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Originally published In Press as doi:10.1074/jbc.M600522200 on July 11, 2006

J. Biol. Chem., Vol. 281, Issue 37, 27216-27228, September 15, 2006
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Mapping the GRIF-1 Binding Domain of the Kinesin, KIF5C, Substantiates a Role for GRIF-1 as an Adaptor Protein in the Anterograde Trafficking of Cargoes*Formula

Miriam J. Smith12, Karine Pozo1, Kieran Brickley, and F. Anne Stephenson3

From the Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom

Received for publication, January 18, 2006 , and in revised form, July 10, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{gamma}-Aminobutyric acid, type A (GABAA) receptor interacting factor-1 (GRIF-1) and N-acetylglucosamine transferase interacting protein (OIP) 106 are both members of a newly identified coiled-coil family of proteins. They are kinesin-associated proteins proposed to function as adaptors in the anterograde trafficking of organelles to synapses. Here we have studied in more detail the interaction between the prototypic kinesin heavy chain, KIF5C, kinesin light chain, and GRIF-1. The GRIF-1 binding site of KIF5C was mapped using truncation constructs in yeast two-hybrid interaction assays, co-immunoprecipitations, and co-localization studies following expression in mammalian cells. Using these approaches, it was shown that GRIF-1 and the KIF5C binding domain of GRIF-1, GRIF-1-(124-283), associated with the KIF5C non-motor domain. Refined studies using yeast two-hybrid interactions and co-immunoprecipitations showed that GRIF-1 and GRIF-1-(124-283) associated with the cargo binding region within the KIF5C non-motor domain. Substantiation that the GRIF-1-KIF5C interaction was direct was shown by fluorescence resonance energy transfer analyses using fluorescently tagged GRIF-1 and KIF5C constructs. A significant fluorescence resonance energy transfer value was found between the C-terminal EYFP-tagged KIF5C and ECFP-GRIF-1, the C-terminal EYFP-tagged KIF5C non-motor domain and ECFP-GRIF-1, but not between the N-terminal EYFP-tagged KIF5C nor the EYFP-KIF5C motor domain and ECFP-GRIF-1, thus confirming direct association between the two proteins at the KIF5C C-terminal and GRIF-1 N-terminal regions. Co-immunoprecipitation and confocal imaging strategies further showed that GRIF-1 can bind to the tetrameric kinesin light-chain/kinesin heavy-chain complex. These findings support a role for GRIF-1 as a kinesin adaptor molecule requisite for the anterograde delivery of defined cargoes such as mitochondria and/or vesicles incorporating beta2 subunit-containing GABAA receptors, in the brain.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{gamma}-Aminobutyric acid, type A (GABAA)4 receptor interacting factor-1 (GRIF-1) was initially identified from rat brain by a yeast two-hybrid screen searching for GABAA receptor clustering and trafficking proteins (1). It was shown to associate at least in vitro with GABAA receptor beta2 subunits. GRIF-1 is highly expressed in excitable tissue, most notably in the brain and in the heart (1). It is the orthologue of the human protein, beta-O-linked N-acetylglucosamine transferase (OGT) interacting protein 98 (OIP98, also termed ALS2CR3, huMilt2, or TRAK2), and it is the homologue of the protein OIP106 (also termed huMilt1 and TRAK1 (2)). GRIF-1 is also probably the orthologue of the Drosophila protein, Milton, a kinesin-associated protein that is involved in the transport of mitochondria to the synapses in retina (3, 4). GRIF-1 and OIP106 have also been shown to aggregate mitochondria following overexpression in mammalian cells (5). Recently, the gene encoding OIP106 (TRAK1), Trak1, was identified as the mutated gene in hyrt mice, an animal model of hypertonia (6). hyrt mice were shown to have a deficit of GABAA receptors; wild-type OIP106 (TRAK1) was shown to co-immunoprecipitate with GABAA receptor {alpha}1 subunits in extracts of mouse brain stem and spinal cord (6). Although the mutant OIP106 was shown to still associate with GABAA receptors, it was concluded that OIP106 (TRAK1) may play a crucial role in regulating endocytic trafficking of receptors and dysfunction disrupts receptor homeostasis leading to hypertonia (6). Thus, GRIF-1, OIP106, and Milton belong to a newly identified family of coiled-coil proteins putatively involved in the trafficking of organelles and GABAA receptors to synapses.

