• Journal of Life Science
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• v.19 no.7
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• pp.859-865
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• 2009

Microtubules, a major cytoskeleton, form parallel arrays in the axon and are oriented with their plus ends toward the cell periphery. Kinesin superfamily proteins (KIFs) are the molecular motors acting in the microtubule-based motilities of organelles in cells. Here, we used the yeast two-hybrid system to identify the protein that interacts with the coiled-coil domain of KIF1A and found a specific interaction with microtubule-destabilizing factor SCG10. SCG10 bound to the amino acid residues between 400 and 820 of KIF1A, but not to other KIFs in the yeast two-hybrid assay. The coiled-coil domain of SCG10 is essential for interaction with KIF1A. In addition, this specific interaction was also observed in the Glutathione S-transferase pull-down assay. An antibody to SCG10 specifically co-immunoprecipitated KIF1A associated with SCG10 from mouse brain extracts. These results suggest that KIF1A motor protein transports SCG10-containing vesicles along microtubules in neurons.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.4
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• pp.199-204
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• 2008

KIF1B${\beta}$ is a member of the Kinesin superfamily proteins (KIFs), which are microtubule-dependent molecular motors that are involved in various intracellular organellar transport processes. KIF1B${\beta}$ is not restricted to neuronal systems, however, is widely expressed in other tissues, even though the function of KIF1B${\beta}$ is still unclear. To elucidate the KIF1B${\beta}$-binding proteins in non-neuronal cells, we used the yeast two-hybrid system, and found a specific interaction of KIF1B${\beta}$ and the sorting nexin (SNX) 17. The C-terminal region of SNX17 is required for the binding with KIF1B${\beta}$. SNX17 protein bound to the specific region of KIF1Bf3 (813-916. aa), but not to other kinesin family members. In addition, this specific interaction was also observed in the Glutathione S-transferase pull-down assay. An antibody to SNX17 specifically co-immunoprecipitated KIF1B${\beta}$ associated with SNX17 from mouse brain extracts. These results suggest that SNX17 might be involved in the KIF1B${\beta}$-mediated transport as a KIF1B${\beta}$ adaptor protein.

• Journal of Life Science
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• v.21 no.8
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• pp.1076-1082
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• 2011

Microtubule-based kinesin motor proteins are used for long-range vesicular transport. KIF5s (KIF5A, KIF5B and KIF5C) mediate the transport of various membranous vesicles along microtubules, but the mechanism behind how they recognize and bind to a specific cargo has not yet been completely elucidated. To identify the interaction protein for KIF5B, yeast two-hybrid screening was performed and a specific interaction with the unconventional myosin Myo9b, an actin-based vesicle transport motor, was found. The GTPase-activating protein (GAP) domain of Myo9s was essential for interaction with KIF5B in the yeast two-hybrid assay. Myo9b bound to the carboxyl-terminal region of KIF5B and to other KIF5 members. In addition, glutathione S-transferase (GST) pull-downs showed that Myo9s specifically interact to the complete Kinesin-I complex. An antibody to KIF5B specifically co-immunoprecipitated KIF5B associated with Myo9s from mouse brain extracts. These results suggest that kinesin-I motor protein interacts directly with actin-based motor proteins in the cell.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.3
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• pp.167-172
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• 2004

The kinesin proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF5 is a heterotetrameric motor that conveys vesicles and plays an important role in neuronal function. Here, we used the yeast two-hybrid system to identify the neuronal protein(s) that interacts with the tail region of KIF5 and found a specific interaction with ${\beta}III$ spectrin. The amino acid residues between 1394 and 1774 of ${\beta}III$ spectrin were required for the interaction with KIF5C. ${\beta}III$ spectrin also bound to the tail region of neuronal KIF5A and ubiquitous KIF5B but not to other kinesin family members in the yeast two-hybrid assay. In addition, these proteins showed specific interactions, confirmed by GST pull-down assay and co-immunoprecipitation. ${\beta}III$ spectrin interacted with GST-KIF5 fusion proteins, but not with GST alone. An antibody to ${\beta}III$ spectrin specifically co-immunoprecipitated KIF5s associated with ${\beta}III$ spectrin from mouse brain extracts. These results suggest that KTF5 motor proteins transport vesicles or organelles that are coated with ${\beta}III$ spectrin.

