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Originally published In Press as doi:10.1074/jbc.M005650200 on July 26, 2000

J. Biol. Chem., Vol. 275, Issue 42, 32854-32860, October 20, 2000
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Kinectin-Kinesin Binding Domains and Their Effects on Organelle Motility*

Lee-Lee OngDagger, Angeline P. C. LimDagger, Connie P. N. Er, Sergei A. Kuznetsov§, and Hanry Yu

From the National University Medical Institutes, Faculty of Medicine, National University of Singapore, Singapore 117597 and § Institut für Zellbiologie und Biosystemtechnnik, Fachbereich Biowissenschaften, Universität Rostock, D-18055 Rostock, Germany

Received for publication, June 27, 2000, and in revised form, July 7, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Intracellular organelle motility involves motor proteins that move along microtubules or actin filaments. One of these motor proteins, kinesin, was proposed to bind to kinectin on membrane organelles during movement. Whether kinectin is the kinesin receptor on organelles with a role in organelle motility has been controversial. We have characterized the sites of interaction between human kinectin and conventional kinesin using in vivo and in vitro assays. The kinectin-binding domain on the kinesin tail partially overlaps its head-binding domain and the myosin-Va binding domain. The kinesin-binding domain on kinectin resides near the COOH terminus and enhances the microtubule-stimulated kinesin-ATPase activity, and the overexpression of the kinectin-kinesin binding domains inhibited kinesin-dependent organelle motility in vivo. These data, when combined with other studies, suggest a role for kinectin in organelle motility.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Minus- and plus-end-directed microtubule (MT)1-based organelle motility is important for active cellular processes, especially in asymmetric cells such as neurons (for reviews, see Refs. 1 and 2). Actin-dependent organelle motility has also been proposed to be important for various short range cellular processes (3, 4). In these active transport systems, motor proteins are regulated to move membrane organelles to specific target compartments (5-10). Among the motor proteins, kinesin or its family members power the plus-end-directed MT-based motility (11, 12), while cytoplasmic dynein or its family members drive the minus-end directed motility (13-15). Myosin-V (or its relatives) is one factor shown to be responsible for the actin-based organelle motility (16-18). This myosin has recently been shown to interact with kinesin (19). Motor proteins transport organelles by anchoring to them via some type of linkage. For minus-end-directed MT-based motility, it has been proposed that cytoplasmic dynein binds to organelles via direct lipid linkage (20), via intermediates such as the dynactin complex (21), or via an Arp1-linked receptor using a classical actin-membrane binding mechanism such as spectrin-ankyrin (22-24). For myosin-V, little information is known about its membrane anchor. In the case of kinesin, a membrane protein (kinectin) has been identified to bind kinesin to membrane organelles (25).

Kinectin is a 160-kDa integral membrane protein identified as a membrane anchor essential for kinesin-dependent organelle motility in vitro (25, 26). A shorter 120-kDa kinectin can bind the 160-kDa kinectin as a heterodimer with the amino-terminal trans-membrane domain of the 160-kDa kinectin anchoring to organelles (27). An anti-kinectin monoclonal antibody, VSP4D, inhibited cytoplasmic dynein as well as kinesin functions in both the motor-membrane binding and the organelle motility assays in vitro (26). Plus- and minus-end-directed organelle motility have also been shown to be coupled in vivo (28). Therefore, kinectin has been proposed to be involved in the regulation of the organelle motility (28). Recently, the kinesin tail, where the cargo-binding domain has been postulated has been reported to activate motor activities of the kinesin head and hereby regulate organelle motility (29-31). On the other hand, organelles that normally are driven by cytoplasmic dynein are not affected in kinesin disruption studies (32, 33). These observations, together with a lack of clearer understanding of the kinectin-motor interaction, support the hypothesis that kinectin might simply anchor kinesin to organelle membrane without an active role in organelle motility. Previous efforts to characterize the kinectin-kinesin interaction with various affinity methods have not been successful, since the interaction measured in vitro seems always weaker than expected (34, 35).2 This has led some to question whether kinectin is truly a kinesin receptor on organelles (36).