Two additional proteins that associate with high affinity with both GRIF-1 and OIP106 have been identified. These proteins are the enzyme, OGT (2),5 and, consistent with the role in trafficking, the molecular motor protein, kinesin (5). Kinesin was also shown to be immunoprecipitated from detergent extracts of Drosophila heads with anti-Milton antibodies, thereby demonstrating, albeit reportedly indirect, an association between Milton and kinesin (3). Kinesins belong to the kinesin superfamily, i.e. KIFs. KIFs are microtubule-based mechanochemical motors that are involved primarily in the anterograde transport of organelles and protein complexes (reviewed in Refs. 7 and 8). They are particularly important in transport processes in neurons where it is necessary to deliver defined cargoes from the cell body along axons and dendrites to pre- and post-synaptic sites. In brain, GRIF-1 associates predominantly with KIF5A, whereas in heart association is predominantly with KIF5B (5). Following overexpression in human embryonic kidney (HEK) 293 cells, GRIF-1 will co-associate with exogenous KIF5C (5). KIF5A, KIF5B, and KIF5C are members of the kinesin-1 family. KIF5B is ubiquitously expressed, whereas KIF5A and KIF5C are only expressed in neurons (7). They are conventional kinesin heavy chains belonging to the kinesin-1 family, and they share at least 60% amino acid identity. It is probable that each has a binding site for GRIF-1 and that GRIF-1 is promiscuous with regard to association with KIF5 subtypes.

Kinesin-1 proteins are tetramers formed by the association of two kinesin heavy chains (KHC or alternatively KIF5) and two kinesin light chains (KLC). The KHC is formed from an N-terminal motor domain that contains the microtubule and ATP binding sites and a C-terminal non-motor domain. This domain includes a neck, a coiled-coil stalk region, and a cargo binding site in its C-terminal region. The KLC interacts with the KHC via the stalk region. Cargoes bind to either the KLC or to the KHC of kinesin-1 proteins. Increasing evidence suggests that this interaction is mediated by an adaptor protein. For example, mitochondria and syntaxin-1-containing vesicles are attached to the KHC cargo binding domain by the adaptor protein, syntabulin, for their transport to synapses (9); JIP-3, a c-Jun N-terminal kinase (JNK) signaling pathway protein, binds to a six tetratricopeptide motif in KLCs to transport the cargo, amyloid precursor protein (APP; 10); KIF17 forms a complex with mLin10 in the transportation of N-methyl-D-aspartate receptor NR2B subunits to the synapse (11, 12); and glutamate receptor interacting protein 1 (GRIP1) is an adaptor protein linking the {alpha}-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of glutamate receptor containing vesicles to KIF5 (13).

