Transport of proteins to the nucleus through a nuclear envelope is controlled mostly via nuclear localization signal (NLS). Nuclear localization signal is rich in positively charged amino acids arginine and lysine. It was observed that activity of this NLS could be regulated through a phosphorylation of serine in its close proximity. Either a phosphorylation of serine or phosphomimetic changes of these "presequences" could represent an important mechanism regulating a localization of protein in cells in relation to a cellular activation. In our laboratory was identified protein - Fragile X mental retardation syndrome 1 neighbor (Fmr1nb), whose cellular localization could be driven by this posttranslational modification.

Dictyostelium is a model eukaryote for the study of a multitude of fundamental cellular processes as well as several human diseases. Despite its extensive study relatively little is known about its nucleolus. Only three nucleolar proteins have been identified. The nucleolus in Dictyostelium is different than that of other eukaryotes since it is neither bipartite nor tripartite, possessing no visible subcompartments at the ultrastructural level. Moreover, it exists as two to four patches adjacent to the inner nuclear envelope instead of within the nucleoplasm. The aim of this study was thus to identify and characterize novel nucleolar proteins in Dictyostelium in order to better understand the structure and function of its nucleolus. Previous work had shown that NumA1, a protein linked to cell cycle in Dictyostelium, localizes to similar intranuclear patches suggesting it may be nucleolar. NumA1-binding partners Ca2+-binding protein (CBP) 4a and puromycin-sensitive aminopeptidase A may therefore also reside in the nucleolus. Based on the function of a potential NumA1 homologue in other organisms, BRG1-associated factor 60a homologue Snf12 and checkpoint kinase 2 (Rad53 in yeast) homologue forkhead-associated kinase (Fhk) A were chosen as potential nucleolar proteins in Dictyostelium that may also be involved in cell cycle events. Using a diversity of approaches, this study found that NumA1, CBP4a, Snf12, and FhkA are nucleolar proteins in Dictyostelium while puromycin-sensitive aminopeptidase A is nucleoplasmic. Several nuclear localization signals (NLSs) were identified in these proteins some of which also act as nucleolar localization signals (NoLSs). These NLS/NoLSs (within NumA1 and Snf12) represent the first NoLSs and first NLS/NoLSs identified in Dictyostelium. Treatment with the rDNA transcription inhibitor AM-D led to the budding of nucleolar CBP4a, Snf12, and FhkA from the nucleus to the cytoplasm, a phenomenon not previously observed in any organism. This study also examined for the first time the redistribution of nucleolar proteins during mitosis, a time when the nucleolus disassembles into its component parts. The nuclear envelope was also shown to become permeable at this time. Finally, multiple nucleolar subcompartments were identified suggesting compartmentalization of different functions in the Dictyostelium nucleolus.

Dictyostelium is a model eukaryote for the study of a multitude of fundamental cellular processes as well as several human diseases. Despite its extensive study relatively little is known about its nucleolus. Only three nucleolar proteins have been identified. The nucleolus in Dictyostelium is different than that of other eukaryotes since it is neither bipartite nor tripartite, possessing no visible subcompartments at the ultrastructural level. Moreover, it exists as two to four patches adjacent to the inner nuclear envelope instead of within the nucleoplasm. The aim of this study was thus to identify and characterize novel nucleolar proteins in Dictyostelium in order to better understand the structure and function of its nucleolus. Previous work had shown that NumA1, a protein linked to cell cycle in Dictyostelium, localizes to similar intranuclear patches suggesting it may be nucleolar. NumA1-binding partners Ca2+-binding protein (CBP) 4a and puromycin-sensitive aminopeptidase A may therefore also reside in the nucleolus. Based on the function of a potential NumA1 homologue in other organisms, BRG1-associated factor 60a homologue Snf12 and checkpoint kinase 2 (Rad53 in yeast) homologue forkhead-associated kinase (Fhk) A were chosen as potential nucleolar proteins in Dictyostelium that may also be involved in cell cycle events. Using a diversity of approaches, this study found that NumA1, CBP4a, Snf12, and FhkA are nucleolar proteins in Dictyostelium while puromycin-sensitive aminopeptidase A is nucleoplasmic. Several nuclear localization signals (NLSs) were identified in these proteins some of which also act as nucleolar localization signals (NoLSs). These NLS/NoLSs (within NumA1 and Snf12) represent the first NoLSs and first NLS/NoLSs identified in Dictyostelium. Treatment with the rDNA transcription inhibitor AM-D led to the budding of nucleolar CBP4a, Snf12, and FhkA from the nucleus to the cytoplasm, a phenomenon not previously observed in any organism. This study also examined for the first time the redistribution of nucleolar proteins during mitosis, a time when the nucleolus disassembles into its component parts. The nuclear envelope was also shown to become permeable at this time. Finally, multiple nucleolar subcompartments were identified suggesting compartmentalization of different functions in the Dictyostelium nucleolus.

