The ribosome receptor, p180, interacts with kinesin heavy chain, KIF5B

The conventional microtubule-dependent motor protein kinesin consists of heavy and light chains both of which have been documented to bind a variety of potential linker or cargo proteins. In this study we employed a yeast two-hybrid assay to identify additional binding partners of the kinesin heavy chain isoform KIF5B. A human brain cDNA library was screened with a bait corresponding to amino acid residues 814-963 of human KIF5B. This screen identified the ribosome receptor, p180, as a KIF5B-binding protein. The sites of interaction are residues 1294-1413 of p180 and the C-terminal half of the cargo binding-domain of KIF5B (residues 867-907). The KIF5B-binding site in p180 is homologous to the previously determined KIF5B-binding site in kinectin. The interacting regions of p180 and KIF5B consist almost entirely of heptad repeats, suggesting the interaction is a coiled-coil. A role for the kinesin/p180 interaction may include mRNA localization and/or transport of endoplasmic reticulum-derived vesicles.     

Diffusion of kinesin motors on cargo can enhance binding and run lengths during intracellular transport

Cellular cargoes, including lipid droplets and mitochondria, are transported along microtubules using molecular motors such as kinesins. Many experimental and computational studies focused on cargoes with rigidly attached motors, in contrast to many biological cargoes that have lipid surfaces that may allow surface mobility of motors. We extend a mechanochemical three-dimensional computational model by adding coupled-viscosity effects to compare different motor arrangements and mobilities. We show that organizational changes can optimize for different objectives: Cargoes with clustered motors are transported efficiently but are slow to bind to microtubules, whereas those with motors dispersed rigidly on their surface bind microtubules quickly but are transported inefficiently. Finally, cargoes with freely diffusing motors have both fast binding and efficient transport, although less efficient than clustered motors. These results suggest that experimentally observed changes in motor organization may be a control point for transport.     

Dynamic distribution of Spatial during mouse spermatogenesis and its interaction with the kinesin KIF17b

The Spatial gene is expressed in highly polarized cell types, such as epithelial cells in the thymus, neurons in the brain and germ cells in the testis. In this study, we report the characterization and distribution of Spatial proteins during mouse spermatogenesis. Besides Spatial-epsilon and -delta, we show that the newly described short isoform Spatial-beta is expressed specifically in round spermatids. Using indirect immunofluorescence, we detected Spatial in the cytosol of the early round spermatid. By the end stages of round spermatids, Spatial is concentrated at the opposite face of the acrosome near the nascent flagellum and in the manchette during the elongation process. Finally in mature sperm, Spatial persists in the principal piece of the tail. Moreover, we found that Spatial colocalizes with KIF17b, a testis-specific isoform of the brain kinesin-2 motor KIF17. This colocalization is restricted to the manchette and the principal piece of the sperm tail. Further, coimmunoprecipitation experiments of native proteins from testis lysates confirmed Spatial-KIF17b association through the long Spatial-epsilon isoform. Together, these findings imply a function of Spatial in spermatid differentiation as a new cargo of kinesin KIF17b, in a microtubule-dependent mechanism specific to the manchette and the principal piece of the sperm tail.     

Modulation of receptor recycling and degradation by the endosomal kinesin KIF16B

Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. We found a kinesin-3, KIF16B, that transports early endosomes to the plus end of microtubules in a process regulated by the small GTPase Rab5 and its effector, the phosphatidylinositol-3-OH kinase hVPS34. In vivo, KIF16B overexpression relocated early endosomes to the cell periphery and inhibited transport to the degradative pathway. Conversely, expression of dominant-negative mutants or ablation of KIF16B by RNAi caused the clustering of early endosomes to the perinuclear region, delayed receptor recycling to the plasma membrane, and accelerated degradation. These results suggest that KIF16B, by regulating the plus end motility of early endosomes, modulates the intracellular localization of early endosomes and the balance between receptor recycling and degradation. We propose that this mechanism could have important implications for signaling.     

