Structural and biochemical characterization of the Importin-beta.Ran.GTP.RanBD1 complex.

Here we present the crystal structure of Importin-beta(1-462).Ran.GTP.RanBD1DeltaN as solved by molecular replacement. HPLC dissociation measurements on this complex show, that the N-terminus of RanBD may be involved in the release of the hydrolysis- and dissociation-block of Ran by Transportin/Importin-beta. We could identify a pair of amino acids which - upon mutation - weaken the interaction between Ran and Importin-beta specifically to allow dissociation without RanBD. These findings support the hypothesis that a ternary complex of Importin-beta.Ran.GTP.RanBD exists in the final step of the export of Importin-beta from the nucleus and that interaction of the N-terminus of RanBD with Ran plays a crucial role in disassembly of this complex.

Comparative proteome analysis of Staphylococcus aureus biofilm and planktonic cells and correlation with transcriptome profiling.

Pathogenic staphylococci can form biofilms in which they show a higher resistance to antibiotics and the immune defense system than their planktonic counterparts, which suggests that the cells in a biofilm have an altered metabolic activity. Here, 2-D PAGE was used to identify secreted, cell wall-associated and cytoplasmic proteins expressed in Staphylococcus aureus after 8 and 48 h of growth. The proteins were separated at pH ranges of 4-7 or 6-11. The protein patterns revealed significant differences in 427 protein spots; from these, 258 non-redundant proteins were identified using ESI-MS/MS. Biofilm cells expressed higher levels of proteins associated with cell attachment and peptidoglycan synthesis, and in particular fibrinogen-binding proteins. Enzymes involved in pyruvate and formate metabolism were upregulated. Furthermore, biofilm cells expressed more staphylococcal accessory regulator A protein (SarA), which corroborates the positive effect of SarA on the expression of the intercellular adhesion operon ica and biofilm growth. In contrast, proteins, such as proteases and particularly immunodominant antigen A (IsaA) and staphylococcal secretory antigen (SsaA), were found in lower amounts. The RNA expression profiling largely supports the proteomic data. The results were mapped onto KEGG pathways.