To address these questions and understand the mechanism of the motor binding to organelles, we have characterized the interacting domains on human kinectin and three known members of the human conventional kinesin family using in vitro and in vivo assays. The kinectin-binding domains on kinesin partially overlap the head-binding domain on the kinesin tail that has been reported to regulate the kinesin motor activities (29). The kinesin-binding domain on kinectin resides in the COOH terminus, and the overexpression of the kinectin-kinesin binding domains in vivo disrupts the kinectin-dependent organelle motility. The kinectin-binding domains also overlap the reported myosin-Va binding domain on kinesin (19). Further, the kinesin-binding domain on kinectin can enhance the microtubule-stimulated kinesin-ATPase activity, supporting the notion that kinectin is the kinesin receptor on organelles not only to anchor kinesin but also to release kinesin from the inactive compact conformation. These and other studies lead us to a better understanding of the kinectin function in organelle motility.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The restriction enzymes used were from Promega (Madison, WI), and all of the other reagents were purchased from Sigma unless otherwise stated.

Construction of the Kinectin Baits-- The human kinectin gene (GenBankTM accession number Z22551) was a generous gift from Dr. Martin Krönke (Christian-Albrechts University of Kiel, Germany). The kinectin bait A (residues 46-444) and bait C (residues 987-1356) were generated by amplifying their respective region using standard polymerase chain reaction protocol (37), and their sequences were verified by sequencing. Bait B (residues 444-1049) was obtained directly by digesting the human kinectin gene with PstI. The three cDNA fragments were directionally subcloned in frame to the GAL4 DNA-binding domain (BD) of the pAS2-1 vector (CLONTECH). The details of the kinectin bait constructs are illustrated in Fig. 1.

Yeast Two-hybrid Screening-- A yeast two-hybrid system (CLONTECH) was used according to the supplied protocol. The kinectin baits were used to screen a human adult brain Matchmaker cDNA library fused to the GAL4 DNA-activation domain (AD) of the pACT2 vector (CLONTECH). The baits and the amplified library were sequentially transformed into yeast strain Y190 (CLONTECH) using the lithium acetate method (38). The transformants were assayed for their expression of his reporter gene by plating them onto the synthetic medium deficient in tryptophan, leucine, and histidine. 25 mM of 3-amino-1,2,4-triazole was included to limit the number of false his positives (39). Colonies that grew successfully in the selective medium were further screened for their expression of the lacZ reporter gene by colony lift assay. The cDNAs in the AD vector of the persistent positive clones were isolated. False positives were further eliminated via testing whether the clones were able to activate both reporter genes with the control bait (BD vector alone).

The primary sequences of the positive clones were determined with an ABI PRISM Big DyeTM Terminator Cycle Sequencing Ready Kit using an ABI PRISMTM 377 DNA sequencer, according to the manufacturer's instructions. The results were analyzed with the LASERGENE (DNASTAR) and the BLAST 2.0 (National Library of Medicine) software.

In Vitro Binding Studies-- Glutathione S-transferase (GST) fusion constructs with the kinectin baits A, B, and C were made by subcloning the corresponding cDNA into the expression vector pGEX4T-1 (Amersham Pharmacia Biotech). The his tag fusion construct of the neuronal kinesin heavy chain (nKHC) was also made by subcloning the nKHC cDNA into an expression vector pRSET (Invitrogen). The fusion proteins were expressed in Escherichia coli strain BL21/pLysS (DE3) (generous gift from Niovi Santama, University of Cyprus and Cyprus Institute of Neurology and Genetics). Protein expression was induced by the addition of isopropyl-beta -D-thiogalactoside. Intact bacterial cells containing the expressed fusion proteins were collected by centrifugation at 6000 × g for 10 min. Protein extracts were obtained by freezing and thawing the cell pellet and resuspended in GST purification buffer (1× phosphate-buffered saline, 50 mM Tris-HCl, pH 8.0, 0.5 mM MgCl2, 0.1% (v/v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride) plus 5 mM dithiothreitol. Equal amounts of the GST fusion proteins with baits A, B, and C were immobilized onto glutathione-agarose beads by end-over-end mixing in a 1.5-ml microcentrifuge tube for 2 h at 4 °C. The beads were washed three times with GST purification buffer to remove unbound proteins. The expressed his-nKHC fusion protein extract was allowed to interact with the GST-bait fusion protein-coated beads for 2 h at 4 °C with end-over-end mixing. Any unbound proteins were removed through extensive washings. The proteins that remained bound to the immobilized GST-baits were released by boiling in SDS gel sample buffer, analyzed by SDS-polyacrylamide gel (12%) electrophoresis (PAGE), and immunoblotting with mouse RGS-his antibody (Qiagen). Antibody binding was detected with goat anti-mouse secondary antibody (Bio-Rad), coupled to alkaline phosphatase according to the manufacturer's specifications.

