cDNA cloning and functional expression of the α-d-galactose-binding lectin frutalin in escherichia coli

cDNA clones encoding frutalin, the α-d-galactose-binding lectin expressed in breadfruit seeds (Artocarpus incisa), were isolated and sequenced. The deduced amino acid sequences indicated that frutalin may be encoded by a family of genes. The NCBI database searches revealed that the frutalin sequence is highly homologous with jacalin and mornigaG sequences. Frutalin cDNA was re-amplified and cloned into the commercial expression vector pET-25b(+) for frutalin production in Escherichia coli. An experimental factorial design was employed to maximise the soluble expression of the recombinant lectin. The results indicated that temperature, time of induction, concentration of IPTG and the interaction between the concentration of IPTG and the time of induction had the most significant effects on the soluble expression level of recombinant frutalin. The optimal culture conditions were as follows: induction with 1 mM IPTG at 22°C for 20 h, yielding 16 mg/l of soluble recombinant frutalin. SDS-PAGE and Western blot analysis revealed that recombinant frutalin was successfully expressed by bacteria with the expected molecular weight (17 kDa). These analyses also showed that recombinant frutalin was mainly produced as insoluble protein. Recombinant frutalin produced by bacteria revealed agglutination properties and carbohydrate-binding specificity similar to the native breadfruit lectin.Fundação para a Ciência e a Tecnologia (FCT

The K/HDEL receptor does not recycle but instead acts as a Golgi-gatekeeper

Accurately measuring the ability of the K/HDEL receptor (ERD2) to retain the ER cargo Amy-HDEL has questioned earlier results on which the popular receptor recycling model is based upon. Here we demonstrate that ERD2 Golgi-retention, rather than fast ER export supports its function. Ligand-induced ERD2 redistribution is only observed when the C-terminus is masked or mutated, compromising the signal that prevents Golgi-to-ER transport of the receptor. Forcing COPI mediated retrograde transport destroys receptor function, but introducing ER-to-Golgi export or cis-Golgi retention signals re-activate ERD2 when its endogenous Golgi-retention signal is masked or deleted. We propose that ERD2 remains fixed as a Golgi gatekeeper, capturing K/HDEL proteins when they arrive and releasing them again into a subdomain for retrograde transport back to the ER. An in vivo ligand:receptor ratio far greater than 100 to 1 strongly supports this model, and the underlying mechanism appears to be extremely conserved across kingdoms

The AP-3 adaptor complex is required for vacuolar function in Arabidopsis

Subcellular trafficking is required for a multitude of functions in eukaryotic cells. It involves regulation of cargo sorting, vesicle formation, trafficking and fusion processes at multiple levels. Adaptor protein (AP) complexes are key regulators of cargo sorting into vesicles in yeast and mammals but their existence and function in plants have not been demonstrated. Here we report the identification of the protein-affected trafficking 4 (pat4) mutant defective in the putative d subunit of the AP-3 complex. pat4 and pat2, a mutant isolated from the same GFP imaging-based forward genetic screen that lacks a functional putative AP-3 ß, as well as dominant negative AP-3 µ transgenic lines display undistinguishable phenotypes characterized by largely normal morphology and development, but strong intracellular accumulation of membrane proteins in aberrant vacuolar structures. All mutants are defective in morphology and function of lytic and protein storage vacuoles (PSVs) but show normal sorting of reserve proteins to PSVs. Immunoprecipitation experiments and genetic studies revealed tight functional and physical associations of putative AP-3 ß and AP-3 d subunits. Furthermore, both proteins are closely linked with putative AP-3 µ and s subunits and several components of the clathrin and dynamin machineries. Taken together, these results demonstrate that AP complexes, similar to those in other eukaryotes, exist in plants, and that AP-3 plays a specific role in the regulation of biogenesis and function of vacuoles in plant cells