Casein kinase 1alpha interacts with retinoid X receptor and interferes with agonist-induced apoptosis

Agonists of retinoid X receptors (RXRs), which include the natural 9-cis-retinoic acid and synthetic analogs, are potent inducers of growth arrest and apoptosis in some cancer cells. As such, they are being used in clinical trials for the treatment and prevention of solid tumors and are used to treat cutaneous T cell lymphoma. However, the molecular mechanisms that underlie the anti-cancer effects of RXR agonists remain unclear. Here, we show that a novel pro-apoptotic pathway that is induced by RXR agonist is negatively regulated by casein kinase 1alpha (CK1alpha). CK1alpha associates with RXR in an agonist-dependent manner and phosphorylates RXR. The ability of an RXR agonist to recruit CK1alpha to a complex with RXR in cells correlates inversely with its ability to inhibit growth. Remarkably, depletion of CK1alpha in resistant cells renders them susceptible to RXR agonist-induced growth inhibition and apoptosis. Our study shows that CK1alpha can promote cell survival by interfering with RXR agonist-induced apoptosis. Inhibition of CK1alpha may enhance the anti-cancer effects of RXR agonists.     

A biosensor of local kinesin activity reveals roles of PKC and EB1 in KIF17 activation

We showed previously that the kinesin-2 motor KIF17 regulates microtubule (MT) dynamics and organization to promote epithelial differentiation. How KIF17 activity is regulated during this process remains unclear. Several kinesins, including KIF17, adopt compact and extended conformations that reflect autoinhibited and active states, respectively. We designed biosensors of KIF17 to monitor its activity directly in single cells using fluorescence lifetime imaging to detect Förster resonance energy transfer. Lifetime data are mapped on a phasor plot, allowing us to resolve populations of active and inactive motors in individual cells. Using this biosensor, we demonstrate that PKC contributes to the activation of KIF17 and that this is required for KIF17 to stabilize MTs in epithelia. Furthermore, we show that EB1 recruits KIF17 to dynamic MTs, enabling its accumulation at MT ends and thus promoting MT stabilization at discrete cellular domains.     

Association of the kinesin motor KIF1A with the multimodular protein liprin-alpha

Liprin-alpha/SYD-2 is a multimodular scaffolding protein important for presynaptic differentiation and postsynaptic targeting of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid glutamate receptors. However, the molecular mechanisms underlying these functions remain largely unknown. Here we report that liprin-alpha interacts with the neuron-specific kinesin motor KIF1A. KIF1A colocalizes with liprin-alpha in various subcellular regions of neurons. KIF1A coaccumulates with liprin-alpha in ligated sciatic nerves. KIF1A cofractionates and coimmunopreciptates with liprin-alpha and various liprin-alpha-associated membrane, signaling, and scaffolding proteins including alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors, GRIP/ABP, RIM, GIT1, and beta PIX. These results suggest that liprin-alpha functions as a KIF1A receptor, linking KIF1A to various liprin-alpha-associated proteins for their transport in neurons.     

The motor domain of testis-enriched kinesin KIF9 is essential for its localization in the mouse flagellum

Kinesin is a molecular motor that moves along microtubules. Testis-enriched kinesin KIF9 (Kinesin family member 9) is localized in the mouse sperm flagellum and is important for normal sperm motility and male fertility; however, it is unclear if the motor domain of KIF9 is involved in these processes. In this study, we substituted threonine of the ATP binding motif in the KIF9 motor domain to asparagine (T100N) in mice using the CRISPR/Cas9 system, which is known to impair kinesin motor activity. T100N mutant mice exhibit reduced sperm motility and male fertility consistent with Kif9 knockout mice. Further, KIF9 was depleted in the spermatozoa of T100N mutant mice although the amounts of KIF9 were comparable between wild-type and T100N mutant testes. These results indicate that the motor domain of KIF9 is essential for its localization in the sperm flagellum.     

The Ras-like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin-like protein KIF9

Gem belongs to the Rad/Gem/Kir (RGK) subfamily of Ras-related GTPases, which also comprises Rem, Rem2 and Ges. The RGK family members Ges and Rem have been shown to produce endothelial cell sprouting and reorganization of the actin cytoskeleton upon overexpression. Here we show that high intracellular Gem levels promote profound changes in cell morphology and we investigate how this phenotype arises dynamically. We also show that this effect requires intact microtubules and microfilaments, and that Gem is associated with both cytoskeletal components. In order to investigate the mechanisms of Gem recruitment to the cytoskeleton, we performed a yeast two-hybrid screen and identified a novel kinesin-like protein, termed KIF9, as a new Gem interacting partner. We further show that Gem and KIF9 interact by co-immunoprecipitation. Furthermore, Gem and KIF9 display identical patterns of gene expression in different tissues and developmental stages. The Gem- KIF9 interaction reported here is the first molecular link between RGK family members and the microtubule cytoskeleton.     

