A divergent Sm fold in EDC3 proteins mediates DCP1 binding and P-body targeting

Members of the (L)Sm (Sm and Sm-like) protein family are found across all kingdoms of life and play crucial roles in RNA metabolism. The P-body component EDC3 (enhancer of decapping 3) is a divergent member of this family that functions in mRNA decapping. EDC3 is composed of a N-terminal LSm domain, a central FDF domain, and a C-terminal YjeF-N domain. We show that this modular architecture enables EDC3 to interact with multiple components of the decapping machinery, including DCP1, DCP2, and Me31B. The LSm domain mediates DCP1 binding and P-body localization. We determined the three-dimensional structures of the LSm domains of Drosophila melanogaster and human EDC3 and show that the domain adopts a divergent Sm fold that lacks the characteristic N-terminal α-helix and has a disrupted β4-strand. This domain remains monomeric in solution and lacks several features that canonical (L)Sm domains require for binding RNA. The structures also revealed a conserved patch of surface residues that are required for the interaction with DCP1 but not for P-body localization. The conservation of surface and of critical structural residues indicates that LSm domains in EDC3 proteins adopt a similar fold that has separable novel functions that are absent in canonical (L)Sm proteins.     

Arabidopsis Synaptotagmin SYT1, a Type I Signal-anchor Protein,Requires Tandem C2 Domains for Delivery to the Plasma Membrane

The correct localization of integral membrane proteins to subcellular compartments is important for their functions. Synaptotagmin contains a single transmembrane domain that functions as a type I signal-anchor sequence in its N terminus and two calcium-binding domains (C₂A and C₂B) in its C terminus. Here, we demonstrate that the localization of an Arabidopsis synaptotagmin homolog, SYT1, to the plasma membrane (PM) is modulated by tandem C2 domains. An analysis of the roots of a transformant-expressing green fluorescent protein-tagged SYT1 driven by native SYT1 promoter suggested that SYT1 is synthesized in the endoplasmic reticulum, and then delivered to the PM via the exocytotic pathway. We transiently expressed a series of truncated proteins in protoplasts, and determined that tandem C₂A-C₂B domains were necessary for the localization of SYT1 to the PM. The PM localization of SYT1 was greatly reduced following mutation of the calcium-binding motifs of the C₂B domain, based on sequence comparisons with other homologs, such as endomembrane-localized SYT5. The localization of SYT1 to the PM may have been required for the functional divergence that occurred in the molecular evolution of plant synaptotagmins.     

Characterization of the topology and functional domains of RKTG

RKTG (Raf kinase trapping to Golgi) is exclusively localized at the Golgi apparatus and functions as a spatial regulator of Raf-1 kinase by sequestrating Raf-1 to the Golgi. Based on the structural similarity with adiponectin receptors, RKTG was predicted to be a seven-transmembrane protein with a cytosolic N-terminus, distinct from classical GPCRs (G-protein-coupled receptors). We analysed in detail the topology and functional domains of RKTG in this study. We determined that the N-terminus of RKTG is localized on the cytosolic side. Two short stretches of amino acid sequences at the membrane proximal to the N- and C-termini (amino acids 61-71 and 299-303 respectively) were indispensable for Golgi localization of RKTG, but were not required for the interaction with Raf-1. The three loops facing the cytosol between the transmembrane domains had different roles in Golgi localization and Raf-1 interaction. While the first cytosolic loop was only important for Golgi localization, the third cytosolic loop was necessary for both Golgi localization and Raf-1 sequestration. Taken together, these findings suggest that RKTG is a type III membrane protein with its N-terminus facing the cytosol and multiple sequences are responsible for its localization at the Golgi apparatus and Raf-1 interaction. As RKTG is the first discovered Golgi protein with seven transmembrane domains, the knowledge derived from this study would not only provide structural information about the protein, but also pave the way for future characterization of the unique functions of RKTG in the regulation of cell signalling.     

