José M. Fernández-Ábalos

Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans

Green fluorescent protein (GFP) is a useful reporter to follow the in vivo behaviour of proteins, but the wild‐type gfp gene does not function in many organisms, including many plants and filamentous fungi. We show that codon‐modified forms of gfp, produced for use in plants, function effectively in Aspergillus nidulans both as gene expression reporters and as vital reporters for protein location. To demonstrate the use of these modified gfps as reporter genes we have used fluorescence to follow ethanol‐induced GFP expression from the alcA promoter. Translational fusions with the modified gfp were used to follow protein location in living cells; plant ER‐retention signals targeted GFP to the endoplasmic reticulum, whereas fusion to the GAL4 DNA‐binding domain targeted it to the nucleus. Nuclear‐targeted GFP allowed real‐time observation of nuclear movement and division. These modified gfp genes should provide useful markers to follow gene expression, organelle behaviour and protein trafficking in real time.

Dynamic distribution of BIMGPP1 in living hyphae of Aspergillus indicates a novel role in septum formation

Mutation of bimG, the major protein phosphatase 1 gene in Aspergillus nidulans, causes multiple cell cycle and hyphal growth defects that are associated with overphosphorylation of subcellular components. We have used functional translational fusions with the green fluorescent protein (GFP) to show that BIMG has at least four discrete locations within growing hyphae. Three of these locations, the hyphal tip, the spindle pole body and the nucleus, correlate with previously known requirements for bimGPP1 in mitosis and hyphal growth and are highly dynamic. BIMG‐GFP in the hyphal tip seemed to be associated with the plasma membrane and formed a collar of fluorescence within the apical dome. The distribution of nuclear BIMG‐GFP varied depending on nutritional conditions; on poor medium, it concentrated more in the nucleolus than in the nucleoplasm, whereas on rich medium, it was more evenly distributed between the two nuclear regions. The association of BIMG‐GFP with developing septa was transient, and we present evidence that BIMG phosphatase plays a direct role in septum formation, distinct from its role in mitosis. We conclude that, by being physically present at several sites, the BIMG phosphatase has roles in multiple cellular processes.

Post-translational processing of modular xylanases from Streptomyces is dependent on the carbohydrate-binding module

Xylanases are very often modular enzymes composed of one or more catalytic domains and carbohydrate-binding modules (CBMs) connected by a flexible linker region. Usually, when these proteins are processed they lose their carbohydrate-binding capacity. Here, the role of the linker regions and cellulose- or xylan-binding domains in the processing of Xys1L from Streptomyces halstedii JM8 and Xyl30L from Streptomyces avermitilis UAH30 was studied. Xys1 variants with different linker lengths were tested, these being unable to avoid protein processing. Moreover, several fusion proteins between the Xys1 and Xyl30 domains were obtained and their proteolytic stability was studied. We demonstrate that CBM processing takes place even in the complete absence of the linker sequence. We also show that the specific carbohydrate module determines this cleavage in the proteins studied.