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Bacterial meningitis occurs when bloodborne pathogens invade and penetrate the blood-brain barrier (BBB), provoking inflammation and disease. Group B Streptococcus (GBS), the leading cause of neonatal meningitis, can enter human brain microvascular endothelial cells (hBMECs), but the host response to intracellular GBS has not been characterized. Here we sought to determine whether antibacterial autophagy, which involves selective recognition of intracellular organisms and their targeting to autophagosomes for degradation, is activated in BBB endothelium during bacterial infection. GBS infection resulted in increased punctate distribution of GFP-microtubule-associated protein 1 light chain 3 (LC3) and increased levels of endogenous LC3-II and p62 turnover, two hallmark indicators of active autophagic flux. Infection with GBS mutants revealed that bacterial invasion and the GBS pore-forming β-hemolysin/cytolysin (β-h/c) trigger autophagic activation. Cell-free bacterial extracts containing β-h/c activity induced LC3-II conversion, identifying this toxin as a principal provocative factor for autophagy activation. These results were confirmed in vivo using a mouse model of GBS meningitis as infection with WT GBS induced autophagy in brain tissue more frequently than a β-h/c-deficient mutant. Elimination of autophagy using Atg5-deficient fibroblasts or siRNA-mediated impairment of autophagy in hBMECs led to increased recovery of intracellular GBS. However, electron microscopy revealed that GBS was rarely found within double membrane autophagic structures even though we observed GBS-LC3 co-localization. These results suggest that although autophagy may act as a BBB cellular defense mechanism in response to invading and toxin-producing bacteria, GBS may actively thwart the autophagic pathway.

In Pichia pastoris, the peroxisomal targeting signal 2 (PTS2)-dependent peroxisomal matrix protein import pathway requires the receptor, Pex7, and its co-receptor Pex20. A conserved lysine (Lys(19)) near the N terminus of Pex20 is required for its polyubiquitination and proteasomal degradation, whereas a conserved cysteine (Cys(8)) is essential for its recycling. In this study, we found that Cys(8) is required for the DTT-sensitive mono- and diubiquitination of Pex20. We also show that the PTS2 cargo receptor, Pex7, is required for Pex20 polyubiquitination. Pex4, the E2 ubiquitin-conjugation enzyme, is required for monoubiquitination of Pex20. However, it is also necessary for polyubiquitination of Pex20, making its behavior distinct from the ubiquitination described for other PTS receptors. Unlike the roles of specific RING peroxins in Pex5 ubiquitination, we found that all the RING peroxins (Pex2, Pex10, and Pex12) are required as E3 ubiquitin ligases for Pex20 mono- and polyubiquitination. A model for Pex20 ubiquitination is proposed based on these observations. This is the first description of the complete ubiquitination pathway of Pex20, which provides a better understanding of the recycling and degradation of this PTS2 cargo co-receptor.

We identified a cysteine residue, conserved near the N terminus of Pex5p- and Pex20p-like proteins, that is essential for the cytosolic relocation of peroxisomal Pex20p. Surprisingly, this residue is not completely essential for the function of the protein; its point mutation into a serine in Pex20p(C8S) causes the accumulation of the protein at the peroxisome membrane, but this is quickly followed by its subsequent degradation by an ubiquitin-dependent quality control pathway called RADAR (receptor accumulation and degradation in the absence of recycling). This degradative pathway allows partial growth of the Pex20p(C8S) mutant on peroxisome-requiring medium. Mutation of cysteine 8 (C8S) and lysine 19 (K19R), the target residue of the RADAR pathway within Pex20p, leads to a stable but non-functional protein because it fails to recycle to the cytosol. This suggests a role for Cys-8 in Pex20p recycling and that constitutive degradation of peroxisomal receptors can be a partially functional alternative to receptor recycling. In addition, expression of this mutant protein in wild-type cells confers a dominant-negative, oleate-specific growth defect, which is a useful tool for a better understanding of peroxisomal receptor recycling.

