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

Secretory bulk flow of soluble proteins is efficient and COPII dependent

COPII-coated vesicles, first identified in yeast and later characterized in mammalian cells, mediate protein export from the endoplasmic reticulum (ER) to the Golgi apparatus within the secretory pathway. In these organisms, the mechanism of vesicle formation is well understood, but the process of soluble cargo sorting has yet to be resolved. In plants, functional complements of the COPII-dependent protein traffic machinery were identified almost a decade ago, but the selectivity of the ER export process has been subject to considerable debate. To study the selectivity of COPII-dependent protein traffic in plants, we have developed an in vivo assay in which COPII vesicle transport is disrupted at two distinct steps in the pathway. First, overexpression of the Sari p-specific guanosine nucleotide exchange factor Sec12p was shown to result in the titration of the GTPase Sar1p, which is essential for COPII-coated vesicle formation. A second method to disrupt COPII transport at a later step in the pathway was based on coexpression of a dominant negative mutant of Sar1p (H74L), which is thought to interfere with the uncoating and subsequent membrane fusion of the vesicles because of the lack of GTPase activity. A quantitative assay to measure ER export under these conditions was achieved using the natural secretory protein barley a-amylase and a modified version carrying an ER retention motif. Most importantly, the manipulation of COPII transport in vivo using either of the two approaches allowed us to demonstrate that export of the ER resident protein calreticulin or the bulk flow marker phosphinothricin acetyl transferase is COPII dependent and occurs at a much higher rate than estimated previously. We also show that the instability of these proteins in post-ER compartments prevents the detection of the true rate of bulk flow using a standard secretion assay. The differences between the data on COPII transport obtained from these in vivo experiments and in vitro experiments conducted previously using yeast components are discussed

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