Interdependence of the Ypt/RabGAP Gyp5p and Gyl1p for recruitment to the sites of polarized growth

Gyp5p and Gyl1p are two members of the Ypt/Rab guanosine triphosphatases-activating proteins involved in the control of polarized exocytosis in Saccharomyces cerevisiae . We had previously shown that Gyp5p and Gyl1p colocalize at the sites of polarized growth and belong to the same complex in subcellular fractions enriched in plasma membrane or secretory vesicles. Here, we investigate the interaction between Gyp5p and Gyl1p as well as the mechanism of their localization to the sites of polarized growth. We show that purified recombinant Gyp5p and Gyl1p interact directly in vitro . In vivo , both Gyp5p and Gyl1p are mutually required to concentrate at the sites of polarized growth. Moreover, the localization of Gyp5p and Gyl1p to the sites of polarized growth requires the formins Bni1p and Bnr1p and depends on actin cables. We show that, in a sec6-4 mutant, blocking secretion leads to coaccumulation of Gyp5p and Gyl1p, together with Sec4p. Electron microscopy experiments demonstrate that Gyp5p is associated with secretory vesicles. Altogether, our results indicate that both Gyp5p and Gyl1p access the sites of polarized growth by transport on secretory vesicles. Two polarisome components, Spa2p and Bud6p, are involved in maintaining Gyp5p and Gyl1p colocalized at the sites of polarized growth.     

Choanoflagellate lorica construction and assembly: the nudiform condition. II. Acanthoeca spectabilis Ellis

Acanthoeca spectabilis is one of the most common loricate choanoflagellates found in marine biofilms everywhere. However, it is special for two reasons; firstly, it is probably the most distinctive member of the small nudiform clade of loricate choanoflagellates. Secondly, the lorica chamber of Acanthoeca comprises a closely wound left-handed coil of costae that is unique amongst choanoflagellates. Mathematical analysis of the lorica chamber shows that the helical costae undergo two turns. This species, more than any, demonstrates that the helical coiling of costae can only be achieved by a rotational movement generated by the cell during lorica assembly. Comparison of the lorica morphology of Acanthoeca with that of the closely related genus Polyoeca indicates that the helical costae of Acanthoeca are probably homologous with the outer longitudinal costae of Polyoeca. This is unusual because helical costae are usually the innermost layer of costae. However, since there is no 'true' inner layer in the chamber of Acanthoeca the outer layer of costae are adjacent to the cell surface and therefore available for coiling. In contrast to tectiform choanoflagellates, which number more than one hundred species and inhabit a wide variety of microniches, the six known extant nudiform species must either represent a minor evolutionary development or be the remnants of a previously more extensive radiation.     

Synthesis of new sulfonate and phosphonate derivatives for cation-independent mannose 6-phosphate receptor targeting

The cation-independent mannose 6-phosphate receptor (CI-M6PR) is essential for the endocytosis of proteins bearing the mannose 6-phosphate (M6P) recognition marker. This study described the synthesis of M6P and M6S analogs presenting greater affinity for CI-M6PR than their natural compounds. Moreover, the finding of their lack of cytotoxicity for human cells and of their increased stability in human serum supports the high potential of these isosteric derivatives in therapies requiring CI-M6PR targeting.     

Gardens in urbanizing rural areas reveal an unexpected floral diversity related to housing density

The increasing urbanization of rural areas leads to a strong development of horticultural flora, which is the main source of alien and invasive plants. In order to assess the pool of cultivated species under different urbanization pressures, the diversity and distribution of horticultural flora were studied in 120 Mediterranean gardens belonging to three housing density types. The results showed a great richness and heterogeneity of this flora, and similarities in species composition between gardens of the same housing density types. Twenty-four percent of the cultivated species are well adapted to the Mediterranean climate, and 21 species known to be invasive on the French territory have emanated from gardens. Inventorying areas adjoining gardens would be useful in identifying escaped garden plants and to assess the associated risks for biological diversity. The results also suggested a detailed analysis of the influence of social, economic and regional factors on planting practices, in order to identify the drivers of these original floral patterns.     

Human mitochondrial TyrRS disobeys the tyrosine identity rules

Human tyrosyl-tRNA synthetase from mitochondria (mt-TyrRS) presents dual sequence features characteristic of eubacterial and archaeal TyrRSs, especially in the region containing amino acids recognizing the N1-N72 tyrosine identity pair. This would imply that human mt-TyrRS has lost the capacity to discriminate between the G1-C72 pair typical of eubacterial and mitochondrial tRNATyr and the reverse pair C1-G72 present in archaeal and eukaryal tRNATyr. This expectation was verified by a functional analysis of wild-type or mutated tRNATyr molecules, showing that mt-TyrRS aminoacylates with similar catalytic efficiency its cognate tRNATyr with G1-C72 and its mutated version with C1-G72. This provides the first example of a TyrRS lacking specificity toward N1-N72 and thus of a TyrRS disobeying the identity rules. Sequence comparisons of mt-TyrRSs across phylogeny suggest that the functional behavior of the human mt-TyrRS is conserved among all vertebrate mt-TyrRSs.     