In this report, we have studied the interaction between GRIF-1 and the prototypic kinesin-1, KIF5C, and kinesin light chain. The results obtained provide supporting evidence that GRIF-1 is a kinesin adaptor protein involved in motor-dependent trafficking of organelles and/or proteins. Please note a preliminary report of some of these findings (14).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Constructs and Antibodies—pCISGRIF-1, splice form GRIF-1a, hereafter referred to as GRIF-1, pCMVTag4aGRIF-1 (C-terminal FLAG-tagged GRIF-1), pGADT7GRIF-1-(1-913), pGAD10GRIF-1-(8-633), pGADT7GRIF-1-(124-283), and affinity-purified rabbit anti-GRIF-18-633 antibodies were all as previously described (1). pMBL33 GABAA receptor beta2 intracellular loop (303-427; IL), pcDNAHisMaxKIF5C, and affinity-purified sheep anti-GRIF-1874-889 were as described in Brickley et al. (5). The kinesin heavy-chain KIF5C fragments, corresponding to the motor domain (1-335), the non-motor domain (336-957), coil 1 (336-542), coil 2 (594-804), and the cargo binding domain, coil 3 (827-957), were PCR-amplified from the construct, pBluescriptSKII+KIAA0531 and cloned in-frame into the EcoR1 and SalI restriction sites of the modified mammalian expression vector, pCMVTag4a, to generate the constructs pCMV-KIF5C-(1-335), pCMV-KIF5C-(336-957), pCMV-KIF5C-(336-542), pCMV-KIF5C-(593-804), and pCMV-KIF5C-(827-957) each with an N-terminal c-Myc tag. The same fragments were also cloned into the EcoR1 and SalI restriction sites of the DNA binding domain yeast expression vector, pMBL33, to generate the constructs, pMBL33-KIF5C-(1-335), pMBL33-KIF5C-(336-957), pMBL33-KIF5C-(336-542), pMBL33-KIF5C-(593-804), and pMBL33-KIF5C-(827-957). The kinesin light chain was amplified by PCR from a kinesin light chain cDNA in the pHA vector and subcloned in-frame into pMBL33 using EcoR1/SalI and into pCMVTag4a using EcoR1/SalI to generate an N-terminal c-Myc-tagged KLC. Full-length GRIF-1 was amplified from pCISGRIF-1 by PCR and subcloned in-frame in the EcoRI and SalI restriction sites of pECFP-C1 (BD Biosciences, Clontech, Palo Alto, CA) to generate pECFP-GRIF-1, i.e. GRIF-1 with an N-terminal enhanced cyan fluorescent protein (ECFP) tag. KIF5C was amplified from pcDNAHisMaxKIF5C and subcloned into either the EcoRI and SalI restriction sites of pEYFP-C1 (BD Biosciences, Clontech) to generate pEYFP-KIF5C, i.e. KIF5C with an N-terminal tag, or into the NheI site of pEYFP to generate pKIF5C-enhanced yellow fluorescent protein (EYFP), i.e. KIF5C with a C-terminal EYFP tag. The KIF5C motor domain, KIF5C-(1-335), and KIF5C non-motor domain, KIF5C-(336-957), were amplified by PCR from pcDNAHisMaxKIF5C. KIF5C-(1-335) was subcloned in-frame in the XhoI/EcoRI sites and KIF5C-(336-957) in the NheI site, respectively, of pEYFP to generate pEYFP-KIF5C-(1-335) with an EYFP tag at the N terminus and pEYFP-KIF5C-(336-957) with an EYFP tag at the C-terminal end of KIF5C. All constructs were verified by DNA sequencing (MWG-Biotech AG, Ebersberg, Germany). Further, for all constructs used in yeast two-hybrid interaction assays, the expression of fusion proteins was verified by immunoblotting (data not shown). A schematic of all constructs used is shown in Fig. 1.

Anti-GFP and anti-KIF5C938-957 antibodies were from Abcam Ltd. (Cambridge, UK); anti-c-Myc clone 4A6 antibodies were from Upstate (Charlottesville, VA); and anti-mouse-Ig Alexa Fluor 633 antibodies from Invitrogen. Anti-FLAG antibodies were raised inhouse against the FLAG amino acid sequence, DYKDDDDK, with an N-terminal cysteine coupled to thyroglobulin and affinity-purified for use using CDYKDDDDK covalently coupled via the cysteine to thiopropyl-activated Sepharose.

