M. van den Berg

A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae

In vivo time-lapse microscopy reveals that the number of peroxisomes in Saccharomyces cerevisiae cells is fairly constant and that a subset of the organelles are targeted and segregated to the bud in a highly ordered, vectorial process. The dynamin-like protein Vps1p controls the number of peroxisomes, since in a vps1Δ mutant only one or two giant peroxisomes remain. Analogous to the function of other dynamin-related proteins, Vps1p may be involved in a membrane fission event that is required for the regulation of peroxisome abundance. We found that efficient segregation of peroxisomes from mother to bud is dependent on the actin cytoskeleton, and active movement of peroxisomes along actin filaments is driven by the class V myosin motor protein, Myo2p: (a) peroxisomal dynamics always paralleled the polarity of the actin cytoskeleton, (b) double labeling of peroxisomes and actin cables revealed a close association between both, (c) depolymerization of the actin cytoskeleton abolished all peroxisomal movements, and (d) in cells containing thermosensitive alleles of MYO2, all peroxisome movement immediately stopped at the nonpermissive temperature. In addition, time-lapse videos showing peroxisome movement in wild-type and vps1Δ cells suggest the existence of various levels of control involved in the partitioning of peroxisomes.

The ABC transporter proteins Pat1 and Pat2 are required for import of long-chain fatty acids into peroxisomes of Saccharomyces cerevisiae.

Peroxisomes of Saccharomyces cerevisiae are the exclusive site of fatty acid beta‐oxidation. We have found that fatty acids reach the peroxisomal matrix via two independent pathways. The subcellular site of fatty acid activation varies with chain length of the substrate and dictates the pathway of substrate entry into peroxisomes. Medium‐chain fatty acids are activated inside peroxisomes hby the acyl‐CoA synthetase Faa2p. On the other hand, long‐chain fatty acids are imported from the cytosolic pool of activated long‐chain fatty acids via Pat1p and Pat2p, peroxisomal membrane proteins belonging to the ATP binding cassette transporter superfamily. Pat1p and Pat2p are the first examples of membrane proteins involved in metabolite transport across the peroxisomal membrane.

Overexpression of Pex15p, a phosphorylated peroxisomal integral membrane protein required for peroxisome assembly in S.cerevisiae, causes proliferation of the endoplasmic reticulum membrane

We have cloned PEX15 which is required for peroxisome biogenesis in Saccharomyces cerevisiae. pex15Δ cells are characterized by the cytosolic accumulation of peroxisomal matrix proteins containing a PTS1 or PTS2 import signal, whereas peroxisomal membrane proteins are present in peroxisomal remnants. PEX15 encodes a phosphorylated, integral peroxisomal membrane protein (Pex15p). Using multiple in vivo methods to determine the topology, Pex15p was found to be a tail‐anchored type II (Ncyt–Clumen) peroxisomal membrane protein with a single transmembrane domain near its carboxy‐terminus. Overexpression of Pex15p resulted in impaired peroxisome assembly, and caused profound proliferation of the endoplasmic reticulum (ER) membrane. The lumenal carboxy‐terminal tail of Pex15p protrudes into the lumen of these ER membranes, as demonstrated by its O‐glycosylation. Accumulation in the ER was also observed at an endogenous expression level when Pex15p was fused to the N‐terminus of mature invertase. This resulted in core N‐glycosylation of the hybrid protein. The lumenal C‐terminal tail of Pex15p is essential for targeting to the peroxisomal membrane. Furthermore, the peroxisomal membrane targeting signal of Pex15p overlaps with an ER targeting signal on this protein. These results indicate that Pex15p may be targeted to peroxisomes via the ER, or to both organelles.

Peroxisomal beta-oxidation of polyunsaturated fatty acids in Saccharomyces cerevisiae: isocitrate dehydrogenase provides NADPH for reduction of double bonds at even positions.

The β‐oxidation of saturated fatty acids in Saccharomyces cerevisiae is confined exclusively to the peroxisomal compartment of the cell. Processing of mono‐ and polyunsaturated fatty acids with the double bond at an even position requires, in addition to the basic β‐oxidation machinery, the contribution of the NADPH‐dependent enzyme 2,4‐dienoyl‐CoA reductase. Here we show by biochemical cell fractionation studies that this enzyme is a typical constituent of peroxisomes. As a consequence, the β‐oxidation of mono‐ and polyunsaturated fatty acids with double bonds at even positions requires stoichiometric amounts of intraperoxisomal NADPH. We suggest that NADP‐dependent isocitrate dehydrogenase isoenzymes function in an NADP redox shuttle across the peroxisomal membrane to keep intraperoxisomal NADP reduced. This is based on the finding of a third NADP‐dependent isocitrate dehydrogenase isoenzyme, Idp3p, next to the already known mitochondrial and cytosolic isoenzymes, which turned out to be present in the peroxisomal matrix. Our proposal is strongly supported by the observation that peroxisomal Idp3p is essential for growth on the unsaturated fatty acids arachidonic, linoleic and petroselinic acid, which require 2,4‐dienoyl‐CoA reductase activity. On the other hand, growth on oleate which does not require 2,4‐dienoyl‐CoA reductase, and NADPH is completely normal in Δidp3 cells.

Soil moisture-temperature coupling: A multiscale observational analysis

[1] Land-atmospheric interactions are complex and variable in space and time. On average soil moisture-temperature coupling is expected to be stronger in transition zones between wet and dry climates. During heatwaves anomalously high coupling may be found in areas of soil moisture deficit and high atmospheric demand of water. Here a new approach is applied to satellite andin situobservations towards the characterization of regions of intense soil moisture-temperature coupling, both in terms of climatology and anomalies during heatwaves. The resulting average summertime couplinghot spotsreflect intermediate climatic regions in agreement with previous studies. Results at heatwave-scale suggest a minor role of soil moisture deficit during the heatwave of 2006 in California but an important one in the 2003 event in Western Europe. Progress towards near-real time satellite products may allow the application of the approach to aid prediction and management of warm extremes.

