PEX11alpha is required for peroxisome proliferation in response to 4-phenylbutyrate but is dispensable for peroxisome proliferator-activated receptor alpha-mediated peroxisome proliferation

The PEX11 peroxisomal membrane proteins promote peroxisome division in multiple eukaryotes. As part of our effort to understand the molecular and physiological functions of PEX11 proteins, we disrupted the mouse PEX11alpha gene. Overexpression of PEX11alpha is sufficient to promote peroxisome division, and a class of chemicals known as peroxisome proliferating agents (PPAs) induce the expression of PEX11alpha and promote peroxisome division. These observations led to the hypothesis that PPAs induce peroxisome abundance by enhancing PEX11alpha expression. The phenotypes of PEX11alpha(-/-) mice indicate that this hypothesis remains valid for a novel class of PPAs that act independently of peroxisome proliferator-activated receptor alpha (PPARalpha) but is not valid for the classical PPAs that act as activators of PPARalpha. Furthermore, we find that PEX11alpha(-/-) mice have normal peroxisome abundance and that cells lacking both PEX11alpha and PEX11beta, a second mammalian PEX11 gene, have no greater defect in peroxisome abundance than do cells lacking only PEX11beta. Finally, we report the identification of a third mammalian PEX11 gene, PEX11gamma, and show that it too encodes a peroxisomal protein.     

Arfophilins are dual Arf/Rab 11 binding proteins that regulate recycling endosome distribution and are related to Drosophila nuclear fallout

Arfophilin is an ADP ribosylation factor (Arf) binding protein of unknown function. It is identical to the Rab11 binding protein eferin/Rab11-FIP3, and we show it binds both Arf5 and Rab11. We describe a related protein, arfophilin-2, that interacts with Arf5 in a nucleotide-dependent manner, but not Arf1, 4, or 6 and also binds Rab11. Arfophilin-2 localized to a perinuclear compartment, the centrosomal area, and focal adhesions. The localization of arfophilin-2 to the perinuclear compartment was selectively blocked by overexpression of Arf5-T31N. In contrast, a green fluorescent protein-arfophilin-2 chimera or arfophilin-2 deletions were localized around the centrosome in a region that was also enriched for transferrin receptors and Rab11 but not early endosome markers, suggesting that the distribution of the endosomal recycling compartment was altered. The arfophilins belong to a conserved family that includes Drosophila melanogaster nuclear fallout, a centrosomal protein required for cellularization. Expression of green fluorescent protein-nuclear fallout in HeLa cells resulted in a similar phenotype, indicative of functional homology and thus implicating the arfophilins in mitosis/cytokinesis. We suggest that the novel dual GTPase-binding capacity of the arfophilins could serve as an interface of signals from Rab and Arf GTPases to regulate membrane traffic and integrate distinct signals in the late endosomal recycling compartment.     

First isolation of an aquatic birnavirus from farmed and wild fish species in Australia

During routine sampling and testing, as part of a systematic surveillance program (the Tasmanian Salmonid Health Surveillance Program), an aquatic birnavirus was isolated from 'pin-head' (fish exhibiting deficient acclimatisation on transfer to saltwater) Atlantic salmon Salmo salar, approximately 18 mo old, farmed in net-pens located in Macquarie Harbour on the west coast of Tasmania, Australia. The isolate grows readily in a range of fish cell lines including CHSE-214, RTG-2 and BF-2 and is neutralised by a pan-specific rabbit antiserum raised against infectious pancreatic necrosis virus (IPNV) Ab strain and by a commercial pan-specific IPNV-neutralising monoclonal antibody. Presence of the virus was not associated with gross clinical signs. Histopathological examination revealed a range of lesions particularly in pancreatic tissue. The virus was localised in pancreas sections by immunoperoxidase staining using the polyclonal antiserum and by electron microscopy. Examination by electron microscopy demonstrated that the virus isolated in cell culture (1) belongs to the family Birnaviridae, genus Aquabirnaviridae; (2) was ultrastructurally and antigenically similar to virus identified in the index fish; (3) is related to IPNV. Western blot analysis using the polyclonal rabbit antiserum confirmed the cross-reactions between various aquatic birnavirus isolates. In addition, PCR analysis of isolated viral nucleic acid from the index case indicated that the virus is more closely related to IPNV fr21 and N1 isolates than to other birnavirus isolates available for comparison. Sampling of other fish species within Macquarie Harbour has demonstrated that the virus is present in several other species of fish including farmed rainbow trout Oncorhynchus mykiss, wild flounder Rhombosolea tapirina, cod Pseudophycis sp., spiked dogfish Squalus megalops and ling Genypterus blacodes.     

