recently discovered chloride channels, calcium-activated (CLCA) gene family are thought to contribute to transmembrane trafficking of anions and other cellular functions. Previous northern blot and in situ hybridization studies revealed expression of the murine putative chloride channel mCLCA1 (alias mCaCC) in numerous epithelia and few other cell types. However, the subsequent cloning of mCLCA2 which shares 96% cDNA sequence identity with mCLCA1 suggested that the distribution pattern proposed for mCLCA1 in fact represented the sum of both mRNA species. In this study, a real-time RT-quantitative PCR assay was established to specifically quantify mCLCA1 and mCLCA2 expression in 19 pre- and 44 postnatal murine tissues. Different expression levels of mCLCA1 and mCLCA2 were found in most tissues analyzed. Particularly strong and virtually exclusive expression was found for mCLCA1 in spleen and bone marrow and for mCLCA2 in lactating and involuting mammary glands. In contrast, other tissues including intestine and trachea were found to express equally moderate levels of both homologues. Moreover, mCLCA2, but not mCLCA1, seems to be involved in stage-specific organogenesis in fetal tissues. These results indicate that, in spite of their extremely close sequence homology, mCLCA1 and mCLCA2 are involved in different, yet unidentified pathways.     

Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas

Pseudomonas aeruginosa is the agent of opportunistic infections in immunocompromised individuals and chronic respiratory illnesses in cystic fibrosis patients. Pseudomonas aeruginosa utilizes a type III secretion system for injection of toxins into the host cell cytoplasm through a channel on the target membrane (the 'translocon'). Here, we have functionally and structurally characterized PopB and PopD, membrane proteins implicated in the formation of the P.aeruginosa translocon. PopB and PopD form soluble complexes with their common chaperone, PcrH, either as stable heterodimers or as metastable heterooligomers. Only oligomeric forms are able to bind to and disrupt cholesterol-rich membranes, which occurs within a pH range of 5-7 in the case of PopB/PcrH, and only at acidic pH for PcrH-free PopD. Electron microscopy reveals that upon membrane association PopB and PopD form 80 A wide rings which encircle 40 A wide cavities. Thus, formation of metastable oligomers precedes membrane association and ring generation in the formation of the Pseudomonas translocon, a mechanism which may be similar for other pathogens that employ type III secretion systems.     

ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resistant cancer cells

In this study we show that P-glycoprotein in multidrug-resistant 2780AD human ovarian carcinoma cells and multidrug resistance-associated protein 1 in multidrug-resistant HT29col human colon carcinoma cells are predominantly located in Lubrol-based detergent-insoluble glycosphingolipid-enriched membrane domains. This localization is independent of caveolae, since 2780AD cells do not express caveolin-1. Although HT29col cells do express caveolin-1, the ATP-binding cassette transporter and caveolin-1 were dissociated on the basis of differential solubility in Triton X-100 and absence of microscopical colocalization. While both the multidrug resistance-associated protein 1 and caveolin-1 are located in Lubrol-based membrane domains, they occupy different regions of these domains.     

HtrA1 serine protease inhibits signaling mediated by Tgfbeta family proteins

HtrA1, a member of the mammalian HtrA serine protease family, has a highly conserved protease domain followed by a PDZ domain. Because HtrA1 is a secretory protein and has another functional domain with homology to follistatin, we examined whether HtrA1 functions as an antagonist of Tgfbeta family proteins. During embryo development, mouse HtrA1 was expressed in specific areas where signaling by Tgfbeta family proteins plays important regulatory roles. The GST-pulldown assay showed that HtrA1 binds to a broad range of Tgfbeta family proteins, including Bmp4, Gdf5, Tgfbetas and activin. HtrA1 inhibited signaling by Bmp4, Bmp2, and Tgfbeta1 in C2C12 cells, presumably by preventing receptor activation. Experiments using a series of deletion mutants indicated that the binding activity of HtrA1 required the protease domain and a small linker region preceding it, and that inhibition of Tgfbeta signaling is dependent on the proteolytic activity of HtrA1. Misexpression of HtrA1 near the developing chick eye led to suppression of eye development that was indistinguishable from the effects of noggin. Taken together, these data indicate that HtrA1 protease is a novel inhibitor of Tgfbeta family members.     

The TNL gene Rdr1 confers broad‐spectrum resistance to Diplocarpon rosae

Black spot disease, which is caused by the ascomycete Diplocarpon rosae, is the most severe disease in field‐grown roses in temperate regions and has been distributed worldwide, probably together with commercial cultivars. Here, we present data indicating that muRdr1A is the active Rdr1 gene, a single‐dominant TIR‐NBS‐LRR (Toll/interleukin‐1 receptor‐nucleotide binding site‐leucine rich repeat) (TNL)‐type resistance gene against black spot disease, which acts against a broad range of pathogenic isolates independent of the genetic background of the host genotype. Molecular analyses revealed that, compared with the original donor genotype, the multiple integrations that are found in the primary transgenic clone segregate into different integration patterns in its sexual progeny and do not show any sign of overexpression. Rdr1 provides resistance to 13 different single‐spore isolates belonging to six different races and broad field mixtures of conidia; thus far, Rdr1 is only overcome by two races. The expression of muRdr1A, the active Rdr1 gene, leads to interaction patterns that are identical in the transgenic clones and the non‐transgenic original donor genotype. This finding indicates that the interacting avirulence (Avr) factor on the pathogen side must be widespread among the pathogen populations and may have a central function in the rose–black spot interaction. Therefore, the Rdr1 gene, pyramided with only a few other R genes by sexual crosses, might be useful for breeding roses that are resistant to black spot because the spread of new pathogenic races of the fungus appears to be slow.