A Mutation Associated with CMT2A Neuropathy Causes Defects in Fzo1 GTP Hydrolysis,Ubiquitylation, and Protein Turnover

Charcot-Marie-Tooth disease type 2A (CMT2A) is caused by mutations in the gene MFN2 and is one of the most common inherited peripheral neuropathies. Mfn2 is one of two mammalian mitofusin GTPases that promote mitochondrial fusion and maintain organelle integrity. It is not known how mitofusin mutations cause axonal degeneration and CMT2A disease. We used the conserved yeast mitofusin FZO1 to study the molecular consequences of CMT2A mutations on Fzo1 function in vivo and in vitro. One mutation (analogous to the CMT2A I213T substitution in the GTPase domain of Mfn2) not only abolishes GTP hydrolysis and mitochondrial membrane fusion but also reduces Mdm30-mediated ubiquitylation and degradation of the mutant protein. Importantly, complexes of wild type and the mutant Fzo1 protein are GTPase active and restore ubiquitylation and degradation of the latter. These studies identify diverse and unexpected effects of CMT2A mutations, including a possible role for mitofusin ubiquitylation and degradation in CMT2A pathogenesis, and provide evidence for a novel link between Fzo1 GTP hydrolysis, ubiquitylation, and mitochondrial fusion.     

Over-expression of an FT-homologous gene of apple induces early flowering in annual and perennial plants

The protein encoded by the FLOWERING LOCUS T (FT) gene from Arabidopsis thaliana seems to be the long-searched florigen, and over-expression of FT orthologues resulted in accelerated flower development in annual and perennial plants. In the present study, we isolated two allelic mRNA sequences of an FT-homologous gene from apple, which was designated as MdFT1. Using a SSR motif this gene was mapped on LG 12 of apple. Over-expression of MdFT1 in Arabidopsis and the commercially important tree species poplar and apple itself using the CaMV 35S or the Arabidopsis Suc2 promoter resulted in significant accelerated flowering compared with wild-type plants. Transgenic T₀ plants of Arabidopsis flowered 4–6 days on average earlier than wild-type Arabidopsis under LD conditions. Under short-day conditions Suc2::MdFT1 plants of the T₁-generation flowered after 66 ± 18 days, while wild-type plants flowered about 22 days later. All transgenic Arabidopsis plants showed a normal habit except for the early flowering phenotype. Early flowering was detected 6–10 months after transformation in transgenic polar clones containing MdFT1 driven by the CaMV 35S, whereas plants of the transgenic apple clone T780 set up its first flowers during in vitro cultivation. Based on our results we conclude that MdFT1 is responsible for inducing flowering and that the function of the apple FT1 gene is conserved in annual herbaceous species as well as perennial woody species. Furthermore, we discuss the role of MdFT1 in flower development with regard to the findings of genetic studies on apple.     

Identification of genes differentially expressed during interaction of resistant and susceptible apple cultivars (Malus × domestica) with Erwinia amylovora

Background  

The necrogenic enterobacterium, Erwinia amylovora is the causal agent of the fire blight (FB) disease in many Rosaceaespecies, including apple and pear. During the infection process, the bacteria induce an oxidative stress response with kinetics similar to those induced in an incompatible bacteria-plant interaction. No resistance mechanism to E. amylovora in host plants has yet been characterized, recent work has identified some molecular events which occur in resistant and/or susceptible host interaction with E. amylovora: In order to understand the mechanisms that characterize responses to FB, differentially expressed genes were identified by cDNA-AFLP analysis in resistant and susceptible apple genotypes after inoculation with E. amylovora.     