The peroxisome biogenesis factors pex4p, pex22p, pex1p, and pex6p act in the terminal steps of peroxisomal matrix protein import

Peroxisomes are independent organelles found in virtually all eukaryotic cells. Genetic studies have identified more than 20 PEX genes that are required for peroxisome biogenesis. The role of most PEX gene products, peroxins, remains to be determined, but a variety of studies have established that Pex5p binds the type 1 peroxisomal targeting signal and is the import receptor for most newly synthesized peroxisomal matrix proteins. The steady-state abundance of Pex5p is unaffected in most pex mutants of the yeast Pichia pastoris but is severely reduced in pex4 and pex22 mutants and moderately reduced in pex1 and pex6 mutants. We used these subphenotypes to determine the epistatic relationships among several groups of pex mutants. Our results demonstrate that Pex4p acts after the peroxisome membrane synthesis factor Pex3p, the Pex5p docking factors Pex13p and Pex14p, the matrix protein import factors Pex8p, Pex10p, and Pex12p, and two other peroxins, Pex2p and Pex17p. Pex22p and the interacting AAA ATPases Pex1p and Pex6p were also found to act after Pex10p. Furthermore, Pex1p and Pex6p were found to act upstream of Pex4p and Pex22p. These results suggest that Pex1p, Pex4p, Pex6p, and Pex22p act late in peroxisomal matrix protein import, after matrix protein translocation. This hypothesis is supported by the phenotypes of the corresponding mutant strains. As has been shown previously for P. pastoris pex1, pex6, and pex22 mutant cells, we show here that pex4Delta mutant cells contain peroxisomal membrane protein-containing peroxisomes that import residual amounts of peroxisomal matrix proteins.     

Bacillus thuringiensisδ-Endotoxin Proteins Show a Correlation in Toxicity and Short Circuit Current Inhibition Against Helicoverpa zea

Pesticidal activity of Bacillus thuringiensisδ-endotoxins, Cry1Aa, Cry1Ab, Cry1Ac, and Cry2A, was determined by using the force-feeding bioassay method to 4^th instar larvae of Helicoverpa zea. H. zea was susceptible to Bt toxins in the order Cry1Ac > Cry1Ab > Cry1Aa > Cry2A with 63.60, 89.04, 159.65, and 375.78 ng/larvae respectively. The abilities of selected Bacillus thuringiensis toxins to inhibit short circuit current (I_SC) in midgut epithelia of H. zea were also investigated by voltage clamp assay. The voltage-clamp studies were conducted on isolated midguts, measuring the inhibition of short circuit current (I_SC) by activated toxin. A Cry1Aa toxin dilution of 33.3 and 500 ng/ml resulted in inhibition of I_SC of −2.29 μA/min (lag time 15 min) and −4.48 μA/min (lag time, 2 min) respectively. The Cry1Ab dilution of 25 ng/ml inhibited I_SC to −1.39 μA/min, a lag time of 14 min, and 333.3 ng/ml dilution resulted in decay of I_SC−2.49 μA/min, lag time 1 min respectively. The Cry1Ac lower dilution 16.7 ng/ml inhibited I_SC to −1.39 μA/min, lag time 4 min, and a high dilution 333.3 ng/ml decay I_SC to −2.44 μA/min, lag time 1 min. The inhibition of I_SC (−1.10 μA/min, lag time 25) at lower dilution (33.3 ng/ml) and high dilution (500 ng/ml), decay (−2.38 μA/min, lag time 5 min), showed a correlation between toxin concentration and inhibitory response with Cry2A toxin. The lag time decreased with increasing concentration of toxin applied, which is additional evidence of dose response besides direct correlation of toxicity assays and I_SC. Received: 7 April 2000 / Accepted: 2 May 2000     

Hot water treatment and insecticidal coatings for disinfesting limes of mealybugs (Homoptera: Pseudococcidae)