SUMO is a small ubiquitin-like modifier. The fluorescent fused SUMO (active for sumoylation) localized in the nucleus, while C-terminal truncated SUMO (inactive for sumoylation) diffused in the cytoplasm. Daxx is a SUMO target protein, locates predominantly in the nucleus. It has been identified as a component of the PODs. During extracellular stimulation, Daxx could be recruited to the cytoplasm with the existence of Ask1. Therefore, it is a shuttle protein. Daxx should contain nuclear localization signal (NLS) and nuclear export signal (NES) motifs. To identify the NES and NLS motifs on Daxx, Daxx were truncated into four segments. Several amino acids on the predicted NES and NLS motifs were mutated. Our results showed that the truncated Daxx fragments D1 (containing NES) and D4 (containing NLS2) could be translocated into nucleus independently. However, either NES or NLS2 mutants disrupted their translocation into nucleus. It indicated that both NES and NLS2 motif of Daxx were involved in the nuclear transport. Nevertheless the co-transfection of SUMOs and Daxx showed that the interactions between SUMO active form and Daxx mutants and between inactive SUMO and Daxx wild type rescued the nuclear transport function of Daxx mutants and inactive SUMO. Therefore, SUMO may play a role in the nuclear transport of Daxx by either sumoylation or interaction with Daxx in cytoplasm, and Daxx may recruit inactive SUMOs into nucleus by interaction.

The BEACH family proteins are conserved in all eukaryotes and are important for membrane trafficking. Defects in specific BEACH proteins have been linked to severe human disorders. For example, loss of human LYST protein causes the Chediak-Higashi Syndrome (CHS), a lethal disorder that affects lysosomal function. I postulate that different classes of BEACH proteins contribute distinct cellular functions in specific organelles. Based on this functional specificity, I hypothesize that each class of BEACH proteins must localize to their respective organelle where they are known to function. Unfortunately, the localization of most mammalian BEACH proteins is not known and no localization signal has been determined for any BEACH protein. Previous work showed that the Dictyostelium LvsA protein localizes and functions on the contractile vacuole while LvsB localizes and functions on the lysosome. Thus, Dictyostelium is a good model system to understand how BEACH proteins localize to specific organelles. Using a knock-in approach and parasexual techniques, I generated a collection of LvsA truncation mutants tagged with GFP and expressed them in different cell lines. Hence I can test the ability of each mutant protein to localize on contractile vacuoles by fluorescence microscopy. I show here that LvsA requires two regions to localize on the contractile vacuole: the N-terminal 140-457 amino acids and the BEACH. In addition, the expression of the N-terminal 651 amino acids of LvsA causes a dominant negative effect suggesting a possible functional protein-protein interaction within this region. Furthermore, sequence alignment analysis shows that this N-terminal region is only conserved within each class of BEACH family proteins. This finding supports our hypothesis and suggests that diversity within the N-terminal region may be due to the specialized targeting sequences of each class of BEACH proteins. Taken together, these results suggest that the conserved BEACH domain may serve as a general localization sequence while the N-terminal segment is responsible for targeting these proteins to their distinct organelles. This study will facilitate the identification of localization signals in other BEACH proteins which is important to dissect the molecular mechanism of their respective functions. / text

Transport of proteins to the nucleus through a nuclear envelope is controlled mostly via nuclear localization signal (NLS). Nuclear localization signal is rich in positively charged amino acids arginine and lysine. It was observed that activity of this NLS could be regulated through a phosphorylation of serine in its close proximity. Either a phosphorylation of serine or phosphomimetic changes of these "presequences" could represent an important mechanism regulating a localization of protein in cells in relation to a cellular activation. In our laboratory was identified protein - Fragile X mental retardation syndrome 1 neighbor (Fmr1nb), whose cellular localization could be driven by this posttranslational modification.

Non-claret disjunctional (Ncd) is a kinesin-14 microtubule motor protein involved in the assembly and stability of meiotic and mitotic spindles in Drosophila oocytes and early embryos, respectively. Ncd functions by cross-linking microtubules through the tail and motor domains. It was originally believed that the role of the Ncd tail domain was to only statically bind microtubules. However, the Ncd tail domain has recently been shown to have properties that stabilize and bundle microtubules, and contribute to the overall motility of the Ncd protein. Continued characterization of the Ncd tail domain is essential to understanding the complete role of Ncd in cell division. This work explored the regulatory function and microtubule binding properties of the Ncd tail domain. Ncd activity is regulated during interphase by nuclear sequestration. GFP-Ncd fusion proteins, containing full length Ncd, individual Ncd domains, or combinations of Ncd domains, were used to identify the presence of a nuclear localization signal (NLS) in the Ncd polypeptide. The nuclear localization of only the GFP fusion proteins containing the Ncd tail sequence indicates that the NLS is contained within the tail domain. Subsequent, experiments performed with GFP fusion proteins containing segments of the tail domain indicate that essential NLS amino acid segments may span the length of the tail domain. Attempts to characterize the microtubule binding properties of the Ncd tail domain, using bacterially expressed MBP-Ncd tail-stalk, were unsuccessful. MBP-Ncd tail-stalk proteins aggregated under binding assay conditions, preventing an accurate determination of the stoichiometric binding relationship between Ncd and the tubulin dimer. / Master of Science