Mammalian Cell Transfection and Preparation of Detergent-solubilized Extracts of Transfected Cells—For immunoprecipitation assays, HEK 293 cells were transfected with combinations of FLAG-tagged pCMVGRIF-1-(1-913) together with c-Myc-tagged pCMV-KIF5C-(1-335), pCMV-KIF5C-(336-957), pCMV-KIF5C-(336-542), pCMV-KIF5C-(593-804), or pCMV-KIF5C-(827-957), using the calcium phosphate method using a 1:1 ratio with a total of 10 µg of DNA per 250-ml culture flask. In pCISGRIF-1, pEYFP-KIF5C, and pCM-VKLC triple transfections, a ratio of 1:1:3 was used with a total of 10 µg of DNA per 250-ml culture flask. For the characterization of the ECFP- and EYFP-tagged proteins, HEK 293 cells were transfected with either pCISGRIF-1 plus pEYFP-KIF5C; pCISGRIF-1 plus pKIF5C-EYFP; pCISGRIF-1 plus pEYFP-KIF5C-(336-957); pCISGRIF-1 plus pEYFP-KIF5C-(1-335); or pcDNAHismaxKIF5C plus pECFP-GRIF-1 and pECFP-GRIF-1 plus pEYFP-KIF5C or pECFP-GRIF-1 plus pKIF5C-EYFP all using a 1:1 ratio with a total of 10 µg of DNA per 250-ml culture flask. Cells were harvested 24-48 h post-transfection, and cell homogenates were either analyzed by immunoblotting or, alternatively, transfected cell homogenates were solubilized with 10 mM HEPES, pH 7.5, 145 mM NaCl, 1 mM EGTA, 0.1 mM MgCl2, benzamidine (1 µg/ml), bacitracin (1 µg/ml), soybean trypsin inhibitor (1 µg/ml), chicken egg trypsin inhibitor (1 µg/ml), and phenylmethylsulfonyl fluoride (1 mM) and 1% (v/v) Triton X-100 for 60 min at 4 °C. Detergent-solubilized extracts were collected following centrifugation for 40 min at 4 °C at 100 000 x g (6). For confocal microscopy studies, HEK 293 cells or COS-7 cells were plated onto poly-D-lysine (0.1 mg/ml)-coated coverslips and transfected using the calcium phosphate method and plasmid DNA ratios as above. Transfections using pDsRed1Mito to visualize mitochondria used a ratio of 1 pDsRed1-Mito:19 ECFP-GRIF-1 for double transfections, and 1 pDsREd1Mito:8 ECFP-GRIF-1:8 EYFP-KIF5C for triples.


Figure 1
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  		FIGURE 1.
A schematic diagram showing the kinesin and GRIF-1 constructs used in this study. A, KIF5C and kinesin light chain constructs used for the yeast two-hybrid and immunoprecipitation studies; B, KIF5C-EYFP constructs used for the imaging and FRET studies; C, GRIF-1 constructs used for the imaging and FRET studies depicting also, the kinesin binding domain, GRIF-1-(124-283); D, a summary of the nomenclatures in the literature for the two proteins encoded by the GRIF-1 coiled-coil gene family.

 
Immunoblotting—Immunoblotting was performed as previously described using 25-50 µg of protein/sample precipitated using the chloroform/methanol method and SDS-PAGE under reducing conditions in either 7.5% or 12.5% polyacrylamide slab minigels (1, 5). Rabbit and mouse horseradish-linked secondary antibodies (Amersham Biosciences) were used at a final dilution of 1:2000, and immunoreactivities were detected using the ECL Western blotting system.

Immunoprecipitation Assays—Detergent-solubilized extracts of transfected HEK 293 cells (diluted to 1 mg of protein/ml with a final Triton X-100 concentration of 0.5% (v/v); 20 ml) were incubated for polyclonal antibodies, with either affinity-purified rabbit anti-FLAG antibodies (10 µg), protein A-purified non-immune rabbit Ig (10 µg) as control or, affinity-purified sheep anti-GRIF-1874-889 antibodies (10 µg), protein G-purified non-immune sheep Ig as control for 1.5 h at 37 °C (6). Protein A (rabbit polyclonal antibodies) or protein G (sheep polyclonal antibodies) Sepharose (20 µl) was added, and samples were incubated for 1 h at 37°C. Immune pellets were collected by centrifugation for 3 s at 10,000 x g and washed with 3 x 1.5 ml of 10 mM HEPES, pH 7.5, 145 mM NaCl, 1 mM EGTA, 0.1 mM MgCl2, benzamidine (1 µg/ml), bacitracin (1 µg/ml), soybean trypsin inhibitor (1 µg/ml), chicken egg trypsin inhibitor (1 µg/ml), phenylmethylsulfonyl fluoride (1 mM), and 0.5% (v/v) Triton X-100 and then were analyzed by immunoblotting.