In Vitro Binding of Cytosolic Kinesin with Kinectin Baits-- Kinectin baits, A, B, and C, were coupled onto glutathione-agarose as described above. Mouse brain cytosolic fractions were prepared as follows. The brain tissues were rinsed once in PBS and frozen in liquid nitrogen and stored at -80 °C until use. The frozen tissues were thawed quickly in one volume of prewarmed PMEE' buffer (35 mM PIPES, pH 7.4, 5 mM MgSO4, 1 mM EGTA, 5 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 10 µg/ml p-tosyl-L-arginine methyl ester, 10 µg/ml tosylphenylalanyl chloromethyl ketone, 1 µg/ml leupeptin, 1 mM dithiothreitol. After complete thawing, the tissue was homogenized with 10 strokes of a Dounce homogenizer, incubated for 15 min on ice, and homogenized with 10 more strokes. This was repeated twice. The homogenate was centrifuged at 16,000 rpm for 15 min at 4 °C. The supernatant (S1) was collected and centrifuged again in a Beckman SW55.1 rotor for 30 min at 35,000 rpm and 4 °C. The resulting supernatant (S2) was adjusted to 1 mM GTP and 20 µM taxol and incubated for 15 min at 37 °C to polymerize the endogenous microtubules. The assembled microtubules were removed by centrifugation for 15 min at 35,000 rpm and 22 °C. The final supernatant (S3 cytosol) was then used as the source of the cytosolic motor kinesin. S3 cytosol was added to the GST-bait fusion protein coated beads and incubated with end-over-end mixing for 2 h at 4 °C. Unbound proteins were removed through extensive washings. The proteins that remained bound to the immobilized GST-baits were released by boiling in SDS gel sample buffer, analyzed by 7.5% SDS-PAGE, and immunoblotting with SUK-4 anti-kinesin heavy chain monoclonal antibody (40) and a goat anti-mouse secondary antibody (Bio-Rad), coupled to alkaline phosphatase according to the manufacturer's specifications.

Mapping Studies for Neuronal Kinesin Heavy Chain-- A series of truncated fragments of the nKHC (N1 to N13) were constructed (Fig. 4) by performing polymerase chain reaction. The amplified fragments were subcloned into the AD vector, and their interaction with the kinectin bait C was assayed using the yeast two-hybrid system as described above. A total of 50 yeast transformants were picked from each truncated clone to assay for the activation of the his and lacZ reporter genes. Clones were considered as positive only when more than 90% of the transformants activated both reporter genes.

Mapping Studies of Human Kinectin-- Ubiquitous kinesin heavy chain (uKHC) (GenBankTM accession number X65873) was provided by Dr. Ronald Vale (Howard Hughes Medical Institute, UCSF). uKHC bait was constructed by amplifying a region between residues 602 and 963 using polymerase chain reaction and subcloned into the BD vector. A series of truncated fragments of the kinectin were constructed (Fig. 1), and their interaction with the uKHC was assayed using the yeast two-hybrid system as described above.

Microtubule-stimulated Kinesin-ATPase Assay with Truncated Kinectin Fragments-- Bovine brain tubulin and kinesin were purified as described previously (41). Microtubule-activated ATPase activity of kinesin in the presence of truncated kinectin fragments at the final concentrations of 0.1 or 0.2 mg/ml was measured at 37 °C as described previously (42, 43). The final concentrations of taxol-stabilized microtubules, kinesin, and kinectin fragments in ATPase assay were 1.0 mg/ml, 15 µg/ml and 0.1 or 0.2 mg/ml, respectively.