Rho localization in cells and tissues

Rho family small GTPases regulate cytoskeletal organization. Although their spatiotemporal activities appear to be important for cellular morphogenesis, there has been little characterization of the localization of Rho family GTPases in cells and tissues. Here we show precise localization of Rho subfamily proteins in mammalian cultured cells and tissues through evaluation of anti-Rho antibodies and fixation protocols. Although Rho is not a structural protein but functions as a switching molecule, it often localizes at several distinct domains or structures of cells. In cultured epithelial cells, Rho was highly accumulated at lateral membranes. However, in fibroblastic cells, Rho appeared to be distributed evenly in the cytoplasm. Rho concentration at the cleavage furrow at cytokinesis was generally observed. In A431 cells, Rho translocation from the cytoplasm to elongating microvilli at the apical membrane within 30 s after EGF stimulation was clearly demonstrated. Also, Myc- or GFP-tagged RhoA did not always reflect the localization of endogenous Rho, indicating a drawback of protein-tagging methods for localization research. In mouse tissues, Rho localization differed depending on cell type, probably reflecting the functional differences of each cell type.     

The pleckstrin homology domain: An intriguing multifunctional protein module

Pleckstrin homology (PH) domains are a family of compact protein modules defined by sequences of roughly 100 amino acids. These domains are common in vertebrate, Drosophila, C. elegans and yeast proteins, suggesting an early origin and fundamental importance to eukaryotic biology. Many enzymes which have important regulatory functions contain PH domains, and mutant forms of several such proteins are implicated in oncogenesis and developmental disorders. Numerous recent studies show that PH domains bind various proteins and inositolphosphates. Here I discuss PH domains in detail and conclude that they form a versatile family of membrane binding and protein localization modules.     

Novel interactions of fission yeast kinesin 8 revealed through in vivo expression of truncation alleles

Fission yeast expresses two kinesin 8s, klp5+ and klp6+, which are important for diverse cellular functions: mitosis, meiosis, and the maintenance of normal cell morphology. During vegetative growth these motors display complex localization patterns, moving from the cytoplasm during interphase to the kinetochores in early mitosis, the interpolar spindle in anaphase B, and then back into the cytoplasm. We have expressed GFP-tagged alleles of domains from these motors, seeking the signals required for their localizations. The tail of Klp5p localized to the interphase nucleus, more specifically to telomeres. Addition of the neck re-directed this fragment to microtubules in the cytoplasm. Klp6-tail and the neck-tail domains of both motors localized at microtubule ends. Klp6-neck-tail localized to the spindle in early mitosis but to the pole-proximal ends of the spindle in anaphase B. The Klp5-motor and motor-neck localized to microtubules, often causing them to bundle. Over-expression of Klp6-motor or motor-neck resulted in shorter microtubules. These localization patterns were no different when constructs were expressed in strains lacking either or both of the endogenous, full-length proteins. Our results indicate that the localization signals for these kinesins are not derived from simple amino acid sequences but from complex interactions among multiple domains of each motor.     

Molecular dissection of a yeast septin: distinct domains are required for septin interaction,localization, and function

The septins are a family of cytoskeletal proteins present in animal and fungal cells. They were first identified for their essential role in cytokinesis, but more recently, they have been found to play an important role in many cellular processes, including bud site selection, chitin deposition, cell compartmentalization, and exocytosis. Septin proteins self-associate into filamentous structures that, in yeast cells, form a cortical ring at the mother bud neck. Members of the septin family share common structural domains: a GTPase domain in the central region of the protein, a stretch of basic residues at the amino terminus, and a predicted coiled-coil domain at the carboxy terminus. We have studied the role of each domain in the Saccharomyces cerevisiae septin Cdc11 and found that the three domains are responsible for distinct and sometimes overlapping functions. All three domains are important for proper localization and function in cytokinesis and morphogenesis. The basic region was found to bind the phosphoinositides phosphatidylinositol 4-phosphate and phosphatidylinositol 5-phosphate. The coiled-coil domain is important for interaction with Cdc3 and Bem4. The GTPase domain is involved in Cdc11-septin interaction and targeting to the mother bud neck. Surprisingly, GTP binding appears to be dispensable for Cdc11 function, localization, and lipid binding. Thus, we find that septins are multifunctional proteins with specific domains involved in distinct molecular interactions required for assembly, localization, and function within the cell.     