Interaction of mouse TTC30/DYF-1 with multiple intraflagellar transport complex B proteins and KIF17

Intraflagellar transport (IFT) is a microtubule based system that supports the assembly and maintenance of cilia. Genetic and biochemical studies have identified two distinct complexes containing multiple proteins that are part of the IFT machinery. In this study we prepared mouse pituitary cells that expressed an epitope-tagged IFT protein and immuno-purified the IFT B complex from these cells. Mass spectrometry analysis of the isolated complex led to identification of a number of well known components of the IFT B complex. In addition, peptides corresponding to mouse tetratricopeptide repeat proteins, TTC30A1, TTC30A2 and TTC30B were identified. The mouse Ttc30A1, Ttc30A2, Ttc30B genes are orthologs of Caenorhabditis elegans dyf-1, which is required for assembly of the distal segment of the cilia. We used co-immunoprecipitation studies to provide evidence that, TTC30A1, TTC30A2 or TTC30B can be incorporated into a complex with a known IFT B protein, IFT52. We also found that TTC30B can interact with mouse KIF17, a kinesin which participates in IFT. In vitro expression in a cell-free system followed by co-immunoprecipitation also provided evidence that TTC30B can directly interact with several different IFT B complex proteins. The findings support the view that mouse TTC30A1, TTC30A2 and TTC30B can contribute to the IFT B complex, likely through interactions with multiple IFT proteins and also suggest a possible link to the molecular motor, KIF17 to support transport of cargo during IFT.     

Structural perturbation of the transmembrane region interferes with calcium binding by the Ca2+ transport ATPase

The Ca(2+)-ATPase of sarcoplasmic reticulum reacts with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl) carbodiimide (NCD4) yielding a fluorescence labeling that interferes with calcium binding to activating and transport sites of the enzyme and, thereby, with Ca(2+)-dependent ATPase activity. On the other hand, the catalytic site does not appear altered, as revealed by the normal occurrence of Ca(2+)-independent reactions, such as enzyme phosphorylation with Pi in the reverse direction of the catalytic cycle. This reaction is not inhibited by Ca2+ in the labeled enzyme, while it is inhibited in the native enzyme. The NCD4 reaction which is involved in functional inactivation occurs in the membrane-bound portion of the ATPase. Sodium dodecyl sulfate solubilization of hydrophobic peptides, electrophoresis, and microsequencing of transblotted electrophoretic bands revealed that the fluorescent NCD4 label resides in a segment of tryptic fragment A1, intervening between Glu231 and Glu309. This segment includes two transmembrane helices, and does not include the domain involved in the phosphoryl transfer reaction during catalytic activity. This specific labeling does not occur when the NCD4 derivatization procedure is carried out in the presence of Ca2+ concentrations that also prevent functional inactivation. Fluorescence characterization by steady state and intensity decay measurements shows only negligible energy transfer between the NCD4 label and fluorescein isothiocyanate label of Lys515, indicating that the NCD4 label is unlikely to reside within the extramembranous region of the ATPase. On the other hand, the fluorescence emission of intrinsic tryptophan residues clustered within or near the transmembrane region of the ATPase, is distinctly affected by NCD4 label specifically bound to the ATPase, and NCD4 label nonspecifically bound to the sarcoplasmic reticulum membrane. The combined sequencing and spectroscopic observations indicate that derivatization with NCD4 induces a perturbation within or near the transmembrane region of the ATPase (at a relatively large distance from the catalytic site) that interferes with specific calcium binding. This is in agreement with experiments (Clarke et al., 1989) demonstrating that mutations of any of six amino acids within the transmembrane region of the ATPase interfere with enzyme activation by Ca2+.     

The PX-domain protein SNX17 interacts with members of the LDL receptor family and modulates endocytosis of the LDL receptor

Sorting nexins (SNXs) comprise a family of proteins characterized by the presence of a phox-homology domain, which mediates the association of these proteins with phosphoinositides and recruits them to specific membranes or vesicular structures within cells. Although only limited information about SNXs and their functions is available, they seem to be involved in membrane trafficking and sorting processes by directly binding to target proteins such as certain growth factor receptors. We show that SNX17 binds to the intracellular domain of some members of the low-density lipoprotein receptor (LDLR) family such as LDLR, VLDLR, ApoER2 and LDLR-related protein. SNX17 resides on distinct vesicular structures partially overlapping with endosomal compartments characterized by the presence of EEA1 and rab4. Using rhodamine-labeled LDL, it was possible to demonstrate that during endocytosis, LDL passes through SNX17-positive compartments. Functional studies on the LDLR pathway showed that SNX17 enhances the endocytosis rate of this receptor. Our results identify SNX17 as a novel adaptor protein for LDLR family members and define a novel mechanism for modulation of their endocytic activity.     