In Vivo Kinesin-dependent Organelle Motility Assay-- The kinesin-binding domain on kinectin (KNT+, residues 1188-1288) and a larger fragment containing the domain (K1, residues 987-1356) were subcloned into the pEGFP-C vector for optimal expression in mammalian cells (CLONTECH). KNT- (residues 1049-1146) that showed no interaction with the uKHC bait in yeast two-hybrid screening2 and the pEGFP-C vector alone were used as controls. In addition, the kinectin-binding domain on uKHC (uKHC+, residues 833-900) that interacts with kinectin and a control domain that does not bind kinectin (uKHC-, residues 735-801) were also cloned into the pEGFP-C vector. COS7 cells were cultured and transfected as described above. Transfected cells were identified by GFP expression. Lysosome-specific staining was obtained by incubating cells with 50 nM LysoTracker DND-99 (Molecular Probes, Inc., Eugene, OR) in normal Ringer's solution for 30 min. Lysosome redistribution upon acidification as described previously (44) was assayed. Cells were subsequently fixed with 4% paraformaldehyde, mounted in FluorSave (Calbiochem), and imaged using a Carl Zeiss LSM410 laser-scanning confocal microscope. A total of 90 cells were observed for each clone for their lysosome redistribution. The results were tabulated based on the mean percentage of cells in each of the four categories, namely clustered around cell center, clustered centrally with radiating tubules, dispersed throughout cell, or dispersed with peripheral organelle clusters (44, 45).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Neuronal Kinesin Heavy Chain Interacts with Kinectin-- We performed a yeast two-hybrid assay to screen for proteins that interact with kinectin. Three overlapping kinectin fragments, A, B, and C, were constructed as baits (Fig. 1). Most of the positive clones identified were those that interact with the kinectin bait C. A total of 14 positive clones were identified from the screening of approximately 1.8 × 106 transformants. Among them, two were nKHC cDNAs (GenBankTM accession number U06698). The two clones (2.5 and 1.1 kilobases) code for two nKHC fragments with residues 284-1032 and 601-957, respectively. Both nKHC fragments interacted with the kinectin bait C but not with the control baits (A or B or BD vector alone). Bait C encodes the kinectin fragment (residues 987-1356) that does not contain the predicted leucine zipper motifs (residues 934-962) in the core of a predicted alpha -helical coiled-coil. The leucine zipper motifs reside in the control bait B. Thus, our observed nKHC-bait C interaction is unlikely to be due to nonspecific interaction between the coiled coils.


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  		Fig. 1.   The kinesin-binding domain on kinectin. A, human kinectin baits A (residues 46-444), B (residues 444-1049), and C (residues 987-1356) were constructed by cloning in frame into a GAL4 DNA-BD vector. The baits were transformed separately with the human adult brain cDNA library into yeast strain Y190. The transformants were assayed for their activation of the his and lacZ reporter genes. TM, trans-membrane domain (dark box); LZ, leucine zipper (empty box). The bait C that is also named K1 (residues 987-1356) in mapping studies was truncated by both amino- and COOH-terminal deletion. The interaction of the truncated K1 clones with uKHC (residues 602-963) was assayed by the yeast two-hybrid system. The solid line indicates the cDNA clones whose gene products activated the his and lacZ reporter genes in >90% of the transformants, whereas the discontinued line indicates the clones that activated the reporter genes in <10% of the transformants. B, schematic representation of the kinesin-binding domain on kinectin illustrates the positions of different structural motifs. The hatched area represents the kinesin-binding domain (residues 1188-1288) on kinectin. Structure motifs in this domain include heptad repeats (dotted area), N-linked glycosylation site (CHO), tyrosine kinase phosphorylation site (Y), and casein kinase phosphorylation site (CK2).

To corroborate the yeast two-hybrid data, we performed an in vitro binding study. GST fusion constructs with the kinectin baits A, B, and C were made, and equal amounts of the individual fusion-protein extracts were immobilized onto glutathione-agarose beads. The beads were washed extensively to remove unbound proteins such that only the kinectin baits remained bound to the beads (Fig. 2A). An nKHC fusion construct with a his tag was expressed in E. coli, and the cell extract was allowed to interact with the immobilized GST-bait fusion proteins. The proteins remained bound to the immobilized GST-baits after extensive washings were analyzed by immunoblotting with anti-his antibody (Fig. 2B). Association of the nKHC in vitro with the kinectin bait C, but not with bait A nor B, is consistent with the interactions observed in the yeast two-hybrid assay.


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  		Fig. 2.   In vitro interaction of nKHC with the kinectin bait C. A, SDS-PAGE analysis of the proteins immobilized on glutathione-agarose beads indicates that the GST-bait C fusion protein is the only protein on the beads. B, equal amount of the his-nKHC, his-uKHC, his-KIAA0531 protein extracts was incubated with the bead-immobilized GST-bait fusion proteins. After extensive washings, bound proteins were eluted and fractionated by SDS-PAGE and analyzed in an immunoblot with anti-his antibody. nKHC specifically binds to bait C, but not bait A or bait B. MW, molecular weight marker; TP, total protein; FT, flow-through; W, wash; E, eluate.

The in vitro binding study was also extended to cytosolic kinesin for further assessment of the kinectin-kinesin interaction. Mouse brain cytosol was extracted and incubated with the immobilized GST-bait (kinectin) fusion proteins. The cytosolic proteins remained bound to the immobilized GST-baits after extensive washings were analyzed by immunoblotting with anti-kinesin antibody (Fig. 3). The association of cytosolic kinesin with the kinectin bait C, but not with bait A nor B, supports the observation of specific kinectin-kinesin interaction. Further in an in vivo co-immunoprecipitation experiment, we tagged nKHC with a FLAG tag and co-transfected the FLAG-tagged nKHC with a GST-tagged kinectin bait C into COS7 cells. Immunoprecipitates of the co-transfected COS7 cell extracts via the GST-tagged kinectin bait C on a glutathione-agarose column were analyzed by immunoblotting with anti-FLAG and anti-human kinectin antibodies. The FLAG-nKHC co-precipitates with the GST-bait C.2


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  		Fig. 3.   Interaction of cytosolic kinesin with kinectin baits. GST-kinectin bait A, B, or C was immobilized on glutathione-agarose beads. Mouse brain cytosol was extracted and allowed to interact with the kinectin baits. After extensive washings, bound proteins were eluted and analyzed by SDS-PAGE and immunoblotting with an anti-kinesin antibody. Cytosolic kinesin binds to the kinectin bait C, but not bait A or bait B. TP, total protein; FT, flow-through; W, wash; E, eluate.

Characterization of the Kinectin-binding Domain on Neuronal Kinesin Heavy Chain-- To identify the minimally sufficient domain of nKHC that interacts with kinectin, we have constructed a series of truncated fragments of nKHC (Fig. 4) and assayed for their interaction with the kinectin bait C using the yeast two-hybrid assay. Clones N1, N2, N3, N4, N5, N6, N9, N10, and N11 could interact with bait C as indicated by the activation of the his and lacZ reporter genes, whereas N7, N8, N12, and N13 could not. A minimally sufficient kinectin-binding domain on nKHC, clone nKHC+ that encodes a 54-amino acid fragment (residues 836-890), was deduced from these results, and its interaction with the kinectin bait C was experimentally confirmed using the yeast two-hybrid assay.


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  		Fig. 4.   The kinectin-binding domain on nKHC. The nKHC (residues 601-957) clone was truncated by both amino- and COOH-terminal deletions. The interaction of the truncated clones with the kinectin bait C was tested by the yeast two-hybrid assay. The solid line indicates the cDNA clones whose gene products activated the his and lacZ reporter genes in >90% of the transformants, whereas the discontinued line indicates the clones that activated the reporter genes in <10% of the transformants.

Interaction Sites of Three Human Conventional Kinesin Heavy Chains with Kinectin-- From our yeast two-hybrid screening for kinectin-binding proteins, we have identified another member of the human conventional kinesin heavy chains (KIAA0531) as a kinectin-binding protein. KIAA0531 (GenBankTM accession number AB011103) was reported as one of the 100 large human proteins whose genes were sequenced. KIAA0531 shares 76% of sequence homology with the human uKHC. The mouse homolog of KIAA0531 is KIF5C, whereas the mouse homologs of the other two members of the human conventional kinesin heavy chains (nKHC and uKHC) are KIF5A and KIF5B, respectively (46). Therefore, KIAA0531 has also been called the human KIF5C, which means the third member of the human conventional kinesin heavy chains.

The kinectin-binding domain on KIAA0531 was characterized, experimentally tested for its interaction with the kinectin baits (Fig. 2), and fine mapped (residues 828-897) as described above for nKHC. The protein sequences of the kinectin-binding domains on nKHC (nKHC+) and KIAA0531 were aligned, and they share 93% sequence homology. The kinectin-binding domain of the uKHC was also characterized, tested experimentally for its interaction with the kinectin baits, and fine mapped (residues 833-900) as described above for nKHC. Indeed, this region of the uKHC that shares 91% sequence homology with nKHC and 98% with KIAA0531 also interacts with the kinectin bait C (Fig. 2). Therefore, the same highly conserved domains of all three members of the human conventional kinesin heavy chains interact with kinectin.

The COOH Terminus of Human Kinectin Interacts with Kinesin-- Kinectin is an elongated molecule with multiple domains (47-49). The amino terminus has a trans-membrane domain in the 160-kDa form but not the 120-kDa form of kinectin (27). There are up to six variable domains in the middle to tail (COOH terminus) of kinectin (49). Multiple modification (glycosylation, myristoylation, and phosphorylation) sites in or near these domains were postulated (48, 49) based on sequence data as well as demonstrated experimentally (50). To understand whether and how these modifications modulate kinesin binding to kinectin and involvement in organelle motility, it is important to characterize the kinesin-binding domain on kinectin.

