In vivo biosynthesis of vacuolar proteinases in proteinase mutants of Saccharomyces cerevisiae

The molecular forms of proteinase A, proteinase B and Car?ypeptidase Y, enzymes of the lysosome like yeast vacuole, were studied in mutants (Wolf,D.H. and Fink,G.R. (1975) J. Bacteriol. 123, 1150–1156;Wolf,D.H. and Ehmann,C. (1978) FEBS Lett. 92, 121–124;Mechler,B. and Wolf,D.H. (1981) Eur. J. Biochem. 121, 47–52) defective in genes, which appear to be structural genes of the respective enzymes. According to the immunochemical reactivity of proteinase protein, mutants could be divided into three classes: 1) Mutants harboring no immunoreactive proteinase material. 2) Mutants synthesizing proteinase precursor molecules of similar size as wild type, which are transferred into mature proteins, which are, however, completely inactive. 3) Mutants synthesizing proteinase precursor-like proteins, which are not processed into the mature proteins. As measured in the car?y-peptidase Y mutant strain the mutant precursor car?ypeptidase Y is rapidly degraded. A pleiotropic mutation (pep4-3) resulting in low activities of five vacuolar enzymes had been shown to accumulate pro-car?ypeptidase Y like immunoreactive material (Hemmings,B.A., Zubenko,G.S., Hasilik,A., and Jones,E.W. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 435–439). We found that this mutant is also defective in processing the proteinase B precursor, whereas no cross-reactive proteinase A molecule could be detected under the conditions employed.     

In vitro biosynthesis, core glycosylation and membrane integration of opsin

A membrane-integrated , core-glycosylated form of bovine opsin was synthesized in vitro when bovine retina mRNA was translated in a wheat germ cell-free system supplemented with dog pancreas microsomal vesicles; glycosylation and integration of opsin into membranes were coupled to translation. Proteolysis with themolysin was used to probe the orientation of opsin within the dog pancreas microsomal membrane, and to compare it with that of opsin in rod cell disk membranes isolated from bovine retina. Intact microsomal or disk vesicles were required for production of discrete, membrane-associated thermolysin fragments of opsin; no discrete opsin fragments were detected when membranes were incubated with thermolysin in the presence of the nonionic detergent, Triton X-100. The major opsin fragments produced by themosylin treatment of intact microsomal vesicles resembled those from disk vesicles in their size, oligosaccharide content, and order of appearance. In each case, the first cleavage of opsin took place at the COOH-terminus, generating a glycosylated fragment, O’, which was only slightly smaller than intact opsin. Both the microsomal and disk membrane forms of O’ were next cleaved internally; glycosylated fragments of similar sizes in both cases were detected which were derived from the NH(2)-terminal portion of O’. Several smaller NH(2)-terminal fragments of opsin were detected only in thermolysin-treated microsomal membranes, and not in disk membranes. The data suggest that the topology of opsin integrated into dog pancreas microsomal vesicles is similar to that in rod cell disk vesicles, although not identical. In each case, the glycosylated NH(2)-terminal region of opsin is located within the lumen of the vesicle, while discrete COOH-terminal and internal segments of opsin apparently emerge at the outer, cytoplasmic face of the membrane. Thus, opsin in the heterologous microsomal membrane, like its counterpart in the native disk membrane, may cross the bilayer at least three times. The internal domain of the polypeptide that emerges at the outer membrane surface is apparently more highly exposed in the case of opsin in microsomal membranes, evidenced by the additional internal thermolysin cleavage sites detected.     

When the control is lacking—the role of tumour suppressor genes in cancer development

The potential to genetically dissect tumorigenesis provides the major reason to study this process in the fruit flyDrosophila. Over the last 30 years genetic analysis has identified some 55 genes in which recessive mutations cause the appearance of specific tumours during development in tissues such as the imaginal discs, the brain hemispheres, the hematopoietic organs or the gonads, Since the normal allele acts dominantly over the mutated allele, these genes are designated as tumour suppressor genes. The estimate of the number of genes that can be mutated to tumour formation may be, however, much higher ranging between I00 to 200. The challenge before this field is how best to identify these genes and elucidate their function. Current molecular procedures, such as mutagenesis mediated by P-element transposon, provide new ways for tagging any gene of interest inDrosophila and thus for cloning it rapidly. Function of the gene product can be inferred by comparing its amino acid sequence with sequences of proteins with known function or can be determined by histochemical and biochemical investigations. Progress in the understanding of tumour suppression inDrosophila is most advanced in the case of genes regulating cell growth in imaginal discs. The imaginal discs are small groups of cells displaying a strong apical-basal polarity and form folded sacs of epithelia which grow throughout the larval life and give rise to the adult tegument during metamorphosis. Tumour suppressor genes regulating cell growth of imaginal discs, such as thelethal(2)giant larvae (l(2)g1),lethal(1)discs large-1 andexpanded genes, were found to encode proteins localized in domains of cell to cell contact on the plasma membrane and were thus thought to maintain cell adhesion. However, recent studies of l(2)gl have revealed that the l(2)gl protein is a component of the normal cytoskeleton which can participates to the cytoskeletal matrix underlaying the plasma membrane. These findings indicate that the changes in cell shape and the loss of apical-basal polarity in imaginal disc cells result primarily from alterations in the cytoskeleton structure. Furthermore the neoplastic growth of the mutated cells may be caused by the disorganization of an intracellular communication system that ultimately controls cell proliferation and/or cell differentiation.     

