Energization of vacuolar transport in plant cells and its significance under stress.

The plant vacuole is of prime importance in buffering environmental perturbations and in coping with abiotic stress caused by, for example, drought, salinity, cold, or UV. The large volume, the efficient integration in anterograde and retrograde vesicular trafficking, and the dynamic equipment with tonoplast transporters enable the vacuole to fulfill indispensible functions in cell biology, for example, transient and permanent storage, detoxification, recycling, pH and redox homeostasis, cell expansion, biotic defence, and cell death. This review first focuses on endomembrane dynamics and then summarizes the functions, assembly, and regulation of secretory and vacuolar proton pumps: (i) the vacuolar H(+)-ATPase (V-ATPase) which represents a multimeric complex of approximately 800 kDa, (ii) the vacuolar H(+)-pyrophosphatase, and (iii) the plasma membrane H(+)-ATPase. These primary proton pumps regulate the cytosolic pH and provide the driving force for secondary active transport. Carriers and ion channels modulate the proton motif force and catalyze uptake and vacuolar compartmentation of solutes and deposition of xenobiotics or secondary compounds such as flavonoids. ABC-type transporters directly energized by MgATP complement the transport portfolio that realizes the multiple functions in stress tolerance of plants.

Elements of transcriptional machinery are compatible among plants and mammals.

In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-κB in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-κB such as its inhibitor IκB isoform α that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-κB in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-κB signaling pathways. The system successfully enabled the controlled manipulation of NF-κB activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e.g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway.

Multivalent binding of formin-binding protein 21 (FBP21)-tandem-WW domains fosters protein recognition in the pre-spliceosome.

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome.