Gene name - Kinesin associated protein 3
Synonyms - DmKAP
Cytological map position - 10B3
Function - cytoskeleton
Symbol - Kap3
FlyBase ID: FBgn0028421
Genetic map position - 1-
Classification - non-motor accessory subunit of Kinesin II
Cellular location - cytoplasmic
|Chen, G. Y., Arginteanu, D. F. and Hancock, W. O. (2015). Processivity of the kinesin-2 KIF3A results from rear head gating and not front head gating. J Biol Chem 290: 10274-10294. PubMed ID: 25657001
The kinesin-2 family motor KIF3A/B works together with dynein to bidirectionally transport intraflagellar particles, melanosomes, and neuronal vesicles. Compared with kinesin-1, kinesin-2 is less processive, and its processivity is more sensitive to load, suggesting that processivity may be controlled by different gating mechanisms. This study used stopped-flow and steady-state kinetics experiments, along with single-molecule and multimotor assays to characterize the entire kinetic cycle of a KIF3A homodimer that exhibits motility similar to that of full-length KIF3A/B. Upon first encounter with a microtubule, the motor rapidly exchanges both mADP and mATP. When adenosine 5'-[(β,γ)-imido]triphosphate was used to entrap the motor in a two-head-bound state, exchange kinetics were unchanged, indicating that rearward strain in the two-head-bound state does not alter nucleotide binding to the front head. A similar lack of front head gating was found when intramolecular strain was enhanced by shortening the neck linker domain from 17 to 14 residues. In single-molecule assays in ADP, the motor dissociates at 2.1 s-1, 20-fold slower than the stepping rate, demonstrating the presence of rear head gating. In microtubule pelleting assays, the KD(Mt) is similar in ADP and ATP. The data and accompanying simulations suggest that, rather than KIF3A processivity resulting from strain-dependent regulation of nucleotide binding (front head gating), the motor spends a significant fraction of its hydrolysis cycle in a low affinity state but dissociates only slowly from this state. This work provides a mechanism to explain differences in the load-dependent properties of kinesin-1 and kinesin-2.
|Ahmed, Z., Mazumdar, S. and Ray, K. (2019). Kinesin associated protein, DmKAP, binding harnesses the C-terminal ends of the Drosophila kinesin-2 stalk heterodimer. Biochem Biophys Res Commun. PubMed ID: 31784087
The heterotrimeric kinesin-2 consists of two distinct motor subunits and an accessory protein, KAP, which binds to the coiled-coil stalk domains and one of the tail domains of the motor subunits. Genetic studies revealed that KAP is essential for the kinesin-2 functions in cilia, flagella, and axon. However, the structural significance of the KAP binding on kinesin-2 assembly and stability is not known. Uusing the Fluorescence Lifetime assay this study showed that DmKAP binding selectively reduces the distance between the C-terminal ends of Drosophila kinesin-2 stalk heterodimer. Insertion of a missense mutation (E551K) in the Drosophila kinesin-2alpha stalk fragment, which was shown to reduce the structural dynamics of the stalk heterodimer earlier, also reduced the distances at both the N- and C-terminal ends of the stalk heterodimer independent of DmKAP. The zipping effect, particularly at the N-terminal end of the mutant stalk heterodimer, is further enhanced in the presence of DmKAP. Together, these results suggest that the KAP binding could alter the structural dynamics of kinesin-2 stalk heterodimer at the C-terminal end and stabilize the association between the stalk domains.
Kinesin II-mediated anterograde intraflagellar transport (IFT) is essential for the assembly and maintenance of flagella and cilia in various cell types. Kinesin associated protein (KAP) is identified as the non-motor accessory subunit of Kinesin II, but its role in the corresponding motor function has not been characterized. Mutations in the Drosophila KAP (DmKap) gene eliminate the sensory cilia as well as the sound-evoked potentials of Johnston's organ (JO) neurons. Ultrastructure analysis of these mutants reveals that the ciliary axonemes are absent. Mutations in Klp64D, which codes for a Kinesin II motor subunit in Drosophila, show similar ciliary defects. All these defects are rescued by exclusive expression of DmKAP and KLP64D/KIF3A in the JO neurons of respective mutants. Furthermore, reduced copy number of the DmKap gene was found to enhance the defects of hypomorphic Klp64D alleles. Unexpectedly, however, both the DmKap and the Klp64D mutant adults produce vigorously motile sperm with normal axonemes. It is concluded that KAP plays an essential role in Kinesin II function, which is required for the axoneme growth and maintenance of the cilia in Drosophila type I sensory neurons. However, the flagellar assembly in Drosophila spermatids does not require Kinesin II and is independent of IFT (Sarpal, 2003).
The type I sense organs of Drosophila, namely, the chordotonal organs, the mechanosensory bristles, and the taste and olfactory sensilla, are innervated by bipolar sensory neurons, each with a single dendritic cilium containing 9+0 axonemal organization of microtubules. Each chordotonal organ neuron has a single long cilium, the assembly of which begins from the distal basal bodies in the dendrite; each cilium is attached to a tube-shaped dendritic cap at the apex. [Type I external mechanosensory bristles include three support cells and one neuron. The innermost support cell (sheath cell) forms a tubular extension around the sensory process and produces an extracellular dendritic cap that covers the cilium tip (Keil, 1997)]. Such chordotonal organs are found in the second antennal segment, where they are required for hearing and are termed Johnston's organ (JO), and in various other parts of the body, where they are required for proprioception. Mutants with auditory defects have defective dendritic cilia in JO neurons. The mechanisms that form and maintain such ciliary structures are only beginning to be elucidated (Sarpal, 2003 and references therein).
Extensive studies in Chlamydomonas and other organisms have shown that flagellar and ciliary proteins are synthesized in the cell body and are then transported in preassembled IFT complexes to the distal tip of the flagella by a mechanism called 'intraflagellar transport' (IFT). This process is essential for the assembly and maintenance (via turnover) of flagella. Members of the Kinesin II family of motor proteins and cytoplasmic Dynein motors are known to play important roles in IFT (see Rosenbaum, 2002, for a review; Sarpal, 2003 and references therein).
Kinesin II holoenzyme was purified from sea urchin embryos as a heterotrimer of two dissimilar motor subunits and a third non-motor accessory subunit called 'kinesin-associated protein'(KAP; Cole, 1993). The motor subunits contain a globular plus end-directed, microtubule-dependent ATPase domain at the N terminus, and they associate with each other via a coiled-coil stalk domain in the middle. The KAP subunit is estimated to bind to the C-terminal tail domains of the motor subunits (Marszalek, 2000b). Support for the trimeric composition of Kinesin II has been provided in various vertebrate and invertebrate organisms, and this motor has been implicated in a variety of intracellular transport processes in vivo (Sarpal, 2003 and references therein).
Kinesin II is the motor for the anterograde IFT, and studies with different types of ciliated cells from Chlamydomonas to humans have shown that it is essential for ciliogenesis as well as for flagellar growth and maintenance. For example, conditional mutations in the fla10 gene of Chlamydomonas block anterograde IFT in the flagella when grown at nonpermissive temperatures. As a result, the flagella gradually reduce and eventually disappear (Cole, 1996). FLA10 is homologous to the Kinesin II motor subunit, which is associated with the IFT complex subunits in the flagella. Disruption of Kinesin II activity in sea urchin cilia (Morris, 1997) and Tetrahymena flagella (Brown, 1999) are also shown to affect axonemal assembly. Similarly, mutations in the osm-3 locus of Caenorhabditis elegans cause defective chemotaxis behavior, and the distal segments of the dendritic cilia of the chemosensory neurons are absent (Shakir, 1993; Starich, 1995; Tabish, 1995). These studies suggested that Kinesin II is required for the transport of essential ciliary components in these neurons. In addition, mouse KIF3A and KIF3B play an important role in the assembly of motile cilia in embryonic nodal cells (Nonaka, 1998). KIF3A is also localized to the connecting cilia of photoreceptor neurons in the retina, which have 9+0 organization of microtubules in the axoneme (Beech, 1996; Muresan, 1997; Whitehead, 1999), and KIF3A has been implicated in the transport of opsin and arrestin to the outer photoreceptor compartment (Marszalek, 2000a). Although these pieces of evidence strongly indicate that Kinesin II is a good candidate for the transport of components required for the assembly and maintenance of eukaryotic cilia and flagella, little is known about the role of KAP in this process. An in vivo analysis in C. elegans with GFP-tagged OSM-6 and KAP has shown that the two proteins transport along the sensory cilium at a rate similar to the in vitro rate of Kinesin II (Orozco, 1999); this finding indicates that KAP is associated with the IFT complex in the cilium (Sarpal, 2003).