The rad21 gene of Schizosaccharomyces pombe is involved in the repair of double-strand breaks in DNA and is essential for mitotic growth (Birkenbihl, R. P., and Subramani, S.(1992) Nucleic Acids Res. 20, 6605-6611). We show that the [Medline] Rad21 protein migrates with an aberrantly slow mobility, has a thrombin cleavage site, and is multiply phosphorylated mainly at serine residues. The expression of the rad21 mRNA and the Rad21 protein is cell cycle-regulated, with the peak of mRNA and protein expression occurring near the G1 to S transition. Following translation of the protein, hypophosphorylated forms of the protein appear. However, the most phosphorylated form of Rad21 appears only later in the cell cycle (in S to G2). Analysis of the radiosensitive mutant rad21-45 revealed that the mutant protein is permanently hypophosphorylated. The Rad21 protein is nuclear during the cell cycle. The nuclear localization signal was identified in the C-terminal third of the protein. Upon repression of the Rad21 protein expressed from the repressible nmt1 promoter, the unphosphorylated and hypophosphorylated forms of Rad21 disappeared first. When the concentration of the most highly phosphorylated form of Rad21 sank under a critical level, the cells underwent aberrant mitoses. They exhibited loss of proper nuclear organization and abnormal septation.

Saccharomyces cerevisiae peroxisomal 3-ketoacyl-CoA thiolase is a soluble matrix protein that does not end in a consensus peroxisomal targeting signal-1. The amino terminus of S. cerevisiae peroxisomal thiolase is conserved in 6 of 11 residues with the amino terminus of rat thiolase B, shown to act as a peroxisomal targeting signal-2 (Swinkels, B.W., Gould, S.J., Bodnar, A.G., Rachubinski, R.A., and Subramani, S. (1991) EMBO J. 10, 3255-3262). Unlike mammalian peroxisomal thiolases, there is no extensive cleavage of S. cerevisiae thiolase upon import into peroxisomes. We demonstrate by in vivo expression that the amino-terminal 16 amino acids of S. cerevisiae thiolase are necessary and sufficient for targeting to peroxisomes. This result implies that yeast, like mammalian cells, can target proteins to the peroxisomal matrix by at least two different routes. We also demonstrate by targeted mutagenesis and in vivo expression of mutated thiolase genes that three amino acids conserved in the amino termini of all known thiolases are critical for efficient targeting of S. cerevisiae thiolase to peroxisomes.

A peroxisome proliferator-responsive element is located in the 5'-flanking region of the gene encoding rat hydratase-dehydrogenase, the second enzyme of the peroxisomal beta-oxidation pathway. DNase I footprint analysis with nuclear extracts from proliferator-responsive rat H4IIEC3 cells revealed two protected regions within the 196-base pair peroxisome proliferator-responsive element. Both regions contained multiple copies of a motif related to the consensus steroid hormone receptor binding half-site TGACCT, suggesting that peroxisome proliferator-dependent activation of this gene is mediated via peroxisome proliferator-activated receptors. Region II contains three TGACCT-like motifs in a direct repeat array. An oligonucleotide corresponding to this region was sufficient to confer responsiveness to the peroxisome proliferator ciprofibrate onto a heterologous promoter, as determined by transient transfection assays. Gel retardation assays demonstrated that nuclear factors bound to the hydratase-dehydrogenase oligonucleotide. Mutation of a single G residue within the second repeat motif abolished factor binding and consequently the ability of the element to respond to ciprofibrate, directly demonstrating that factor binding is necessary for peroxisome proliferator responsiveness. These results are discussed in the context of our current understanding of the mechanism of the coordinated transcriptional induction of the genes encoding peroxisomal beta-oxidation enzymes by peroxisome proliferators.

DNA, isolated from Simian virus 40 (SV40), has been encapsulated in large (0.4-micrometer diameter) unilamellar phospholipid vesicles. The procedure used for liposome preparation encapsulated the SV40 DNA at high efficiency (30 to 50% entrapment) and did not alter the physical or biological properties of the DNA molecules. The biological activity of the liposome-entrapped viral DNA was determined by plaque assays on a permissive monkey cell line. The infectivity of liposome-entrapped SV40 DNA was enhanced at least 100-fold over that of free naked DNA. Importantly, the infectivity of vesicle-entrapped DNA was resistant to DNase digestion, dependent on the amount of DNA encapsulated per vesicle and on the vesicle lipid composition. Liposomes composed of phosphatidylserine were the most efficient for delivery of DNA to cells (1.8 x 10(3) plaque-forming units/micrograms of DNA). Following the incubation of DNA-containing liposomes with cells, their infectivity could be enhanced an additional 10- to 200-fold by exposing the cells to high concentrations of polyethylene glycol or glycerol. Under these conditions the infectivity of liposome-encapsulated SV40 DNA (3 x 10(5) plaque-forming units/microgram) was comparable with values reported using the calcium phosphate method. In addition to providing a sensitive assay for monitoring and optimizing the delivery of vesicle contents to cells, the liposome-mediated delivery of nucleic acids may have potential for increasing the efficiency of DNA delivery to cells and for extending the number of cell types which can be transformed or transfected.