Immunolocalisation of phospholipase D1 on tubular vesicular membranes of endocytic and secretory origin

We have examined the localisation of overexpressed phospholipase D1 (PLD1) using antibodies against its amino- and carboxyl-terminal domains. PLD1 overexpressed in COS-7 cells showed variable distribution by immunofluorescence but was mainly in punctate structures in the perinuclear region and at the plasma membrane. Downregulation by an anti-sense plasmid resulted in almost exclusively perinuclear distribution in punctate structures that contained immunoreactivity for the endogenous KDEL receptor and the early endosomal antigen EEA1 protein. Influenza haemagglutinin (HA) and HA-derived mutants designed to locate primarily to secretory or endocytic membranes were present in PLD1-positive membranes. Immunofluorescence analysis in permanent CHO cell lines that express PLD1 inducibly confirmed the presence of PLD1 on both endocytic and secretory membranes. Analysis of PLD1 distribution by immunocytochemistry and electron microscopy of intact CHO cells and of isolated membranes revealed that PLD1 was present in tubulovesicular elements and multivesicular bodies. Some of these were close to the Golgi region whereas others stained positive for endocytic cargo proteins. Morphometric analysis assigned the majority of PLD1 immunoreactivity on endosomal membranes and a smaller amount on membranes of secretory origin. PLD1, via signals that are currently not understood, is capable of localising in tubulovesicular membranes of both endocytic and secretory origin.     

Nucleotide excision repair/TFIIH helicases RAD3 and SSL2 inhibit short-sequence recombination and Ty1 retrotransposition by similar mechanisms

Eukaryotic genomes contain potentially unstable sequences whose rearrangement threatens genome structure and function. Here we show that certain mutant alleles of the nucleotide excision repair (NER)/TFIIH helicase genes RAD3 and SSL2 (RAD25) confer synthetic lethality and destabilize the Saccharomyces cerevisiae genome by increasing both short-sequence recombination and Ty1 retrotransposition. The rad3-G595R and ssl2-rtt mutations do not markedly alter Ty1 RNA or protein levels or target site specificity. However, these mutations cause an increase in the physical stability of broken DNA molecules and unincorporated Ty1 cDNA, which leads to higher levels of short-sequence recombination and Ty1 retrotransposition. Our results link components of the core NER/TFIIH complex with genome stability, homologous recombination, and host defense against Ty1 retrotransposition via a mechanism that involves DNA degradation.     

A re-examination of the structural and functional consequences of mutation of alanine-128 of the b subunit of Escherichia coli ATP synthase to aspartic acid

The effects of mutation of residue Ala-128 of the b subunit of Escherichia coli ATP synthase to aspartate on the structure of the subunit and its interaction with the F(1) sector were analyzed. Determination of solution molecular weights by sedimentation equilibrium ultracentrifugation revealed that the A128D mutation had little effect on dimerization in the soluble b construct, b(34-156). However, the mutation caused a structural perturbation detected through both a 12% reduction in the sedimentation coefficient and also a reduced tendency to form intersubunit disulfide bonds between cysteine residues inserted at position 132. Unlike the wild-type sequence, the A128D mutant was unable to interact with F(1)-ATPase. These results indicate that the A128D mutation caused a structural change in the C-terminal region of the protein, preventing the binding to F(1) but having little or no effect on the dimeric nature of b.     

Wheat germ translation initiation factor eIF4B affects eIF4A and eIFiso4F helicase activity by increasing the ATP binding affinity of eIF4A

It has been proposed that, during translational initiation, structures in the 5' untranslated region of mRNA are unwound. eIF4A, a member of the DEAD box family of proteins (those that contain a DEAD amino acid sequence), separately or in conjunction with other eukaryotic initiation factors, utilizes the energy from ATP hydrolysis to unwind these structures. As a step in defining the mechanism of helicase activity in the wheat germ protein synthesis system, we have utilized direct fluorescence measurements, ATPase assays, and helicase assays. The RNA duplex unwinding activity of wheat germ eIF4A is similar to other mammalian systems; however, eIF4F or eIFiso4F is required, probably because of the low binding affinity of wheat germ eIF4A for mRNA. Direct ATP binding measurements showed that eIF4A had a higher binding affinity for ADP than ATP, resulting in a limited hydrolysis and procession along the RNA in the helicase assay. The addition of eIF4B resulted in a change in binding affinity for ATP, increasing it almost 10-fold while the ADP binding affinity was approximately the same. The data presented in this paper suggest that eIF4F or eIFiso4F acts to position the eIF4A and stabilize the interaction with mRNA. ATP produces a conformational change which allows a limited unwinding of the RNA duplex. The binding of eIF4B either prior to or after hydrolysis allows for increased affinity for ATP and for the cycle of conformational changes to proceed, resulting in further unwinding and processive movement along the mRNA.     