Desethylamiodarone interferes with the binding of co-activator GRIP-1 to the beta 1-thyroid hormone receptor

Ligand binding to the thyroid hormone nuclear receptor beta1 (TRbeta(1)) is inhibited by desethylamiodarone (DEA), the major metabolite of the widely used anti-arrhythmic drug amiodarone. Gene expression of thyroid hormone (triiodothyronine, T(3))-regulated genes can therefore be affected by amiodarone due to less ligand binding to the receptor. Previous studies have indicated the possibility of still other explanations for the inhibitory effects of amiodarone on T(3)-dependent gene expression, probably via interference with receptor/co-activator and co-repressor complex. The binding site of DEA is postulated to be on the outside surface of the receptor protein overlapping the regions where co-activator and co-repressor bind. Here we show the effect of a drug metabolite on the interaction of TRbeta(1) with the co-activator GRIP-1 (glucocorticoid receptor interacting protein-1). The T(3)-dependent binding of GRIP-1 to the TRbeta(1) is disrupted by DEA. A DEA dose experiment showed that the drug metabolite acts like an antagonist under 'normal' conditions (at 10(-7) M T(3) and 5x10(-6)-->10(-3) M DEA), but as an agonist under extreme conditions (at 0 and 10(-9) M T(3) and >10(-4) M DEA). To our knowledge, these results show for the first time that a metabolite of a drug which was not devised for this purpose can interfere with nuclear receptor/co-activator interaction.     

Tight binding of influenza virus hemagglutinin to its receptor interferes with fusion pore dilation

Deletion of oligosaccharide side chains near the receptor binding site of influenza virus A/USSR/90/77 (H1N1) hemagglutinin (HA) enhanced the binding of HA to erythrocyte receptors, as was also observed with A/FPV/Rostock/34 (H7N1). Correlated with the enhancement of binding activity, the cell fusion activity of HA was reduced. A mutant HA in which three oligosaccharide side chains were deleted showed the highest level of binding and the lowest level of fusion among the HAs tested. The cell fusion activity of the oligosaccharide deletion mutant of HA, however, was drastically elevated when the binding activity was reduced by deletion of four amino acids adjacent to the receptor binding site. Thus, a reciprocal relationship was observed between the receptor binding and the cell fusion activities of H1/USSR HA. No difference was observed, however, in lipid mixing activity, so-called hemifusion, between wild-type (WT) and oligosaccharide deletion mutant HAs. Soluble dye transfer testing showed that even the HA with the lowest cell fusion activity was able to form fusion pores through which a small molecule such as calcein could pass. However, electron microscopic studies revealed that a large molecule such as hemoglobin hardly passed through the fusion pores formed by the mutant HA, whereas hemoglobin did efficiently pass through those formed by the WT HA. These results suggested that interference in the process of dilation of fusion pores occurs when the binding of HA to the receptor is too tight. Since the viral nucleocapsid is far larger than hemoglobin, appropriate receptor binding affinity is important for virus entry.     

On the binding mechanism of the peptide receptor of the oligopeptide transport system of Lactococcus lactis

Lactococcus lactis degrades exogenous proteins such as beta-casein to peptides of 4-30 amino acids, and uses these as nitrogen sources. The binding protein or receptor (OppA(Ll)) of the oligopeptide transport system (Opp) of L.LACTIS: has the unique capacity to bind peptides from five up to at least 20 residues. To study the binding mechanism of OppA(Ll), nonameric peptides were used in which the cysteine at position 1, 3, 4, 5, 6, 7 or 9 was selectively labeled with either bulky and non-fluorescent or bulky and fluorescent groups. Also, nonameric peptides with a non-natural residue, azatryptophan, at positions 3 or 7 were used. The fluorescence of azatryptophan reports on the polarity of the environment. The studies indicate that the binding protein encloses the first six amino acids of the peptide, whereas the remaining residues stick out and interact with the surface of the binding protein. The peptide binding mechanism of OppA(Ll) is discussed in relation to known three-dimensional structures of members of this class of proteins, and an adaptation of the general binding mechanism is proposed.