In the present study we report on the identification of the new KIF1 binding protein KBP. The association of KBP with KIF1Bα was verified in vitro, in 293 cells transiently overexpressing both proteins and in untransfected BHK cells. Anti-sense experiments revealed that KBP is required for a proper distribution of mitochondria in the cell. Finally, we showed that KBP increases KIF1Bα motility in vitro.
The bait used in the yeast two-hybrid screen that led to the identification of KBP contained the sequence from the motor domain to the PTPD1 binding region (amino acids 261–800). Additional data from binding experiments of KBP to KIF1C and KIF1Bα deletion mutants allowed us to narrow down the binding sequence of KIF1B/C for KBP to the carboxyl-terminal region of the motor domain. A high amino acid sequence homology of 90% between KIF1C and KIF1Bα in this region supports the conclusion that KBP can bind to both proteins, and a manuscript describing the role of KBP for KIF1C is in preparation. It is important to note that the binding region of KBP is located close to the K-loop, a region in KIF1 KLPs that increases the affinity of the motor domain for MT [24
]. For KIF1B, several splice variants involving or located next to the K-loop are known [13
], and it will be interesting to check whether the presence of these variants has an effect on interaction with KBP [12
]. An interaction with the KIF1Bβ variant would open a role for KBP as a player in neurodegenerative diseases. Recently, the role of the K-loop in a 679 amino acid construct of the rat orthologue of KIF1C was analysed [25
]. In in vitro experiments this protein was not chemically processive, the K-loop increased the affinity for MT and stabilized a weak binding mode with ADP in the active site. The hypothesis was formed that rat KIF1C could work in teams of motor proteins, which could generate processive movement of attached organelles. This would be a similar mechanism as proposed for KIF1A [22
]. In our motility assays we observed that KBP increased the motility of beads in extracts from KIF1Bα overexpressing cells. When the motor protein was overexpressed with KBP, it traveled average distances of 2–3 μm and maximally 6 μm, while with KBPΔ251–281 we observed KIF1Bα moving an average of 1 μm. These experiments do not contradict the data of Rogers et al. and Klopfenstein et al. since it is possible that KBP facilitates grouping of motor proteins and thus increases the motility. However, it also indicates an alternative mechanism where associated proteins like KBP could increase the motility of single molecules of KIF1B.
Numerous proteins have been identified that interact with KLPs [26
]. These proteins may determine the localization of the KLP complex or which cargo is transported. Very little, however, is known about associating proteins that regulate either the cargo binding of the KLP or its activity. Such a KLP associating protein that is required for the KLP function has been described in yeast. The MT minus-end directed motor protein Kar3p is important for meiosis I in S. cerevisiae
, and a knock out leads to an arrest in prophase I [28
]. The associating protein Cik1p targets Kar3p to MT, implicating this as a reason why loss of Cik1p also impairs meiosis, although the effect is less severe [29
]. Several studies describe a regulatory role of the carboxyl-terminus in conventional kinesin processivity. Kinesin light chains were reported to inhibit the binding of the heavy chain to MT [30
]. Further, the kinesin tail domain by itself has an ATPase inhibiting role that is relieved by cargo binding [31
]. One function of the light chains and the carboxyl-terminus of the heavy chain may thus be to ensure that kinesin is active only in the presence of its cargo. However, most of these studies have been done in vitro and cannot account for effects of regulating proteins.
The drastic consequences of the loss of KBP expression for mitochondria localization could also indicate that KBP is targeting KIF1Bα to the mitochondria. However, KIF1Bα is attached to mitochondria also after depletion of KBP (Fig. ). In addition, KLPs normally bind their cargo with carboxyl-terminal sequences, whereas KBP interacts with KIF1Bα amino-terminal sequences. Therefore, we do not think that KBP is important for the cargo – kinesin connection. Alternatively and mentioned before, KBP may regulate the KIF1Bα activity, i.e., in the absence of KBP, KIF1B is not or less able to move mitochondria.
Only two widely expressed KLPs have been reported to transport mitochondria, KIF1Bα and KIF5B, while KIF5A and KIF5C are expressed predominantly in neuronal tissues [34
]. KIF5B did not interact with KBP, and it should not be affected by KBP depletion. Thus, even if there is some expression of KIF5B, a major role of KIF1Bα for mitochondria transport in NIH3T3 cells is likely. Recently, the Drosophila
protein MILTON was described that associates with the KIF5 ortholog kinesin heavy chain and may play a role similar to KBP since its mutation impairs MT-plus end directed mitochondrial transport in axons and synapses [35
]. In addition to KLPs, dynein motor proteins probably can transport mitochondria in cells [36
], and this transport would occur in a MT minus-end direction. When force moving mitochondria to the MT plus-ends is reduced, a perinuclear localization of the mitochondria would result. On the other hand, a complete depolymerization of MTs by nocodazole treatment led to a diffusion of mitochondria throughout the cytoplasm [21
], demonstrating the specificity of the consequences of KBP depletion.