DNA circles with cruciforms from Isospora (Toxoplasma) gondii

We have isolated a closed circular duplex DNA fraction from the unicellular parasite Isospora (Toxoplasma) gondii and examined the purified DNA by electron microscopy. A major part of this circular DNA consists of 12-micron circles containing a cruciform with 0.5-micron tails. We also found 23-micron circles with the properties expected of head-to-tail dimers of the 12-micron circles. Some of these dimers have two cruciforms with 0.4-micron tails, some have one cruciform with 0.8-micron tails. When ethidium bromide was diffused into the DNA solution, circles with tails were replaced by twisted circles without tails. Direct mixing of the DNA with high ethidium bromide concentrations (5 micrograms/ml) gave rise to highly twisted circles with tails. This proves that the tailed circles are covalently continuous and indicates that ethidium bromide blocks branch migration. The 0.5-micron tails are part of a 1.7-micron palindrome, which was visualized by spreading denatured DNA under snap-back conditions. We argue that the cruciform is not present in vivo and that the 12-micron circles may represent the mitochondrial DNA of Toxoplasma.

IgE and IgG cross-reactivity among Lol p I and Lol p II/III. Identification of the C-termini of Lol p I, II, and III as cross-reactive structures.

In this study, the homologous C‐termini of Lol p I Lol p II, and Lol p III were shown to contain cross‐reactive B‐cell epitopes. This was demonstrated by inhibition studies with purified Lol p I, II, and III and synthetic peptides of their C‐termini. It was ruled out that the observed cross‐reactivity was caused by cross‐contamination of the purified allergens. Both human IgE and IgG bound to the C‐terminus of Lol p I. These antibodies were cross‐reactive with Lol p II and, more specifically, with its C‐terminus. Within a small panel of allergic patients, no cross‐reactivity with Lol p III was found. A hyperimmune polyclonal rabbit antiserum against Lol p I also recognized the Lol p I C‐terminus. As for human antibodies, cross‐reactivity with Lol p II and its C‐terminus was demonstrated. Cross‐reactivity with Lol p III was demonstrated with C‐terminal peptides, but not with native Lol p III. A polyclonal rabbit antiserum against Lol p II bound to the C‐terminal peptides of both Lol p II and III. This binding was inhibited with Lol p I, confirming that cross‐reactive structures exist not only on the C‐termini of Lol p II and Lol p I, but also of Lol p III and Lol p I. The existence of cross‐reactivity between Lol p I and Lol p II and III possibly contributes to the frequently observed cosensitization for these allergens in grass‐pollen‐allergic patients.

Characterization of dendritic cells, isolated from normal and stimulated lymph nodes of the rat

SummaryNon-lymphoid dendritic cells were isolated from normal and paratyphoid vaccine-stimulated lymph nodes draining the rat skin. They were studied using enzymecytochemical, immunocytochemical and electron-microscopical methods. These cells had an irregular outline and an eccentrically situated nucleus. All showed acid phosphatase activity in a central area and expressed Ia antigen on the plasma membrane. Birbeck granules were exclusively present in dendritic cells isolated from lymph nodes in the induction phase of the immune response. This observation concurs with the presence of Birbeck granules in interdigitating cells in situ during the same period of the immune response. It is concluded that the dendritic cells are the in-vitro equivalents of the non-actively phagocytizing population of interdigitating cells.

Uptake and elimination of PCDD/PCDF congeners by fish after aqueous exposure to a fly-ash extract from a monicipal incimerator

Abstract After aqueous exposure of fish to a fly-ash extract only a few highly chlorinated dibenzo-p-dioxins and dibenzofurans are accumulated. The estimated uptake rate constants of the accumulated congeners are comparable to those of PCBs or other hydrophobic chemicals. The elimination rate constants however seem to be relatively high. For the 2,3,7,8 TeCDD and 2,3,7,8 TeCDF a slow elimination is found if compared to other PCDDs and PCDFs.

Mitochondrial minicircles in the free-living bodonid Bodo saltans contain two gRNA gene cassettes and are not found in large networks

In trypanosomatids, the majority of the guide (g) RNAs that provide the information for U-insertion/deletion RNA editing are encoded by minicircles that are catenated into large networks. In contrast, in the distantly related cryptobiid Trypanoplasma borreli, gRNA genes appear to reside in large 180-kb noncatenated DNA circles. To shed light on the evolutionary history and function of the minicircle network, we have analyzed minicircle organization in the free-living bodonid Bodo saltans, which is more closely related to trypanosomatids than T. borreli. We identified 1.4-kb circular DNAs as the B. saltans equivalent of minicircles via sequence analysis of 4 complete minicircles, 14 minicircle fragments, and 14 gRNAs. We show that each minicircle harbors two gRNA gene cassettes of opposite polarity residing in variable regions of about 200 nt in otherwise highly conserved molecules. In the conserved region, B. saltans minicircles contain a putative bent helix sequence and a degenerate dodecamer motif (CSB-3). Electron microscopy, sedimentation, and gel electrophoresis analyses showed no evidence for the existence of large minicircle networks in B. saltans, the large majority of the minicircles being present as circular and linear monomers (85-90%) with small amounts of catenated dimers and trimers. Our results provide the first example of a kinetoplastid species with noncatenated, gRNA gene-containing minicircles, which implies that the creation of minicircles and minicircle networks are separate evolutionary events.