Bioinformatics and functional analysis of an Entamoeba histolytica mannosyltransferase necessary for parasite complement resistance and hepatical infection

The glycosylphosphatidylinositol (GPI) moiety is one of the ways by which many cell surface proteins, such as Gal/GalNAc lectin and proteophosphoglycans (PPGs) attach to the surface of Entamoeba histolytica, the agent of human amoebiasis. It is believed that these GPI-anchored molecules are involved in parasite adhesion to cells, mucus and the extracellular matrix. We identified an E. histolytica homolog of PIG-M, which is a mannosyltransferase required for synthesis of GPI. The sequence and structural analysis led to the conclusion that EhPIG-M1 is composed of one signal peptide and 11 transmembrane domains with two large intra luminal loops, one of which contains the DXD motif, involved in the enzymatic catalysis and conserved in most glycosyltransferases. Expressing a fragment of the EhPIG-M1 encoding gene in antisense orientation generated parasite lines diminished in EhPIG-M1 levels; these lines displayed reduced GPI production, were highly sensitive to complement and were dramatically inhibited for amoebic abscess formation. The data suggest a role for GPI surface anchored molecules in the survival of E. histolytica during pathogenesis.     

Effect of primary mild stresses on resilience and resistance of the nitrate reducer community to a subsequent severe stress

The factors regulating soil microbial stability (e.g. resistance and resilience) are poorly understood, even though microorganisms are essential for ecosystem functioning. In this study, we tested whether a functional microbial community subjected to different primary mild stresses was equally resistant or resilient to a subsequent severe stress. The nitrate reducers were selected as model community and analysed in terms of nitrate reduction rates and genetic structure by narG PCR-restriction fragment length polymorphism fingerprinting. Heat, copper and atrazine were used as primary stresses and mercury at a high concentration as a severe stress. None of the primary stresses had any significant impact on the nitrate reducer community. Although primary stress with heat, copper or atrazine had no effect on the resilience of the nitrate reducer activity to mercury stress, pre-exposure to copper, another heavy metal, resulted in increased resilience. In contrast, the resistance of both structure and activity of the nitrate reducer community to severe mercury stress was not affected by any of the primary stresses tested. Our experiment suggests that the hypothetical effect of an initial stress on the response of a microbial community to an additional stress is complex and may depend on the relatedness of the two consecutive stresses and the development of positive cotolerance.     

Non-discriminating and discriminating aspartyl-tRNA synthetases differ in the anticodon-binding domain

In most organisms, tRNA aminoacylation is ensured by 20 aminoacyl-tRNA synthetases (aaRSs). In eubacteria, however, synthetases can be duplicated as in Thermus thermophilus, which contains two distinct AspRSs. While AspRS-1 is specific, AspRS-2 is non-discriminating and aspartylates tRNA(Asp) and tRNA(Asn). The structure at 2.3 A resolution of AspRS-2, the first of a non-discriminating synthetase, was solved. It differs from that of AspRS-1 but has resemblance to that of discriminating and archaeal AspRS from Pyrococcus kodakaraensis. The protein presents non-conventional features in its OB-fold anticodon-binding domain, namely the absence of a helix inserted between two beta-strands of this fold and a peculiar L1 loop differing from the large loops known to interact with tRNA(Asp) identity determinant C36 in conventional AspRSs. In AspRS-2, this loop is small and structurally homologous to that in AsnRSs, including conservation of a proline. In discriminating Pyrococcus AspRS, the L1 loop, although small, lacks this proline and is not superimposable with that of AspRS-2 or AsnRS. Its particular status is demonstrated by a loop-exchange experiment that renders the Pyrococcus AspRS non-discriminating.     

Inhibitor of apoptosis 2 and TAK1-binding protein are components of the Drosophila Imd pathway

The Imd signaling cascade, similar to the mammalian TNF-receptor pathway, controls antimicrobial peptide expression in Drosophila. We performed a large-scale RNAi screen to identify novel components of the Imd pathway in Drosophila S2 cells. In all, 6713 dsRNAs from an S2 cell-derived cDNA library were analyzed for their effect on Attacin promoter activity in response to Escherichia coli. We identified seven gene products required for the Attacin response in vitro, including two novel Imd pathway components: inhibitor of apoptosis 2 (Iap2) and transforming growth factor-activated kinase 1 (TAK1)-binding protein (TAB). Iap2 is required for antimicrobial peptide response also by the fat body in vivo. Both these factors function downstream of Imd. Neither TAB nor Iap2 is required for Relish cleavage, but may be involved in Relish nuclear localization in vitro, suggesting a novel mode of regulation of the Imd pathway. Our results show that an RNAi-based approach is suitable to identify genes in conserved signaling cascades.