Characterization of a PIAS4 homologue from zebrafish: insights into its conserved negative regulatory mechanism in the TRIF, MAVS, and IFN signaling pathways during vertebrate evolution

Members of the protein inhibitor of activated STAT (PIAS) family are key regulators of various human and mammalian signaling pathways, but data on their occurrence and functions in ancient vertebrates are limited. This study characterizes for the first time to our knowledge a PIAS4 homologue (PIAS4a) from zebrafish. Structurally, this zebrafish PIAS4a (zfPIAS4a) shares a number of conserved functional domains with mammalian PIAS4 proteins, including the scaffold attachment factor A/B/acinus/PIAS box, PINIT, and RING-finger-like zinc-binding domains and a highly acidic domain in the C-terminal region. Subcellular localization analysis shows that zfPIAS4a is a nuclear-localized protein and that the C terminus of the molecule harbors strict nuclear localization signals. Functionally, zfPIAS4a expression can be dramatically induced by the stimulation of polyinosinic-polycytidylic acid and zebrafish IFN1. It acts as a critical negative regulator of the TIR domain-containing adapter inducing IFN-β, mitochondrial antiviral signaling (MAVS), and IFN signaling pathways, and it is the first PIAS protein that plays a role in the MAVS-mediated pathway to be identified. The structure and functionality of PIAS4 seem highly conserved from zebrafish to mammals, making zebrafish an attractive model for screens designed to uncover genes involved in IFN- and inflammatory cytokine-induced signaling pathways. This study provides preliminary evidence that the PIAS regulatory mechanism already existed in fish during vertebrate evolution. It presents valuable clues for improving the understanding of not only the negative regulation of cytokine signaling in fish but also the evolutionary history of the PIAS family from fish to mammals as a whole.     

Identification of 40LoVe, a Xenopus hnRNP D family protein involved in localizing a TGF-beta-related mRNA during oogenesis

Asymmetric distribution of cellular components underlies many biological processes, and the localization of mRNAs within domains of the cytoplasm is one important mechanism of establishing and maintaining cellular asymmetry. mRNA localization often involves assembly of large ribonucleoproteins (RNPs) in the cytoplasm. Using an RNA affinity chromatography approach, we investigated localization RNP formation on the vegetal localization element (VLE) of the mRNA encoding Vg1, a Xenopus TGF-beta family member. We identified 40LoVe, an hnRNP D family protein, as a specific VLE binding protein from Xenopus oocytes. Interaction of 40LoVe with the VLE strictly correlates with the ability of the RNA to localize, and antibodies against 40LoVe inhibit vegetal localization in vivo in oocytes. Our results associate an hnRNP D protein with mRNA localization and have implications for several functions mediated by this important protein family.     

Heteromeric interactions required for abundance and subcellular localization of human CDC50 proteins and class 1 P4-ATPases

Members of the P(4) family of P-type ATPases (P(4)-ATPases) are believed to function as phospholipid flippases in complex with CDC50 proteins. Mutations in the human class 1 P(4)-ATPase gene ATP8B1 cause a severe syndrome characterized by impaired bile flow (intrahepatic cholestasis), often leading to end-stage liver failure in childhood. In this study, we determined the specificity of human class 1 P(4)-ATPase interactions with CDC50 proteins and the functional consequences of these interactions on protein abundance and localization of both protein classes. ATP8B1 and ATP8B2 co-immunoprecipitated with CDC50A and CDC50B, whereas ATP8B4, ATP8A1, and ATP8A2 associated only with CDC50A. ATP8B1 shifted from the endoplasmic reticulum (ER) to the plasma membrane upon coexpression of CDC50A or CDC50B. ATP8A1 and ATP8A2 translocated from the ER to the Golgi complex and plasma membrane upon coexpression of CDC50A, but not CDC50B. ATP8B2 and ATP8B4 already displayed partial plasma membrane localization in the absence of CDC50 coexpression but displayed a large increase in plasma membrane abundance upon coexpression of CDC50A. ATP8B3 did not bind CDC50A and CDC50B and was invariably present in the ER. Our data show that interactions between CDC50 proteins and class 1 P(4)-ATPases are essential for ER exit and stability of both subunits. Furthermore, the subcellular localization of the complex is determined by the P(4)-ATPase, not the CDC50 protein. The interactions of CDC50A and CDC50B with multiple members of the human P(4)-ATPase family suggest that these proteins perform broader functions in human physiology than thus far assumed.     