• Journal of Life Science
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• v.22 no.12
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• pp.1608-1613
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• 2012

A conventional kinesin, KIF5/Kinesin-I, transports various cargoes along the microtubule through interaction between its light chain subunit and the cargoes. Kinesin light chains (KLCs) interact with many different cargoes using their tetratricopeptide repeat (TPR) domain, but the mechanism underlying recognition and binding of a specific cargo has not yet been completely elucidated. We used the yeast two-hybrid assay to identify proteins that interact with the TPR domain of KLC1. We found an interaction between the TPR domain of KLC1 and an amyloid precursor protein (APP)-binding protein PAT1 (protein interacting with APP tail 1). The yeast two-hybrid assay demonstrated that the TPR domain-containing region of KLC1 mediated binding to the C-terminal tail region of PAT1. PAT1 also bound to KLC2 but not to kinesin heavy chains (KIF5A, KIF5B, and KIF5C) in the yeast two-hybrid assay. These protein-protein interactions were also observed in the glutathione S-transferase (GST) pull-down assay and by co-immunoprecipitation. Anti-PAT1 antibody as well as anti-APP anti-body co-immunoprecipitated KLC and KHCs associated with PAT1 from mouse brain extracts. These results suggest that PAT1 could mediate interactions between Kinesin-I and APP containing vesicles.

• Journal of Life Science
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• v.24 no.1
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• pp.14-19
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• 2014

Kinesin-1 consists of two heavy chains (KHCs), also called KIF5s, and two light chains (KLCs) that form a heterotetrameric complex. Here, we demonstrate the binding of a neuronal KHC, KIF5A, to the carboxyl (C)-terminal tail region of Fig4 (also known as Sac3), a phosphatase that removes the 5-phosphate from phosphatidylinositol-3,5-bisphosphate ($PtdIns(3,5)P_2$). Fig4 bound to the C-terminal region of KIF5A but not to other KHCs (KIF5B and KIF5C) and KLC1 in yeast two-hybrid assays. The interaction was further confirmed in a glutathione S-transferase pull-down assay and by co-immunoprecipitation. Anti-KIF5A antibody co-immunoprecipitated Fig4 with KIF5A from mouse brain extracts. These results suggest that kinesin-1 could transport the Fig4-associated protein complex or cargo in cells.

• Journal of Life Science
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• v.26 no.6
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• pp.698-704
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• 2016

The intracellular transport of organelles and protein complexes is mediated by kinesin superfamily proteins (KIFs). The first kinesin, kinesin 1, was identified as a molecular motor protein that moves various organelles and protein complexes along the microtubule rails in cells. Kinesin 1 is a tetramer of two heavy chains (KHCs, also called KIF5s) and two kinesin light chains (KLCs). KIF5s interact with many different proteins through their tail region, but their binding proteins have not yet been fully identified. To identify the interaction proteins for KIF5A, we performed yeast two-hybrid screening and found a specific interaction with ferritin heavy chain (Frt-h), which has a role in iron storage and detoxification. Frt-h bound to the amino acid residues between 800 and 940 of KIF5A and to other KIF5s in the yeast two-hybrid assay. The coiled-coil domain of Frt-h is essential for interaction with KIF5A. In addition, ferritin light chain (Frt-l) interacted with KIF5s in the yeast two-hybrid assay. In addition, these proteins showed specific interactions in the glutathione S-transferase (GST) pull-down assay. An antibody to KHC specifically co-immunoprecipitated Frt-h and Frt-l from mouse brain extracts. These results suggest the kinesin 1 motor protein may transport the ferritin complex in cells.