Hot water immersion and insecticidal coatings were tested to determine if they could be used to disinfest Persian limes, Citrus latifolia Tanaka, of the mealybug pests Planococcus citri Risso and Pseudococcus odermatti Miller & Williams. A 20-min 49 degrees C hot water immersion treatment is effective in killing mealybugs and all other arthropods tested found externally on limes, or under the calyx. No insects or mites were found to survive after the 20-min hot water treatment. In this test, 7,200 limes were treated with 1,308 insects killed and zero survivors. Treatment at 49 degrees C for 20 min did not significantly affect quality when treated fruit were compared with untreated control fruit. Four coatings were tested at a 3% rate: two petroleum-based oils (Ampol and Sunspray oil), a vegetable oil (natural oil), and a soap (Mpede). The coatings gave up to 94% kill (Ampol) of mealybugs, which is not sufficient to provide quarantine security. The coatings might be effective as a postharvest dip before shipment.     

Functional role of anthocyanins in the leaves of Quintinia serrata A. Cunn

The protective functions that have been ascribed to anthocyanins in leaves can be performed as effectively by a number of other compounds. The possibility that anthocyanins accumulate most abundantly in leaves deficient in other phytoprotective pigments has been tested. Pigment concentrations and their histological distribution were surveyed for a sample of 1000 leaves from a forest population of Quintinia serrata, which displays natural polymorphism in leaf colour. Eight leaf phenotypes were recognized according to their patterns of red coloration. Anthocyanins were observed in almost all combinations of every leaf tissue, but were most commonly located in the vacuoles of photosynthetic cells. Red leaves contained two anthocyanins (Cy-3-glc and Cy-3-gal), epicuticular flavones, epidermal flavonols, hydroxycinnamic acids, chlorophylls, and carotenoids. Green leaves lacked anthocyanins, but had otherwise similar pigment profiles. Foliar anthocyanin levels varied significantly between branches and among trees, but were not correlated to concentrations of other pigments. Anthocyanins were most abundant in older leaves on trees under canopies with south-facing gaps. These data indicate that anthocyanins are associated with photosynthesis, but do not serve an auxiliary phytoprotective role. They may serve to protect shade-adapted chloroplasts from brief exposure to high intensity sunflecks.     

Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5

Many proteins contain targeting signals within their sequences that specify their delivery to particular organelles. The peroxisomal targeting signal-1 (PTS1) is a C-terminal tripeptide that is sufficient to direct proteins into peroxisomes. The PTS1 sequence closely approximates Ser-Lys-Leu-COO-. PEX5, the receptor for PTS1, interacts with the signal via a series of tetratricopeptide repeats (TPRs) within its C-terminal half. Here we report the crystal structure of a fragment of human PEX5 that includes all seven predicted TPR motifs in complex with a pentapeptide containing a PTS1 sequence. Two clusters of three TPRs almost completely surround the peptide, while a hinge region, previously identified as TPR4, forms a distinct structure that enables the two sets of TPRs to form a single binding site. This structure reveals the molecular basis for PTS1 recognition and demonstrates a novel mode of TPR-peptide interaction.     

Identification of further important residues within the Glut4 carboxy-terminal tail which regulate subcellular trafficking

The insulin-responsive glucose transporter, Glut4, exhibits a unique subcellular distribution such that in the absence of insulin >95% of the protein is stored within intracellular membranes. In response to insulin, Glut4 exhibits a large mobilisation to the plasma membrane. Studies of the amino acid motifs which regulate the unique trafficking of Glut4 have identified several key residues within the soluble cytoplasmic N- and C-terminal domains of Glut4. Of particular note is a Leu-498Leu-499 motif within the C-terminal domain that has been proposed to regulate both internalisation from the plasma membrane and sorting to an insulin-sensitive compartment. In this study, we have examined the role of the adjacent amino acids (Glu-491, Gln-492 and Glu-493) by their sequential replacement with Ala. Our results are consistent with the notion that Glu-491 and Glu-493 play an important role in the sub-endosomal trafficking of Glut4, as substitution of these residues with Ala results in increased levels of these proteins at the cell surface, reduced insulin-stimulated translocation and increased susceptibility to endosomal ablation. These residues, together with other identified sequences within the C-terminus of Glut4, are likely to be crucial targeting elements that regulate Glut4 subcellular distribution.