Using a yeast two-hybrid assay, we have established above that uKHC interacts with the kinectin bait C (residues 987-1356). To identify the minimally sufficient domain of kinectin that interacts with uKHC, we have constructed a series of truncated kinectin fragments (Fig. 1) and assayed for their interaction with an uKHC-bait (residues 602-963) that contains the kinectin-binding domain (residues 833-900). Among the 14 clones, 10 were positive as indicated by the activation of the his and lacZ reporter genes in the yeast two-hybrid assay (Fig. 1A). Clones K1, K5, K6, K7, K8, K9, K12, K13, and K14 could interact with the uKHC-bait, whereas K2, K3, K4, K10, and K11 could not. The minimally sufficient kinesin-binding domain KNT+ (residues 1188-1288) on kinectin was deduced from these results, and its interaction with the uKHC-bait was confirmed experimentally with the yeast two-hybrid assay.

The Kinesin-binding Domain on Kinectin Enhances the Microtubule-stimulated Kinesin-ATPase Activity-- Since the COOH terminus of kinectin can bind to the kinesin tail where the head and tail of kinesin interact with each other to maintain the inactive compact conformation, we hypothesize that kinectin is the postulated cargo receptor for kinesin activation, so we tested whether the kinesin-binding domain on kinectin can activate the microtubule-stimulated kinesin-ATPase activity. When purified KNT+ (kinesin-binding domain, residues 1188-1288) and KNT- (negative control, residues 1049-1146) were added to an assay measuring the microtubule-stimulated kinesin-ATPase activity, KNT+ significantly (74 ± 10%) enhanced the kinesin-ATPase activities whereas KNT- did not (Fig. 5).


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  		Fig. 5.   Kinesin-binding domain on kinectin enhances the microtubule-stimulated kinesin-ATPase activity. Microtubule-stimulated kinesin-ATPase activity is significantly (74 ± 10%) enhanced by KNT+ but not by KNT-. The empty bar represents control activity where no kinectin fragment is added. The slash-shaded bar represents activity in the presence of KNT-, and the dot-shaded bar represents activity in the presence of KNT+. The error bar represents S.D.

The Sites of Kinectin-Kinesin Interaction Are Important for Kinesin-dependent Organelle Motility-- To further investigate the physiological relevance of our characterization of the sites of kinectin-kinesin interaction, we have overexpressed kinectin and uKHC fragments in COS7 cells and analyzed their effects on the disruption of the kinesin-dependent organelle motility. In our studies, cDNAs encoding the kinectin fragments K1 (residues 987-1356), KNT+ (kinesin-binding domain, residues 1188-1288), and KNT- (residues 1049-1146) (Fig. 1) were cloned into the pEGFP-C vector. cDNAs encoding the uKHC fragments uKHC+ (kinectin-binding domain, residues 833-900) and uKHC- (residues 735-801) were also cloned into the pEGFP-C vector. The cDNAs in the vector and the pEGFP-C vector alone were introduced into COS7 cells. The cells were subjected to brief acetate treatment to change the medium pH from 7.2 to 6.9, which has been reported to cause redistribution of lysosomes to the cell periphery (44). Such redistribution is kinesin-dependent, since mutation of the kinesin motor domain inhibited the redistribution (45).

When the lysosome redistribution upon acidification was quantified, the controls (untransfected cells or cells transfected with KNT- or uKHC- in pEGFP-C vector or pEGFP-C vector alone) showed the typical lysosome redistribution, with 90, 90, 84, and 86% of the lysosomes spreading throughout the cytoplasm, respectively, or near the cell periphery (Fig. 6 and Table I). In cells overexpressing K1, KNT+, and uKHC+ in pEGFP-C vector, there were only 19, 33, and 30% of the lysosomes redistributing throughout the cytoplasm or near the cell periphery upon acidification. The inhibition of the lysosome redistribution upon acidification was slightly more pronounced in K1-transfected than in KNT+- or uKHC+-transfected COS7 cells. Nonetheless, overexpression of the kinectin and uKHC fragments that contain the interaction sites inhibited the lysosome redistribution, confirming that the characterized sites of kinectin-kinesin interaction are important for the kinesin-dependent organelle motility.


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  		Fig. 6.   An example of the overexpression of binding domains in COS7 cells and disruption of lysosome redistribution upon acetate treatment. Lysosome distribution after exposure to 70 mM Ringer's acetate was followed with 50 nM LysoTracker and observed by confocal microscopy. Transfected cells were identified with GFP expression (green). Four phenotypes were typically observed: 1) lysosomes tightly accumulate around the cell center (A and A'); 2) central clustering of lysosomes (B and B'); 3) lysosomes disperse to the cell periphery (C and C'); or 4) lysosomes distribute throughout the entire cytoplasm (D and D'). K1- and KNT+-transfected cells have their plus-end-directed transport inhibited so that their lysosomes remain in the cell center (A, A', B, and B'). In GFP- and KNT--transfected control cells, their plus-end directed transport is not inhibited, and their lysosomes disperse to the cell periphery or throughout the entire cytoplasm upon acetate treatment (C, C', D, and D'). Bar, 100 µm.