• Journal of Life Science
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• v.33 no.1
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• pp.1-7
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• 2023

Intracellular cargo transport is mediated by molecular motor proteins, such as kinesin and cytoplasmic dynein. Kinesins make up a large subfamily of molecular motors. Kinesin-1 is a plus-end-directed molecular motor protein that moves various cargoes, such as organelles, protein complexes, and mRNAs, along a microtubule track. It consists of the kinesin superfamily protein (KIF) 5A, 5B, and 5C (also called kinesin heavy chains) and kinesin light chains (KLCs). Kinesin-1 interacts with many different binding proteins through its carboxyl (C)-terminal region of KIF5s and KLCs, but their binding proteins have not yet been fully identified. In this study, a yeast two-hybrid assay was used to identify the proteins that interact with the KIF5A specific C-terminal region. The assay revealed an interaction between KIF5A and glutamate-rich 4 (ERICH4). ERICH4 bound to the KIF5A specific the C-terminal region but did not interact with the C-terminal region of KIF5B or KIF3A (a motor protein of kinesin-2). In addition, KIF5A did not interact with another isoform, ERICH1. Glutathione S-transferase (GST) pull-downs showed that KIF5A interacts with GST-ERICH4 and GST-ERICH4-amino (N)-terminal but not with GST-ERICH4-C or GST alone. When co-expressed in HEK-293T cells, ERICH4 co-localized with KIF5A and co-immunoprecipitated with KIF5A and KLC but not KIF3B. Together, our findings suggest that ERICH4 is capable of binding to KIF5A and that it may serve as an adaptor protein that links kinesin-1 with cargo.

• Journal of Life Science
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• v.18 no.8
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• pp.1059-1065
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• 2008

KIF21A is a member of the Kinesin superfamily proteins (KIFs), which are microtubule-dependent molecular motors, anterograde axonal transporters of cargoes. Recently, congenital fibrosis of the extraocular muscles 1 (CFEOM1) has been shown to result from a small number of recurrent heterozygous missense mutations of KIF21A. CFEOM1 results from the inability of mutated KIF21A to successfully deliver cargoes to the development of the occulo-motor neuron or neuromuscular junction. Here, we used an yeast two-hybrid system to identify a protein that interacts with the WD-40 repeat domain of KIF21A and found a specific interaction with Purkinje cell protein-2 (Pcp-2), a small protein also known as L7. Pcp-2 protein bound to the WD-40 domain of KIF21A and KIF21B but not to other KIFs in yeast two-hybrid assays. In addition, this specific interaction was also observed in the glutathione S-transferase pull-down assay. An antibody to Pcp-2 specifically co-immunoprecipitated KIF21A associated with Pcp-2 from mouse brain extracts. These results suggest that Pcp-2 may be involved in the KIF21A-mediated transport as a KIF21A adaptor protein.

• Journal of Life Science
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• v.23 no.8
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• pp.978-983
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• 2013

Kinesin-II, a molecular motor, consists of two different motor subunits, KIF3A and KIF3B, and one large kinesin superfamily-associated protein 3 (KAP3), forming a heterotrimeric complex. KAP3 is associated with the tail domains of motor subunits. However, its exact role remains unclear. Here, we demonstrated KAP3 binding to the carboxyl (C)-terminal tail region of HS-associated protein X-1 (HAX-1). HAX-1 bound to the C-terminal region of KAP3, but not to KIFs (KIF3A, KIF3B, and KIF5B) and the kinesin light chain (KLC) in the yeast two-hybrid assays. The interaction was further confirmed in the glutathione S-transferase (GST) pull-down assay and by co-immunoprecipitation. Anti- HAX-1 antibody as well as anti-KIF3A antibody co-immunoprecipitated KIF3B and KAP3 from mouse brain extracts. These results suggest that KAP3 could mediate the interaction between Kinesin-II and HAX-1.