Combinatorial approaches to functional models for galactose oxidase

The copper enzyme galactose oxidase (GOase, EC 1.1.3.9) catalyses the oxidation of D-galactose and other primary alcohols in air to the corresponding aldehydes and hydrogen peroxide. The current mechanistic hypothesis for this two-electron redox reaction involves a Cu(I)/Cu(II) couple and the reversible oxidation of a ligating phenolate (tyrosine residue of the Tyr272-Cys228 conjugate) to a phenoxyl radical. Our approaches to functional models for galactose oxidase comprise both the use of low-molecular-weight copper complexes of a Schiff-base and sulfonamide ligands, and the synthesis/screening of combinatorial libraries. With regard to the latter, we have synthesized (by the IRORI-directed synthesis approach) peptide libraries carrying either His or the redox-active amino acids Tyr, mod-Cys (a model for the Tyr272-Cys228 conjugate) or TOAC (a TEMPO-derived alpha-amino acid) at four variable positions. After incubation with copper ions, the catalytically active library members were identified by specially designed screening methods.     

tRNA-balanced expression of a eukaryal aminoacyl-tRNA synthetase by an mRNA-mediated pathway

Aminoacylation of transfer RNAs is a key step during translation. It is catalysed by the aminoacyl-tRNA synthetases (aaRSs) and requires the specific recognition of their cognate substrates, one or several tRNAs, ATP and the amino acid. Whereas the control of certain aaRS genes is well known in prokaryotes, little is known about the regulation of eukaryotic aaRS genes. Here, it is shown that expression of AspRS is regulated in yeast by a feedback mechanism that necessitates the binding of AspRS to its messenger RNA. This regulation leads to a synchronized expression of AspRS and tRNA(Asp). The correlation between AspRS expression and mRNA(AspRS) and tRNA(Asp) concentrations, as well as the presence of AspRS in the nucleus, suggests an original regulation mechanism. It is proposed that the surplus of AspRS, not sequestered by tRNA(Asp), is imported into the nucleus where it binds to mRNA(AspRS) and thus inhibits its accumulation.     

Differential sensitization of cancer cells to doxorubicin by DHA: a role for lipoperoxidation

Polyunsaturated fatty acids have been reported to enhance the cytotoxic activity of several anticancer drugs. In the present study, we observed that doxorubicin chemosensitization of breast cancer cell lines by docosahexaenoic acid (DHA, a long-chain omega-3 polyunsaturated fatty acid) was cell-line selective, affecting MDA-MB-231 and MCF-7 dox (a doxorubicin-resistant cell line) but not the parental MCF-7 cell line. DHA supplementation led to an increase in membrane phospholipid DHA level, but did not induce changes in intracellular [(14)C]doxorubicin accumulation. In MDA-MB-231, doxorubicin efficacy enhancement by DHA was linked to an increase in malondialdehyde level, a final product of lipid peroxidation. DHA elicited by itself a 3.7-fold malondialdehyde level increase, additive to that induced by doxorubicin. Addition of doxorubicin to DHA further increased the glutathione level, indicative of the generation of an oxidative stress. In contrast to MDA-MB-231, doxorubicin did not increase the malondialdehyde level in MCF-7, although DHA induced lipid peroxidation. Therefore in MCF-7, lipid peroxidation induced by DHA itself was not sufficient to trigger an oxidative stress and to subsequently increase sensitivity to doxorubicin. These data indicate that the differential effect of DHA among cells on drug toxicity results from a differential oxidative response to doxorubicin. Chemosensitization through fatty acids appears as a new promising adjuvant therapeutic paradigm, since omega-3 fatty acids are physiological molecules found in food and are nontoxic in vivo.     

Proteinase-3 induces procaspase-3 activation in the absence of apoptosis: potential role of this compartmentalized activation of membrane-associated procaspase-3 in neutrophils

In the present study, we provide evidence that procaspase-3 is a novel target of proteinase 3 (PR3) but not of human neutrophil elastase (HNE). Human mast cell clone 1 (HMC1) and rat basophilic leukemia (RBL) mast cell lines were transfected with PR3 or the inactive mutated PR3 (PR3S203A) or HNE cDNA. In both RBL/PR3 and HMC1/PR3, a constitutive activity of caspase-3 was measured with DEVD substrate, due to the direct processing of procaspase-3 by PR3. No caspase-3 activation was observed in cells transfected with the inactive PR3 mutant or HNE. Despite the high caspase-3 activity in RBL/PR3, no apoptosis was detected as demonstrated by an absence of 1) phosphatidylserine externalization, 2) mitochondria cytochrome c release, 3) upstream caspase-8 or caspase-9 activation, or 4) DNA fragmentation. In vitro, purified PR3 cleaved procaspase-3 into an active 22-kDa fragment. In neutrophils, the 22-kDa caspase-3 activation fragment was present only in resting neutrophils but was absent after apoptosis. The 22 kDa fragment was specific of myeloid cells because it was absent from resting lymphocytes. This 22-kDa fragment was not present when neutrophils were treated with pefabloc, an inhibitor of serine proteinase. Like in HMC1/PR3, the 22-kDa caspase-3 fragment was restricted to the plasma membrane compartment. Double immunofluorescence labeling after streptolysin-O permeabilization further showed that PR3 and procaspase-3 could colocalize in an extragranular compartment. In conclusion, our results strongly suggest that compartmentalized PR3-induced caspase-3 activation might play specific functions in neutrophil survival.