Investigating nucleo-cytoplasmic shuttling of the human DEAD-box helicase DDX3

The human DEAD-box helicase DDX3 is a multi-functional protein involved in the regulation of gene expression and additional non-conventional roles as signalling adaptor molecule that are independent of its enzymatic RNA remodeling activity. It is a nucleo-cytoplasmic shuttling protein and it has previously been suggested that dysregulation of its subcellular localization could contribute to tumourigenesis. Indeed, both tumour suppressor and oncogenic functions have been attributed to DDX3. In this study, we investigated the regulation of DDX3’s nucleocytoplasmic shuttling. We confirmed that an N-terminal conserved Nuclear Export Signal (NES) is required for export of human DDX3 from the nucleus, and identified three regions within DDX3 that can independently facilitate its nuclear import. We also aimed to identify conditions that alter DDX3’s subcellular localisation. Viral infection, cytokine treatment and DNA damage only induced minor changes in DDX3’s subcellular distribution as determined by High Content Analysis. However, DDX3’s nuclear localization increased in early mitotic cells (during prophase) concomitant with an increase in DDX3 expression levels. Our results are likely to have implications for the proposed use of (nuclear) DDX3 as a prognostic biomarker in cancer.     

Interaction of the nuclear localizing cytolytic granule serine protease granzyme B with importin alpha or beta: modulation by the serpin inhibitor PI-9

Conditional on perforin-dependent delivery to the nucleus of target cells, the cytolytic granule serine protease granzyme B (GrB) plays a central role in eliciting the nuclear events of apoptosis, as shown by the fact that reducing GrB nuclear entry prevents nuclear apoptosis. Apart from a requirement for cytosolic factors and lack of dependence on the guanine-nucleotide-binding protein Ran, little is known regarding the nuclear import pathway of GrB. In this study we use quantitative yeast two-hybrid and direct binding assays to show that GrB can be recognized independently by either of the nuclear import receptor family members importin (IMP) alpha and beta1, but that these proteins either alone or in combination cannot replace exogenous cytosol to reconstitute GrB nuclear import in vitro. Whereas antibodies to IMP(alpha) inhibit transport, indicating that IMP(alpha) is required for GrB nuclear import, those to IMP(beta) enhance transport, implying that IMP(beta) inhibits GrB nuclear import; consistent with this, the addition of recombinant IMP(beta) but not IMP(alpha) reduces maximal nuclear accumulation in the presence of cytosol. Intriguingly, complexation of GrB with its specific serpin inhibitor PI-9 was found to prevent recognition by IMP(beta) but not by IMP(alpha), and eliminate the apparent requirement for IMP(alpha) for nuclear import. We conclude that GrB nuclear import exhibits complex regulation by IMPs; that heterodimerization with PI-9 can modulate the interaction has implications for protection against apoptosis.     

Homologous protein import machineries in chloroplasts and cyanelles

The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria, especially in the presence of a peptidoglycan wall between the inner and outer envelope membranes. However, it is now clear that cyanelles are in fact primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario, cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high gene content of rhodoplasts and the peptidoglycan wall of cyanelles. This means that the import apparatuses of all primary plastids, i.e. those from glaucocystophytes, red algae, green algae and higher plants, should be homologous. If this is the case, then transit sequences should be similar and heterologous import experiments feasible. Thus far, heterologous in vitro import has been shown in one direction only: precursors from C. paradoxa were imported into isolated pea or spinach chloroplasts. Cyanelle transit sequences differ from chloroplast stroma targeting peptides in containing in their N-terminal domain an invariant phenylalanine residue which is shown here to be crucial for import. In addition, we now demonstrate that heterologous precursors are readily imported into isolated cyanelles, provided that the essential phenylalanine residue is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor/channel showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved, explaining the efficient heterologous import of native precursors from C. paradoxa.     