                              
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Table I
Effects of overexpressing kinectin-kinesin binding domains in COS7 cells
KNT- and uKHC- are negative controls from the regions of the molecules that are not involved in the kinectin-kinesin interaction as determined by the yeast two-hybrid assay. KNT+ is the kinesin-binding domain on kinectin, and K1 is a larger COOH-terminal fragment containing KNT+. uKHC+ is the kinectin-binding domain on uKHC.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In our search for kinectin-associated proteins using yeast two-hybrid, direct binding, and co-immunoprecipitation assays, we have identified members of the conventional kinesin family as kinectin-associated proteins. This provided independent evidence to the original experiments with the kinesin affinity approach (25) to confirm the interaction between kinectin and kinesin. This helps to settle a controversy on the role of kinectin as the kinesin receptor (36). We have also constructed a series of deletion mutants of kinectin and kinesin and characterized the minimally sufficient sites for the kinectin-kinesin interaction. The kinectin-binding domain on kinesin resides near the COOH terminus within a region called the "coiled-coil tail," which is adjacent to the globular tail at the extreme COOH terminus (51). This coiled-coil tail has been postulated as the cargo-binding domain on kinesin (34). In other words, kinectin binds to the region where cargoes bind. This is consistent with the proposed role of kinectin as the membrane receptor for kinesin binding to membrane cargoes.

The resolution of the fine mapping of the kinectin-binding domain on nKHC was restricted by the qualitative nature of the yeast two-hybrid assay. Further deletion at the single amino acid level gave a gradient of results with respect to whether the two reporter genes were activated.2 Therefore, the minimally sufficient kinectin-binding domain boundaries were deduced from N6, N7, N11, and N12 (Fig. 4). The N6 and N11 showed positive in >90% of the transfected yeast cells in activating the two reporter genes, whereas N7 and N12 showed positive in <10% of the transfected cells. A more sensitive assay is needed to more precisely determine the boundaries of the binding domain.

The kinectin-binding domain on kinesin is highly conserved for all three members of the human conventional kinesin family. This is the only highly conserved region on kinesin other than the motor domain on the kinesin head (51). Therefore, it is not surprising that all three members of the human conventional kinesin family interact with kinectin in our assays. Whether the slight variants near the edge of the characterized kinectin-binding domain on kinesin contribute to variations in the interaction between the different kinesins and kinectin has to be defined when the precise boundaries of the kinectin-binding domain are defined at the single amino acid level with a more quantitative assay.

The characterized kinectin-binding domain on kinesin partially overlaps two other important domains (Fig. 7). One is the myosin-Va binding domain (19) that overlaps 23 residues on the amino edge of the kinectin-binding domain on kinesin (Fig. 7). The interaction of myosin-Va and kinesin has been postulated as a key link between the microtubule- and actin-based organelle motility (19). The other important domain is the kinesin head-binding domain on the kinesin tail that overlaps 12 residues on the COOH edge of the kinectin-binding domain on kinesin (Fig. 7). The kinesin tail has been shown to interact with the kinesin globular head to inhibit the motor activities of the head (29). It was reported that deletion or mutation of the kinesin tail constitutively activated the kinesin motor activities (29, 30). The implication was that the cargo binding to the kinesin tail could prevent the kinesin head-tail interaction and thereby derepress the kinesin motor activities (30). Since kinectin is the cargo anchor for kinesin and the kinectin-binding domain on the kinesin tail overlaps its head-binding domain and the myosin-Va binding domain (Fig. 7), we suggest that kinectin plays a bigger role in the organelle motility than simply anchoring kinesin. Indeed, we have observed that the kinesin-binding domain on kinectin can significantly enhance the microtubule-stimulated kinesin-ATPase activity (Fig. 5). We have also observed that the overexpression of the sites of kinectin-kinesin interaction in COS7 cells disrupted the lysosome redistribution upon acidification (Fig. 6 and Table I), which is a well established kinesin-dependent organelle motility assay in vivo (44, 45).