Cyk3, a novel SH3-domain protein, affects cytokinesis in yeast

Cytokinesis requires the wholesale reorganization of the cytoskeleton and secretion to complete the division of one cell into two. In the budding yeast Saccharomyces cerevisiae, the IQGAP-related protein Iqg1 (Cyk1) promotes cytokinetic actin ring formation and is required for cytokinesis and viability [1-3]. As the actin ring is not essential for cytokinesis or viability, Iqg1 must act by another mechanism [4]. To uncover this mechanism, a screen for high-copy suppressors of the iqg1 lethal phenotype was performed. CYK3 suppressed the requirement for IQG1 in viability and cytokinesis without restoration of the actin ring, demonstrating that CYK3 promotes cytokinesis through an actomyosin-ring-independent pathway. CYK3 encodes a novel SH3-domain protein that was found in association with the actin ring and the mother-bud neck. cyk3 null cells had misshapen mother-bud necks and were deficient in cytokinesis. In the cyk3 null strain, actin rearrangements associated with cytokinesis appeared normal, suggesting that the phenotype reflects a defect in secretory targeting or septal synthesis. Deletion of either cyk3 or hof1 alone results in a mild cytokinetic phenotype [5-7], but deletion of both genes resulted in lethality and a complete cytokinetic block, suggesting overlapping function. Thus, Cyk3 appears to be important for cytokinesis and acts potentially downstream of Iqg1.     

High copy numbers of multiple transposable element families in an Australian population of Drosophila simulans

Sudden mobilization of transposable elements in Drosophila is a well-reported phenomenon but one that usually affects no more than a few elements (one to four). We report here the existence of a D. simulans natural population (Canberra) from Australia, which had high copy numbers for various transposable elements (transposons, LTR retrotransposons and non-LTR retrotransposons). The impact of transposable elements on the host genome and populations is discussed.     

Novel cathepsin D inhibitors block the formation of hyperphosphorylated tau fragments in hippocampus

Lysosomal disturbances may be a contributing factor to Alzheimer's disease. We used novel compounds to test if suppression of the lysosomal protease cathepsin D blocks production of known precursors to neurofibrillary tangles. Partial lysosomal dysfunction was induced in cultured hippocampal slices with a selective inhibitor of cathepsins B and L. This led within 48 h to hyperphosphorylated tau protein fragments recognized by antibodies against human tangles. Potent nonpeptidic cathepsin D inhibitors developed using combinatorial chemistry and structure-based design blocked production of the fragments in a dose-dependent fashion. Threshold was in the submicromolar range, with higher concentrations producing complete suppression. The effects were selective and not accompanied by pathophysiology. Comparable results were obtained with three structurally distinct inhibitors. These results support the hypothesis that cathepsin D links lysosomal dysfunction to the etiology of Alzheimer's disease and suggest a new approach to treating the disease.     

Sample size for collecting seeds in germplasm conservation: the case of the Lima bean (Phaseolus lunatus L.)

 The design of optimum sampling strategies integrating criteria of efficiency relevant to multilocus models and many target populations has been investigated with respect to the number of plants and the number of seeds per plant to be sampled for a Lima bean (Phaseolus lunatus L.) gene pool. This study, using five populations and six polymorphic enzyme loci, shows that the number of plants rather than the number of seeds collected per plant primarily determines the success of seed sampling, suggesting that plant number plays an essential part in maintaining the allelic multiplicity of predominantly selfing species like Lima bean. According to the results, it appears that among Lima bean populations an efficient sampling procedure is achieved by collecting 1–4 seeds from 200 to 300 plants. These sample sizes will retain 8–10 alleles, regardless of their frequencies. When we consider polymorphism at the 5% level, it is expected that sampling 10–80 plants will collect combinations of 4–8 alleles. Based on data from genetic and demographic studies, we suggest an efficient sampling scheme for Lima bean germplasm at both population and geographical levels. Received: 10 March 1998 / Accepted: 1 April 1998