In Arabidopsis thaliana Substrate Recognition and Tissue- as Well as Plastid Type-Specific Expression Define the Roles of Distinct Small Subunits of Isopropylmalate Isomerase.

In Arabidopsis thaliana, the heterodimeric isopropylmalate isomerase (IPMI) is composed of a single large (IPMI LSU1) and one of three different small subunits (IPMI SSU1 to 3). The function of IPMI is defined by the small subunits. IPMI SSU1 is required for Leu biosynthesis and has previously also been proposed to be involved in the first cycle of Met chain elongation, the first phase of the synthesis of Met-derived glucosinolates. IPMI SSU2 and IPMI SSU3 participate in the Met chain elongation pathway. Here, we investigate the role of the three IPMI SSUs through the analysis of the role of the substrate recognition region spanning five amino acids on the substrate specificity of IPMI SSU1. Furthermore, we analyze in detail the expression pattern of fluorophore-tagged IPMI SSUs throughout plant development. Our study shows that the substrate recognition region that differs between IPMI SSU1 and the other two IMPI SSUs determines the substrate preference of IPMI. Expression of IPMI SSU1 is spatially separated from the expression of IPMI SSU2 and IPMI SSU3, and IPMI SSU1 is found in small plastids, whereas IMPI SSU2 and SSU3 are found in chloroplasts. Our data show a distinct role for IMPI SSU1 in Leu biosynthesis and for IMPI SSU2 and SSU3 in the Met chain elongation pathway.

DNA residence time is a regulatory factor of transcription repression.

Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation.

Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling.

The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca(2+). The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca(2+) and regulation of Ca(2+)-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca(2+) release from intracellular stores; (iii) Ca(2+) entry from the extracellular compartment; and (iv) nuclear translocation of the Ca(2+)-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca(2+) signaling.

The Arabidopsis CstF64-Like RSR1/ESP1 Protein Participates in Glucose Signaling and Flowering Time Control.

Mechanisms for sensing and regulating metabolic processes at the cellular level are critical for the general physiology and development of living organisms. In higher plants, sugar signaling is crucial for adequate regulation of carbon and energy metabolism and affects virtually every aspect of development. Although many genes are regulated by sugar levels, little is known on how sugar levels are measured by plants. Several components of the sugar signaling network have been unraveled and demonstrated to have extensive overlap with hormone signaling networks. Here we describe the reduced sugar response1-1 (rsr1-1) mutant as a new early flowering mutant that displays decreased sensitivity to abscisic acid. Both hexokinase1 (HXK1)-dependent and glucose phosphorylation-independent signaling is reduced in rsr1-1. Map-based identification of the affected locus demonstrated that rsr1-1 carries a premature stop codon in the gene for a CstF64-like putative RNA processing factor, ESP1, which is involved in mRNA 3'-end formation. The identification of RSR1/ESP1 as a nuclear protein with a potential threonine phosphorylation site may explain the impact of protein phosphorylation cascades on sugar-dependent signal transduction. Additionally, RSR1/ESP1 may be a crucial factor in linking sugar signaling to the control of flowering time.

RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J.

The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin-dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence-binding protein J (RBP-J)/CBF-1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP-J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP-J/CBF-1-interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP-J/CBF-1 from the nucleus. Functionally, we show that RITA can reverse a Notch-induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch-mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP-J/CBF-1, where its amounts are limiting.

The inability to disrupt the immunological synapse between infected human T cells and APCs distinguishes HIV-1 from most other primate lentiviruses.

Viruses that infect T cells, including those of the lentivirus genus, such as HIV-1, modulate the responsiveness of infected T cells to stimulation by interacting APCs in a manner that renders the T cells more permissive for viral replication. HIV-1 and other primate lentiviruses use their Nef proteins to manipulate the T cell/APC contact zone, the immunological synapse (IS). It is known that primate lentiviral Nef proteins differ substantially in their ability to modulate cell surface expression of the TCR-CD3 and CD28 receptors critical for the formation and function of the IS. However, the impact of these differences in Nef function on the interaction and communication between virally infected T cells and primary APCs has not been investigated. Here we have used primary human cells to show that Nef proteins encoded by HIV-2 and most SIVs, which downmodulate cell surface expression of TCR-CD3, disrupt formation of the IS between infected T cells and Ag-presenting macrophages or DCs. In contrast, nef alleles from HIV-1 and its simian precursor SIVcpz failed to suppress synapse formation and events downstream of TCR signaling. Our data suggest that most primate lentiviruses disrupt communication between virally infected CD4+ Th cells and APCs, whereas HIV-1 and its SIV precursor have largely lost this capability. The resulting differences in the levels of T cell activation and apoptosis may play a role in the pathogenesis of AIDS.