Phylogenetic Analysis of Components of the Eukaryotic Vesicle Transport System Reveals a Common Origin of Adaptor Protein Complexes 1, 2, and 3 and the F Subcomplex of the Coatomer COPI

Eukaryotic vesicular transport requires the recognition of membranes through specific protein complexes. The heterotetrameric adaptor protein complexes 1, 2, and 3 (AP1/2/3) are composed of two large, one small, and one medium adaptin subunit. We isolated and characterized the cDNA for Arabidopsisγ-adaptin and performed a phylogenetic analysis of all adaptin subunits (proteins) in the context of all known homologous proteins. This analysis revealed (i) that the large subunits of AP1/2/3 are homologous and (ii) two subunits of the heptameric coatomer I (COPI) complex belong to this gene family. In addition, all small subunits and the aminoterminal domain of the medium subunits of the heterotetramers are homologous to each other; this also holds for two corresponding subunits of the COPI complex. AP1/2/3 and a substructure (heterotetrameric, F-COPI subcomplex) of the heptameric COPI had a common ancestral complex (called pre-F-COPI). Since all large and all small/medium subunits share sequence similarity, the ancestor of this complex is inferred to have been a heterodimer composed of one large and one small subunit. The situation encountered today is the result of successive rounds of coordinated gene duplications of both the large and the small/medium subunits, with F-COPI being the first that separated from the ancestral pre-F-COPI. Received: 1 October 1998 / Accepted: 4 January 1999     

Molybdenum co-factor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two-domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins

The molybdenum co-factor (Moco) is an essential part of all eukaryotic molybdoenzymes. It is a molybdopterin and reveals the same principal structure in eubacteria, archaebacteria and eukaryotes. This paper reports the isolation of cnx1 , a cDNA clone of Arabidopsis thaliana which complements the Escherichia coli Moco mutant mogA . The mapping data of this cDNA correlate well with the mapping position of the A. thaliana molybdenum cofactor locus chl6 . As mutants in chl6 are known to be repairable by high concentrations of molybdate, the defective gene is very likely to be involved in the last step of Moco biosynthesis, that is, the insertion of molybdenum into molybdopterin. The protein encoded by cnx1 shows a two-domain structure: the N-terminal domain is homologous to the E. coli Moco protein MoeA, the C-terminal domain is homologous to the E. coli Moco proteins MoaB and MogA, respectively. These homologies show that part of the prokaryotic Moco biosynthetic pathway accomplished by monofunctional proteins in E. coli , is performed by a single multifunctional protein in eukaryotes. In addition Cnx1 is homologous to the eukaryotic proteins Gephyrin, a rat neuroprotein, and Cinnamon, a Drosophila protein with a function in Moco biosynthesis. These proteins also show a two-domain structure but the order of the domains is inversed as compared with Cnx1. Southern analysis indicates the existence of at least one further member, in addition to the cnx1 gene, of this novel gene family in the Arabidopsis genome.     

Simulated hypogravity and proline incorporation into salt-extractable macromolecules from cell walls

Proline [U-¹⁴C] was fed to shoots of intact Tagetes patula grown normally, on horizontal clinostats, or on vertical clinostats rotating at 15 rev/hr. After various periods the incorporation of ¹⁴C into salt-extractable material from the cell walls of stems, petioles, leaves and flowers was determined. The cell walls of the gravity-compensated plants (grown on horizontal clinostats) has the highest amount of salt-extractable radioactivity. A 2- to 9-fold increase was observed in comparison to either the normal or vertical clinostat plant controls. Some physico-chemical properties of the salt-extractable fraction suggest that it consists of highly charged, low MW entities, possibly short chain peptides. On acid hydrolysis this material yields radioactive aspartic acid, glutamic acid and proline. The presence of labelled hydroxyproline is suggested. After acid hydrolysis of the cell walls of leaves, it was found that ca 4 times the amount of ¹⁴C was incorporated in the hypogravity-grown plant compared to the controls. It appears likely that extensibility changes in tissues under simulated hypogravity required additional cell wall protein.     