KLP64D, KLP68D, and DmKAP are predicted to form the Kinesin II holoenzyme in Drosophila, and they are shown to coexpress in ciliated sensory neurons during embryogenesis (Pesavento, 1994; Ray, 1999; Sarpal, 2002). The expression levels are particularly high in the neurons innervating the lateral chordotonal organs as well as the anterior sense organs (Ray, 1999; Sarpal, 2002), and these have well-defined dendritic cilia. This indicates that Drosophila Kinesin II could play an important role in ciliogenesis. Therefore, to define a functional assay to study the role of KAP and other Kinesin II-associated proteins in ciliogenesis, auditory responses of Drosophila carrying mutations in the DmKap and Klp64D genes were studied. This revealed that KAP plays a critical role in Kinesin II function during ciliogenesis in these type I sensory neurons of Drosophila, and the genetic interaction study suggests that DmKAP interacts with a Kinesin II motor subunit in vivo (Sarpal, 2003).
Other studies have shown that vertebrate homologs of KAP protein associate with the Kinesin II motor subunits (Shimizu, 1998; Wedaman 1996; Yamazaki, 1996), which are implicated in ciliogenesis in various cell types (see Marszalek, 2000b for a review). However, the precise role of KAP in this process has been unknown. Mutations in the DmKap locus are shown to be haplo-insufficient in Klp64D hypomorphic backgrounds and enhance both the auditory reception defects as well as the ciliogenesis defects of the Klp64D alleles. This study establishes that KAP plays a critical role in Kinesin II motor function in vivo. A recent study has further shown that mutations in the nompB locus of Drosophila, which encodes an IFT88/Tg737/OSM-5 homologous protein, also affect auditory responses of JO neurons and that mutations in Klp64D reduce the GFP-NOMPB localization in the cilia (Han, 2003). Therefore, the auditory system of Drosophila can be used to further study in vivo interactions between various IFT components (Sarpal, 2003).
Studies in Chlamydomonas have established that the Kinesin II motor subunits associate with a soluble, protein-rich IFT complex, which they transport toward the distal ends of flagella (Kozminski, 1993; Kozminski, 1995). This anterograde IFT seems to play a critical role in maintaining the flagellar length and activity (see Rosenbaum, 2002, for a review). The electron microscopic data presented in this study show that both DmKap and Klp64D gene functions are critical for proper axoneme growth in the dendritic cilia of the JO neurons. This suggests that Kinesin II may transport essential axonemal components into the dendritic cilia for the growth and maintenance of the axoneme structure. Thus, Kinesin II activity in the sensory cilia of Drosophila and in the motile cilia and flagella of other organisms appears to be conserved. In contrast, the spermatogenesis in Drosophila seems to be independent of anterograde IFT. This indicates the presence of hitherto unknown mechanisms of axonemal assembly operating in constructing these unusually long flagella (Sarpal, 2003).
It is concluded that DmKap interacts with Klp64D, and these two gene products are involved in axonemal assembly in the sensory cilia of JO neurons, but not in sperm. The genetic interaction study suggests that DmKAP plays an important role in Kinesin II motor activity in vivo. This work has established a genetic interaction paradigm to further study the in vivo functions of Kinesin II and IFT proteins by using auditory function as an assay (Sarpal, 2003).
See NCBI's Conserved Domain Database for information about pfam05804.3, KAP
date revised: 15 October 2004
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