Phospholipase Cgamma2 modulates integrin signaling in the osteoclast by affecting the localization and activation of Src kinase

Integrin engagement induces a cascade of signaling pathways that include tyrosine phosphorylation of numerous proteins that lead to modulation of the actin cytoskeleton. Src is a major intracellular mediator of integrin-dependent functions, but the mechanism(s) by which Src is regulated in response to integrin signals is not fully understood. Here, we demonstrate an important role for phospholipase C gamma 2 (PLCgamma2) in Src activation in the osteoclast. Through analysis of primary cells from PLCgamma2(-/-) mice, PLCgamma2 was found to be an important regulator of alpha(v)beta(3) integrin-mediated bone osteoclast cell adhesion, migration, and bone resorption. Adhesion-induced PYK2 and Src phosphorylation is decreased in the absence of PLCgamma2, and the interaction of Src with beta(3) integrin and PYK2 is dramatically reduced. Importantly, PLCgamma2 was found to be required for proper localization of Src to the sealing actin ring, and this function required both its catalytic activity and adapter domains. Based on these results, we propose that PLCgamma2 influences Src activation by mediating the localization of Src to the integrin complex and thereby regulating integrin-mediated functions in the osteoclast.     

The characterization of human adenylate kinases 7 and 8 demonstrates differences in kinetic parameters and structural organization among the family of adenylate kinase isoenzymes

Differences in expression profiles, substrate specificities, kinetic properties and subcellular localization among the AK (adenylate kinase) isoenzymes have been shown to be important for maintaining a proper adenine nucleotide composition for many different cell functions. In the present study, human AK7 was characterized and its substrate specificity, kinetic properties and subcellular localization determined. In addition, a novel member of the human AK family, with two functional domains, was identified and characterized and assigned the name AK8. AK8 is the second known human AK with two complete and active AK domains within its polypeptide chain, a feature that has previously been shown for AK5. The full-length AK8, as well as its two domains AK8p1 and AK8p2, all showed similar AK enzyme activity. AK7, full-length AK8, AK8p1 and AK8p2 phosphorylated AMP, CMP, dAMP and dCMP with ATP as the phosphate donor, and also AMP, CMP and dCMP with GTP as the phosphate donor. Both AK7 and full-length AK8 showed highest affinity for AMP with ATP as the phosphate donor, and proved to be more efficient in AMP phosphorylation as compared with the major cytosolic isoform AK1. Expression of the proteins fused with green fluorescent protein demonstrated a cytosolic localization for both AK7 and AK8.     