New GATEWAY vectors for High Throughput Analyses of Protein-Protein Interactions by Bimolecular Fluorescence Complementation

Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation （BiFC） is a suitable technique to investigate the formation of protein complexes and the localization of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N-or C-terminal sub-fragment of S（CFP）3A or Venus, respectively, and enable the performance of multicolor BiFC （mcBiFC）. We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.     

Kairomonal response of the parasitoid Anagyrus spec. nov. near pseudococci to the sex pheromone of the vine mealybug

The occurrence of a kairomonal response of the parasitoid Anagyrus spec. nov. near pseudococci (Hymenoptera: Encyrtidae) to (+)‐(1R,3R)‐cis‐2,2‐dimethyl‐3‐isopropenyl‐cyclobutanemethanol acetate (PcA, namely, planococcyl acetate) and (S)‐(+)‐lavandulyl senecioate (LS), the respective female sex pheromones of its hosts, the citrus mealybug, Planococcus citri (Risso) and the vine mealybug, Planococcus ficus (Signoret) (Homoptera: Pseudococcidae) was investigated. Attraction to the pheromones was tested by employing pheromone traps in field trials and by static air olfactometer bioassays in the laboratory. Female wasps showed a significant response to LS, in both field and olfactometer experiments. No significant response was registered to the sex pheromone of P. citri. Despite the similarity between the structures of LS and its analogue (S)‐(+)‐lavandulyl isovalerate (LI), no significant response to the latter compound was observed. It seems that differences between the structures of the carboxylate moiety of the respective molecules (LS and LI) markedly affect the kairomonal attractiveness to the parasitoid. The kairomonal response of Anagyrus spec. nov. near pseudococci was neither influenced by the host habitat nor by the host species on which it developed. This suggested innate behaviour of Anagyrus spec. nov. near pseudococci, possibly derived from evolutionary relationships between the parasitoid and P. ficus. The practical implications of the results are discussed.     

Tobacco plants that lack expression of functional nitrate reductase in roots show changes in growth rates and metabolite accumulation.

When tobacco is provided with a high nitrate supply, only a small amount of the nitrate taken up by the roots is immediately assimilated inside the roots, while the majority is transported to the leaves where it is reduced to ammonium. To elucidate the importance of root nitrate assimilation, tobacco plants have been engineered that showed no detectable nitrate reductase activity in the roots. These plants expressed the nitrate reductase structural gene nia2 under control of the leaf-specific potato promoter ST-LS1 in the nitrate reductase-mutant Nia30 of Nicotiana tabacum. Homozygous T2-transformants grown in sand or hydroponics with 5.1 mM nitrate had approximately 55-70% of wild-type nitrate reductase acivity in leaves, but lacked nitrate reductase acivity in roots. These plants showed a retarded growth as compared with wild-type plants. The activation state of nitrate reductase was unchanged; however, diurnal variation of nitrate reductase acivity was not as pronounced as in wild-type plants. The transformants had higher levels of nitrate in the leaves and reduced amounts of glutamine both in leaves and roots, while roots showed higher levels of hexoses (3-fold) and sucrose (10-fold). It may be concluded that the loss of nitrate reductase acivity in the roots changes the allocation of reduced nitrogen compounds and sugars in the plant. These plants will be a useful tool for laboratories studying nitrate assimilation and its interactions with carbon metabolism.     

Synthesis and evaluation of 1,4,5,6-tetrahydropyridazine derivatives as influenza neuraminidase inhibitors.

1,4,5,6-Tetrahydropyridazine derivative 15 and its C-5 epimer 19, which possessed side chains similar to GS4071, were synthesized via a hetero Diels-Alder reaction, and evaluated as influenza neuraminidase inhibitors. Compounds 15 and 19 exhibited a microM range of influenza neuraminidase inhibitory activity.