Yeast Two-hybrid Interaction Assays—Yeast two-hybrid assays were carried out using a modified LexA system as described previously (5). The Saccharomyces cerevisiae strain, L40 (MATa his3{Delta}200 trp1-901 leu2-3112 ade2 LYS2::(4lexAop-HIS3) URA3::(8lexAoplacZ) GAL4), was transformed with combinations of GRIF-1 fragments in the activation domain (AD) plasmid, pGADT7, together with each of the KIF5C fragments in the DNA binding domain plasmid, pMBL33. Negative control transformations were carried out using empty AD or DNA binding domain vectors. Positive control transformations were carried out using AD-GRIF-1-(8-633) with pMBL33beta2-IL, i.e. the DNA binding domain vector containing the residues 303-427 of the intracellular loop of the GABAA receptor beta2 subunit. Resulting colonies were assessed for reporter gene activation by growth on nutritional selection agar lacking tryptophan, leucine, and histidine.


Figure 2
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  		FIGURE 2.
GRIF-1 associates with the non-motor domain of KIF5C: demonstration by immunoprecipitation following co-expression of GRIF-1 and KIF5 constructs in HEK 293 cells. HEK 293 cells were transfected with pCISGRIF-1-FLAG and either pCMV-KIF5C-(1-335), pCMV-KIF5C-(336-957), or pCMVKLC, detergent extracts of cell homogenates were prepared 48 h post-transfection, and immunoprecipitation assays were carried out using either affinity-purified anti-FLAG antibodies or non-immune Ig and immune pellets analyzed by immunoblotting using anti-FLAG (20% of immune pellet) and anti-c-Myc (80% of immune pellet) antibodies all as described under "Experimental Procedures." A and B, immunoprecipitation from HEK 293 cells transfected with pCISGRIF-1FLAG and pCMV-KIF5C-(1-335); C and D, immunoprecipitation from HEK 293 cells transfected with pCISGRIF-1FLAG and pCMV-KIF5C-(336-957); and E and F, pCISGRIF-1FLAG and pCMVKLC. In A, C, and E, immunoblots were probed with anti-FLAG antibodies, and in B, D, and F immunoblots were probed with anti-c-Myc antibodies. Gel lanes are: 1, detergent-solubilized transfected HEK 293 cell homogenate; 2, non-immune pellet; 3, anti-FLAG pellet. Arrows denote: A, C, and E, GRIF-1-FLAG, 115 kDa; B, c-Myc-tagged KIF5C-(1-335), 43 kDa; D, c-Myc-tagged KIF5C-(336-957), 98 kDa; and F, c-Myc-tagged KLC, 70 kDa. The positions of molecular mass standards (kDa) are shown on the right. The immunoblots are representative of at least n = 3 immunoprecipitations from n = 3 independent transfections.