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  		Fig. 7.   Kinectin-binding, head-binding, and myosin-Va-binding domains on uKHC. The binding domains for myosin-Va (residues 763-855; thick line), kinectin (residues 833-900; thin line), and kinesin head (residues 889-955; dotted line) were aligned, and the overlapping sequences were boxed.

If kinectin-kinesin interaction is important in organelle motility, then it is likely that the interaction is regulated. Phosphorylation has been proposed to be a way to modulate organelle motility and kinesin association with membrane cargoes in vivo (50). We have identified only one putative modification site (casein kinase 2 phosphorylation) near the edge of the kinectin-binding domain). It is conceivable that either casein kinase 2 phosphorylation of the kinectin-binding domain directly or the association with the kinesin light chains or other kinesin-associated phosphoproteins (52, 53) can potentially modulate the kinesin binding to kinectin. Further experiments will be needed to test these hypotheses.

The regulation of the kinectin-kinesin interaction can also occur on kinectin, since it is an extended molecule with multiple domains (49). Kinectin is also highly phosphorylated, and phosphorylation states can affect organelle motility (50). We have characterized the kinesin-binding domain on kinectin using the same in vitro and in vivo assays as for the kinectin-binding domains on kinesin. The kinesin-binding domain resides near the COOH terminus of kinectin within a region near the COOH-edge of the putative coiled-coils. It covers the last two heptad repeats (17 and 18) with a break of 26 amino acids that can render some potential flexibility in modulating the kinectin-kinesin interaction. These heptads were found throughout most of the kinectin molecule (residues 327-1362) and are essential features for forming alpha -helical coiled-coil structures (47, 48). There are two potential phosphorylation sites for tyrosine kinase and casein kinase 2 and a potential N-glycosylation site in the kinesin-binding domain that may be involved in such a modulation. It is interesting that the kinesin-binding domain on kinectin also resides in the region where variable domains 3 (residues 1177-1200) and 4 (residues 1229-1256) reside (49). We have observed that the kinectin isoforms lacking the two variable domains do exist in specific cell types.2 Whether these kinectin isoforms can still interact with kinesin or other members of the kinesin superfamily is not yet known.

Previous efforts with in vitro affinity approaches such as blot overlay, surface plasma resonance, or direct binding assay to characterize the interaction between kinectin and kinesin have failed to identify the sites of interaction.2 One common observation from these previous efforts is that the measured interaction seems to be weaker (~50× lower affinity)2 compared with the measured kinesin-organelle interaction (34, 35). Such a discrepancy might be due to the lack of post-translational modifications on the bacterially expressed kinectin. Alternatively, regulatory modifications such as phosphorylation or association with other regulatory proteins might play a role in modulating the interaction. Since kinectin-kinesin interaction seems to be playing an important role in organelle motility, it is likely that such an interaction is tightly regulated such that the high affinity interaction only occurs in specific conformation or modification. Since the yeast two-hybrid assay is known to detect weak or transient interactions between proteins (54), it is not surprising that we have been able to use this assay to characterize the sites of kinectin-kinesin interaction. Our characterization of the kinectin-kinesin interaction is consistent with other studies leading to our current understanding of the kinesin-dependent organelle motility.

    ACKNOWLEDGEMENTS

We thank Drs. Gareth Griffiths (EMBL), Shu Wang (Institute of Materials Research and Engineering), and Wing Chan (Institute of Molecular and Cell Biology) for critical review of the manuscript. We thank Dr. Mike Sheetz (Columbia) for encouragement to initiate the project and Dr. Ron Vale (University of California, San Francisco) for providing the human uKHC cDNA. We also acknowledge the excellent technical assistance from other members of the Yu laboratory and the National University Medical Institutes core units and laboratories.

    FOOTNOTES

* The work was supported by National Medical Research Council of Singapore Grant RP6600014 and Human Frontier Science Program Grant RG-227/98.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Research Scholar of the National University of Singapore.

To whom correspondence should be addressed: NUMI, Block MD11 03-02 Clinical Research Center, 10 Medical Dr., Singapore 117597. Tel.: 65-874-8066; Fax: 65-872-7150; E-mail: nmiyuh@nus.edu.sg.

Published, JBC Papers in Press, July 26, 2000, DOI 10.1074/jbc.M005650200

2 L.-L. Ong, A. P. C. Lim, C. P. N. Er, S. A. Kuznetsov, and H. Yu, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: MT, microtubule; BD, binding domain; AD, activation domain; GST, glutathione S-transferase; nKHC, neuronal kinesin heavy chain; PAGE, polyacrylamide gel electrophoresis; uKHC, ubiquitous kinesin heavy chain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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