Yeast PIAS-type Ull1/Siz1 is composed of SUMO ligase and regulatory domains

SUMO (small ubiquitin-like modifier)/Smt3 (suppressor of mif two) is a member of the ubiquitin-related protein family and is known to conjugate with many proteins. In the sumoylation pathway, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme), and E3 (SUMO ligase) functions as an adaptor between E2 and each substrate. Yeast Ull1 (ubiquitin-like protein ligase 1)/Siz1, a PIAS (protein inhibitor of activated STAT)-type SUMO ligase, modifies both cytoplasmic and nuclear proteins. In this paper, we performed a domain analysis of Ull1/Siz1 by constructing various deletion mutants. A novel conserved N-terminal domain, called PINIT, as well as the RING-like domain (SP-RING) were required for the SUMO ligase activity in the in vitro conjugation system and for interaction with Smt3 in an in vitro binding assay. The most distal N-terminal region, which contains a putative DNA-binding SAF-A/B, Acinus, and PIAS (SAP) motif, was not required for the ligase activity but was involved in nuclear localization. A strong SUMO-binding motif was identified, which interacted with Smt3 in the two-hybrid system but was not necessary for the ligase activity. The most distal C-terminal domain was important for stable localization at the bud neck region and thereby for the substrate recognition of septins. Furthermore, the C-terminal half conferred protein instability on Ull1/Siz1. Taken together, we conclude that the SP-RING and PINIT of Ull1/Siz1 are core domains of the SUMO ligase, and the other domains are regulatory for protein stability and subcellular localization.     

Localization of Microtubule-Associated Protein (MAP) 1B in the Postsynaptic Densities of the Rat Cerebral Cortex

1. Although microtubule-associated protein (MAP) 1B and its phosphorylation have been suggested to be important for synapse formation among cortical neurons, the localization of MAP1B in synapses has not yet been confirmed. In this report, we examine the localization of MAP1B in synaptic regions. 2. The localization of MAP1B was observed by immunohistochemical and electron microscopic techniques using specific antibodies against MAP1B. 3. MAP1B immunoreactivities were widely distributed in the cerebral cortex and were observed in the postsynaptic area but not in presynaptic terminals. 4. These synapses were classified as the asymmetrical type. 5. Only some synapses exhibited MAP1B immunoreactivities. MAP1B-immunopositive synapses accounted for about half of the total synapses. 6. Such a localization suggests MAP1B's important roles in synaptic functions.     

Human sperm telomere-binding complex involves histone H2B and secures telomere membrane attachment

Telomeres are unique chromatin domains located at the ends of eukaryotic chromosomes. Telomere functions in somatic cells involve complexes between telomere proteins and TTAGGG DNA repeats. During the differentiation of germ-line cells, telomeres undergo significant reorganization most likely required for additional specific functions in meiosis and fertilization. A telomere-binding protein complex from human sperm (hSTBP) has been isolated by detergent treatment and was partially purified. hSTBP specifically binds double-stranded telomeric DNA and does not contain known somatic telomere proteins TRF1, TRF2, and Ku. Surprisingly, the essential component of this complex has been identified as a specific variant of histone H2B. Indirect immunofluorescence shows punctate localization of H2B in sperm nuclei, which in part coincides with telomeric DNA localization established by fluorescent in situ hybridization. Anti-H2B antibodies block interactions of hSTBP with telomere DNA, and spH2B forms specific complex with this DNA in vitro, indicating that this protein plays a role in telomere DNA recognition. We propose that hSTBP participates in the membrane attachment of telomeres that may be important for ordered chromosome withdrawal after fertilization.     

CalDAG-GEFI integrates signaling for platelet aggregation and thrombus formation

Signaling through the second messengers calcium and diacylglycerol (DAG) is a critical element in many biological systems. Integration of calcium and DAG signals has been suggested to occur primarily through protein kinase C family members, which bind both calcium and DAG. However, an alternative pathway may involve members of the CalDAG-GEF/RasGRP protein family, which have structural features (calcium-binding EF hands and DAG-binding C1 domains) that suggest they can function in calcium and DAG signal integration. To gain insight into the signaling systems that may be regulated by CalDAG-GEF/RasGRP family members, we have focused on CalDAG-GEFI, which is expressed preferentially in the brain and blood. Through genetic ablation in the mouse, we have found that CalDAG-GEFI is crucial for signal integration in platelets. Mouse platelets that lack CalDAG-GEFI are severely compromised in integrin-dependent aggregation as a consequence of their inability to signal through CalDAG-GEFI to its target, the small GTPase Rap1. These results suggest that analogous signaling defects are likely to occur in the central nervous system when CalDAG-GEFI is absent or compromised in function.