 
Confocal Microscopy and FRET Efficiency Determinations— Transfected cells 20-40 h post-transfection were rinsed three times with ice-cold phosphate-buffered saline followed by fixation for 10 min with 4% (w/v) ice-cold paraformaldehyde. Coverslips were rinsed three times with phosphate-buffered saline and mounted onto a microscope slide using 10 µl of mounting solution containing an anti-fading agent (Citifluor, Citifluor Ltd., Leicester, UK). The coverslips were sealed and kept at 4 °C until analysis. For visualization of cells transfected with pCM-VKLC, post-fixation coverslips were incubated with anti-c-Myc antibodies (1:4000) overnight at 37 °C, washed as described (1), and incubated with anti-mouse-Ig Alexa Fluor 633, and again washed as previously described (1). Cells were imaged using an inverted LSM510 META Zeiss confocal microscope in the multitrack mode. EYFP was excited with the {lambda} = 514 nm laser line, and emitted light was collected with a long pass filter LP530. ECFP was excited at {lambda} = 458 nm, and light was collected with a band-pass filter BP 475-525. The absence of bleed-through was checked prior to each experiment by using the Meta software of the microscope. This software enables the decomposition of the mixed emission spectra (i.e. generated from cells co-transfected with both ECFP and EYFP) into the emission of the single fluorescent dyes, i.e. ECFP and EYFP, according to reference spectra generated from cells transfected with either ECFP or EYFP alone. Images were analyzed using the Image Browser software (Zeiss) available with the microscope.

For FRET analyses, again the Meta software mode of the LSM510 META Zeiss confocal microscope was used. FRET efficiency was measured by acceptor photobleaching as described previously by Liu et al. (15) and Nashmi et al. (16). That is, co-transfected HEK 293 cells were firstly imaged with the {lambda} = 458 nm laser to visualize ECFP-tagged proteins. A defined area of co-localization within one cell was selected, and EYFP was photobleached for ~30 s using the {lambda} = 514 nm laser at full power. Cells were imaged post-photobleaching with the {lambda} = 458 nm laser. FRET was measured as the increase of ECFP fluorescence after photobleaching where values were taken at t = 1.6 s prior to photobleaching and t = ~32 s post-photobleaching and were corrected for background fluorescence (usually of the order of ~10%) determined by the imaging of untransfected HEK 293 cells. The relative FRET efficiency was calculated by the following: (1 - [pre-bleached intensity of ECFP/post-bleached intensity of ECFP]) x 100% (14). For each FRET experiment, pseudo-FRET was always determined by applying the photobleaching protocol to cells transfected with pECFP alone. Pseudo-FRET values (typically ~2.9%) were always subtracted from the calculated FRET efficiencies of the test samples. As a further control, an area of the cell that was not photobleached was also analyzed in parallel for FRET to ensure that the FRET efficiency values determined were not artifacts due to the photobleaching protocol. A positive control, an ECFP-EYFP tandem construct linked by two amino acids (17), and a negative control, co-transfection of the separate clones pECFP and pEYFP, were both used to validate the system.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
GRIF-1 Associates with the KIF5C Non-motor Domain: Demonstration by Both Yeast Two-hybrid Interaction Assays and Immunoprecipitation Strategies
We have previously shown that GRIF-1 associates with endogenous kinesin in the brain and in the heart by co-immunoprecipitation of GRIF-1 and kinesin using anti-GRIF-1874-889 antibodies. Association was also demonstrated, again by co-immunoprecipitation, between exogenous GRIF-1 and exogenous KIF5C both co-expressed in HEK 293 cells. Further, yeast two-hybrid interaction assays using GRIF-1 and KIF5C as fish and bait, respectively, suggested that association between the two proteins is probably direct and involves the first coiled-coil domain of GRIF-1, GRIF-1-(124-283) (5). To delineate the GRIF-1 binding domain of KIF5C initially, the KIF5C motor domain, i.e. KIF5C-(1-335), and KIF5C non-motor domain, i.e. KIF5C-(336-957), were subcloned in-frame into either the yeast two-hybrid DNA binding domain vector, pMBL33, or the modified mammalian expression vector, pCMVTag4a, to yield N-terminally c-Myc-tagged fusion proteins. The association between GRIF-1 and KIF5C was then studied by both yeast two-hybrid interaction assays and immunoprecipitation strategies following the co-expression of GRIF-1 and KIF5C in HEK 293 cells. The results are summarized in Table 1 and Fig. 2.


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  		TABLE 1
GRIF-1 interacts directly with the KIF5C non-motor domain: demonstration by yeast two-hybrid interaction assays The yeast strain, L40, was co-transformed with bait and fish constructs and transformants grown on -2 SD, i.e. -W and -L, and -3 SD, i.e. -W, -L, and -H. Colonies from -2 plates were re-streaked onto new plates, and colony growth was determined as described under "Experimental Procedures."