Drosophila snRNP associated protein P11 which specifically binds to heat shock puff 93D reveals strong homology with hnRNP core protein A1.

We have isolated cDNAs coding for a ribonucleoprotein of Drosophila melanogaster that is distinguished by its nearly exclusive presence at only one of the several heat shock puffs in polytene chromosomes of third instar larvae. We determined the nucleotide sequence and deduced the corresponding amino acid sequence. Its coding capacity for a 39 kDa protein is consistent with the size of the protein detected by the monoclonal antibody P11 used for expression cloning. Our results show that the P11 protein belongs to the category of hnRNP proteins of bipartite structure: the amino-terminal half contains two RNA binding domains and the carboxyterminal half is rich in glycine residues. Analysis of the genomic structure revealed two introns located within the coding portion of the gene and a third one in the 3'untranslated region. We detect two different polyadenylation sites as a result of alternative termination-polyadenylation. Its strong sequence homology with hnRNP A1 protein and its previously shown association with snRNP particles indicates that a typical hnRNP protein may also exist in a complex with snRNP particles. The P11 sequence corresponds to the Hrb87F sequence that was recently described by Haynes et al. (1) as hnRNP A related gene.     

Attentional modulation of central odor processing

Two studies were conducted to investigate the influence of attention on the components of the chemosensory event-related potential (CSERP). In the first study the odors linalool and eugenol were delivered to six male subjects, in the second study three male and two female subjects were presented with their own body odor (axillary hair) and the body odor of a same sex donor. In both studies the odors were presented in an oddball paradigm under ignore and attend conditions via a constant- flow olfactometer. In the ignore condition attention was diverted from the odors with a distractor task, while in the attend condition the subjects were asked to respond to the infrequently occurring odor. In both studies the allocation of attention led to a decrease in the latency of the early components (N1, P2, N2) and to an increase in the amplitude of the late positivities. The modulation of the early components suggests that attentional gating in olfaction might already be effective at an early processing level.      

Down-regulation of RpS21, a putative translation initiation factor interacting with P40, produces viable minute imagos and larval lethality with overgrown hematopoietic organs and imaginal discs

Down-regulation of the Drosophila ribosomal protein S21 gene (rpS21) causes a dominant weak Minute phenotype and recessively produces massive hyperplasia of the hematopoietic organs and moderate overgrowth of the imaginal discs during larval development. Here, we show that the S21 protein (RpS21) is bound to native 40S ribosomal subunits in a salt-labile association and is absent from polysomes, indicating that it acts as a translation initiation factor rather than as a core ribosomal protein. RpS21 can interact strongly with P40, a ribosomal peripheral protein encoded by the stubarista (sta) gene. Genetic studies reveal that P40 underexpression drastically enhances imaginal disc overgrowth in rpS21-deficient larvae, whereas viable combinations between rpS21 and sta affect the morphology of bristles, antennae, and aristae. These data demonstrate a strong interaction between components of the translation machinery and showed that their underexpression impairs the control of cell proliferation in both hematopoietic organs and imaginal discs.     

Evaluating eukaryotic secreted protein prediction

Background  

Improvements in protein sequence annotation and an increase in the number of annotated protein databases has fueled development of an increasing number of software tools to predict secreted proteins. Six software programs capable of high throughput and employing a wide range of prediction methods, SignalP 3.0, SignalP 2.0, TargetP 1.01, PrediSi, Phobius, and ProtComp 6.0, are evaluated.     

Valois, a component of the nuage and pole plasm, is involved in assembly of these structures, and binds to Tudor and the methyltransferase Capsuléen

Using the Capsuleen (Csul) methyltransferase as bait in the yeast two-hybrid system, we have identified a novel Drosophila protein containing multiple WD repeats and encoded by the valois (vsl) gene, which acts in pole plasm function. Vls is homologous to human MEP50, which forms a complex with the PRMT5 methyltransferase--the human homologue of Csul. We found that Vls localizes to the nuage in the nurse cells and to the pole plasm in the oocyte. Moreover vls is required for the synthesis and/or stability of Oskar and the localization of Tudor (Tud) in both the nuage and at the posterior pole of the oocyte. Furthermore, we show that Vls and a fragment of Tud interact directly in binding assay. As the PMRT5/MEP50 complex is involved in ribonucleoprotein complex assembly, we hypothesize that the Vls complex may play a similar function in assembling the nuage in nurse cells and the polar granules in the oocyte.     

Stage-specific chromosomal association of <Emphasis Type="Italic">Drosophila</Emphasis> dMBD2/3 during genome activation

The Drosophila gene dMBD2/3 encodes a protein with significant homologies to the mammalian methyl-DNA binding proteins MBD2 and MBD3. These proteins are essential components of chromatin complexes involved in epigenetic gene regulation. Because the available in vitro data on dMBD2/3 are conflicting we have started an in vivo characterization of dMBD2/3. We detected expression of two isoforms specifically during embryonic development. Staining of whole embryos combined with high-resolution confocal microscopy revealed a highly regulated spatial distribution. During the syncytial blastoderm stage, dMBD2/3 formed speckles that localized to the cytoplasm. Shortly after, during the cellular blastoderm stage, the protein entered the nucleus and formed bright foci that associated with DNA. This rapid transition coincided with the activation of the embryonic genome. A similar observation was made during activation of the spermatocyte genome as dMBD2/3 formed distinct foci associated with the activated Y chromosome. Our results indicate that dMBD2/3 forms specialized nuclear compartments to keep certain genes epigenetically silenced during genome activation.