Mitochondrial targeting signals and mature peptides of 3-methylcrotonyl-CoA carboxylase

Inherited deficiency of 3-methylcrotonyl-CoA carboxylase (MCC), an enzyme of leucine degradation, is an organic acidemia detectable by expanded newborn screening with a variable phenotype that ranges from asymptomatic to death in infancy. Here, we show that the two subunits of the enzyme (MCCalpha; MCCbeta) are imported into the mitochondrial matrix by the classical pathway involving cleavable amino-terminal targeting presequences. We identified the cleavage sites (Tyr41/Thr42 and Ala22/Tyr23 for MCCalpha and MCCbeta, respectively) of the targeting signals and the amino-termini of the mature polypeptides of MCC and propionyl-CoA carboxylase, a mitochondrial paralog. The amino-termini containing 39 (MCCalpha) or 20 amino acids (MCCbeta) were both necessary and sufficient for targeting. Structural requirements for mitochondrial import were defined by site-directed mutagenesis. Our studies provide the prerequisite to understand the impact of specific mutations on the clinical phenotype of MCC deficiency.     

Translocation biosensors to study signal-specific nucleo-cytoplasmic transport, protease activity and protein-protein interactions

Regulated nucleo-cytoplasmic transport is crucial for cellular homeostasis and relies on protein interaction networks. In addition, the spatial division into the nucleus and the cytoplasm marks two intracellular compartments that can easily be distinguished by microscopy. Consequently, combining the rules for regulated nucleo-cytoplasmic transport with autofluorescent proteins, we developed novel cellular biosensors composed of glutathione S-transferase, mutants of green fluorescent protein and rational combinations of nuclear import and export signals. Addition of regulatory sequences resulted in three classes of biosensors applicable for the identification of signal-specific nuclear export and import inhibitors, small molecules that interfere with protease activity and compounds that prevent specific protein-protein interactions in living cells. As a unique feature, our system exploits nuclear accumulation of the cytoplasmic biosensors as the reliable readout for all assays. Efficacy of the biosensors was systematically investigated and also demonstrated by using a fully automated platform for high throughput screening (HTS) microscopy and assay analysis. The introduced modular biosensors not only have the potential to further dissect nucleo-cytoplasmic transport pathways but also to be employed in numerous screening applications for the early stage evaluation of potential drug candidates.     

Nur77 nuclear import and its NBRE-binding activity in thymic lymphoma cells are regulated by different mechanisms sensitive to FK506 or HA1004

Thymic lymphoma cells restore their sensitivity to ionomycin-induced apoptosis when treated with FK506 or HA1004. In apoptosis-resistant cells, ionomycin-induced Nur77 strongly binds DNA during the first 2 h of response, in contrast to lymphoma cells treated with ionomycin together with FK506 or HA1004, which undergo massive apoptosis. We show that Nur77 could discriminate between calcium signals sensitive to FK506 and those sensitive to HA1004, as the inhibitors differentially regulate the kinetics of Nur77 nuclear import, and FK506, unlike HA1004, inhibits Nur77 DNA-binding activity. In the presence of HA1004, NBRE binding by Nur77 protein increases with time (6 h vs 2 h), whereas the final outcome of both inhibitors is apoptosis of thymic lymphoma cells.     

Importin alpha/beta-mediated nuclear protein import is regulated in a cell cycle-dependent manner

Functional nuclear proteins are selectively imported into the nucleus by transport factors such as importins alpha and beta. The relationship between the efficiency of nuclear protein import and the cell cycle was measured using specific import substrates for the importin alpha/beta-mediated pathway. After the microinjection of SV40 T antigen nuclear localization signal (NLS)-containing substrates into the cytoplasm of synchronized culture cells at a certain phase of the cell cycle, the nuclear import of the substrates was measured kinetically. Cell cycle-dependent change in import efficiency, but not capacity, was found. That is, import efficiency was found low in the early S, G2/M, and M/G1 phases compared with other phases. In addition, we found that the extent of co-imunoprecipitation of importin alpha with importin beta from cell extracts was strongly associated with import efficiency. These results indicate that the importin alpha/beta-mediated nuclear import machinery is regulated in a cell cycle-dependent manner through the modulation of interaction modes between importins alpha and beta.     

Two novel proteins in the mitochondrial outer membrane mediate beta-barrel protein assembly

Mitochondrial outer and inner membranes contain translocators that achieve protein translocation across and/or insertion into the membranes. Recent evidence has shown that mitochondrial beta-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex. Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial beta-barrel proteins, Tom40, and/or porin in the outer membrane. Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the beta-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of beta-barrel proteins in the outer membrane.