mRNA localisation is a widely used mechanism for achieving temporal-spatial restriction of protein expression and is essential during development to establish cell polarity. mRNA localisation is particularly well studied in the Drosophila egg chamber where gurken mRNA is localised to the dorsal-anterior corner of the oocyte in a Dynein-dependent process that establishes the anterior-posterior and dorsal-ventral axes of the future embryo. An RNA stem-loop called the gurken localisation signal is necessary and sufficient to drive gurken localisation through interactions with a specific complement of protein factors. However, the exact RNA sequence and structural features required to promote each stage of gurken localisation are unknown. Using a live-cell injection assay I have dissected regions of the mRNA signal that are responsible for driving gurken transport and anchoring through their association with Egalitarian, Me31B and Squid proteins. I show the structure of an AU-rich stem and a purine stack are essential for gurken transport, and demonstrate that the size of the internal loop between these stems is important. These features of the localisation signal are essential for recruitment of Egalitarian, which links the mRNA to the Dynein transport machinery. I also show that these mRNA sequence and structural elements are present in several other Dynein-transported mRNAs. The bulge at the distal end of the gurken localisation signal is important for anchoring grk at the dorsal-anterior of the oocyte, possibly through Squid binding, and the proximal third of the signal is essential for recruitment of the translation component Me31B. These studies indicate that the role of the gurken localisation signal in controlling gurken transport, anchoring and translation can be mapped to distinct regions of the signal and provide insights into how the signal carries out these numerous functions at a molecular level. Determining the molecular interactions involved in mRNA localisation improves our understanding of how specificity is generated to direct different mRNAs to distinct regions of the cell to restrict protein expression.

Php4 is a subunit of the CCAAT-binding protein complex that has a negative regulatory function during iron deprivation in the fission yeast Schizosaccharomyces pombe. Under low iron conditions, Php4 fosters the repression of genes encoding iron using proteins. In contrast, under iron-replete conditions, Php4 is inactivated at both transcriptional and post-transcriptional levels. Our group has already described that Php4 is a nucleo-cytoplasmic shuttling protein, which accumulates into the nucleus during iron deficiency. On the contrary, Php4 is exported from the nucleus to the cytoplasm in response to iron abundance. Php4 possesses a leucine-rich NES (93LLEQLEML100) that is necessary for its nuclear export by the exportin Crm1. Our current study aims at understanding the mechanism by which Php4 is imported in the nucleus during iron starvation. Through microscopic analyses using different mutant strains, we showed that the nuclear localization of Php4 is independent of the other subunits of the CCAAT-binding core complex namely Php2, Php3 and Php5. Deletion mapping analysis of Php4 identifies two putative nuclear localization sequences (NLSs) in Php4 (171KRIR174 and 234KSVKRVR240). Using chimeric proteins that consist of GFP fused to Php4, we engineered substitutions of the basic amino acid residues 171AAIA174 and 234ASVAAAA240 and analyzed the functionality of both NLSs. We observed that both monopartite NLSs play critical role for Php4 nuclear localization. We also observed that mutant strains of cut15+, imp1+ or sal3+ exhibited defects in nuclear targeting of Php4, revealing that nuclear accumulation of Php4 is dependent on two karyopherin α (Imp1 and Cut15) and one karyopherin β (Sal3) receptors. Consistently, the Php4-mediated repression activity is abolished in the absence of two functional NLSs. Moreover, loss of Imp1, Cut15 or Sal3 resulted in increased expression of isa1+, which is a target gene of Php4. Co-immunoprecipitation assay (Co-IP) reveals physical interaction of Php4 with Imp1, Cut15 and Sal3 in vitro. Collectively, our results demonstrate that Php4 has two distinct NLS regions responsible for its nuclear localization. Furthermore, karyopherin α and β receptors play a role in the nuclear import of Php4. Because Php4 is essential for growth under low iron conditions, the presence of two NLSs would ensure the protein to reach its nuclear destination when cells undergo a transition from iron-sufficient to iron-limiting conditions.

Tumor suppressor p53-binding protein 1 (53BP1) is an integral part of a sophisticated network of cellular pathways termed as the DNA damage response (DDR). These pathways are specialized in the maintenance of genome integrity. Recently, it was reported that nuclear import of 53BP1 depends on importin ß. Here, I used fluorescence microscopy and co-immunoprecipitation experiments to identify its nuclear localization signal (NLS). Clusters of basic amino acids 1667-KRK-1669 and 1681-KRGRK- 1685 were required for 53BP1 interaction with importin ß and for its nuclear localization. Short peptide containing these two clusters was sufficient for interaction with importin ß and targeting EGFP to the nucleus. Additionally, the effect of 53BP1 phosphorylation at S1678 on its nuclear import was examined. Mimicking the phosphorylation in the 53BP1-S1678D mutant decreased the binding to importin ß and resulted in a mild defect in 53BP1 nuclear import. However, 53BP1 entered the nucleus continuously during the cell cycle, suggesting that CDK-dependent phosphorylation of S1678 probably does not significantly contribute to the regulation of 53BP1 nuclear transport. Taken together, 53BP1 NLS meets the attributes of a classical bipartite NLS. Although no cell cycle-dependent regulation of its import was observed, the...