 


Figure 3
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  		FIGURE 3.
GRIF-1 associates with the KIF5C cargo binding domain: demonstration by immunoprecipitation following co-expression of GRIF-1 and KIF5C truncation constructs in HEK 293 cells. HEK 293 cells were transfected with pCISGRIF-1-FLAG and either pCMV-KIF5C C1-(336-542), pCMV-KIF5C C2-(593-804), or pCMV-KIF5C C3-(827-957), detergent extracts of cell homogenates were prepared 48 h post-transfection, and immunoprecipitation assays were carried out using either affinity-purified anti-FLAG antibodies or non-immune Ig and immune pellets analyzed by immunoblotting using anti-FLAG (20% of immune pellet) and anti-c-Myc (80% of immune pellet) antibodies, all as described under "Experimental Procedures." A and B, immunoprecipitation from HEK 293 cells transfected with pCISGRIF-1FLAG and pCMV-KIF5C-(336-542); C and D, immunoprecipitation from HEK 293 cells transfected with pCISGRIF-1FLAG and pCMV-KIF5C-(593-8-4); E and F, pCISGRIF-1FLAG and pCMV-KIF5C-(827-957). In A, C, and E, immunoblots were probed with anti-FLAG antibodies and in B, D, and F, immunoblots were probed with anti-c-Myc antibodies. Gel lanes are: 1, detergent-solubilized transfected HEK 293 cell homogenate; 2, non-immune pellet; 3, anti-FLAG pellet. Arrows denote: A, GRIF-1-FLAG, 115 kDa; B, c-Myc-tagged KIF5C-(336-542), 37 kDa; C, GRIF-1-FLAG, 115 kDa; D, c-Myc-tagged KIF5C-(593-804), 31 kDa; E, GRIF-1-FLAG, 115 kDa; and F, c-Myc-tagged KIF5C-(827-957), 20 kDa. The positions of molecular mass standards (kDa) are shown on the right. The immunoblots are representative of at least n = 3 immunoprecipitations from n = 3 independent transfections.

 
Co-transformation of yeast with pGADT7GRIF-1-(1-913) with pMBL33KIF5C-(336-957) revealed a significant interaction between the two proteins as determined by nutritional selection on SD media -W, -L, and -H media (Table 1). An interaction was also found between both GRIF-1-(8-633) and GRIF-1-(124-283) and KIF5C-(336-957). This was in contrast to yeast co-transformed with KIF5C-(1-335) and either full-length or truncated GRIF-1 constructs where no evidence for protein-protein association was found (Table 1). The same results were obtained in immunoprecipitation studies. FLAG-tagged GRIF-1 was co-expressed in HEK 293 cells with either c-Myc-tagged KIF5C-(1-335) (43 kDa) or KIF5C-(336-957) (98 kDa), detergent extracts prepared and immunoprecipitation carried out using anti-FLAG antibodies. In each case, anti-FLAG immunoreactivity with ~114 kDa was detected in the immune pellets (Fig. 2). Anti-c-Myc antibody immunoreactivity was, however, only present for GRIF-1/KIF5C-(336-957) transfectants further demonstrating an association between GRIF-1 and the C-terminal KIF5C non-motor domain.

The above experiments were also repeated for KLC (70 kDa). No association between GRIF-1 and KLC was detected by either yeast two-hybrid or co-immunoprecipitation assays (Table 1 and Fig. 2).

GRIF-1 Associates with the KIF5C C3 Cargo Binding Domain: Demonstration by Both Yeast Two-hybrid Interaction Assays and Immunoprecipitation Strategies
The GRIF-1 binding domain was further refined by generating three truncation constructs, KIF5C-(336-542), KIF5C-(593-804), and KIF5C-(827-957), of the KIF5C non-motor domain in both yeast DNA binding domain and mammalian expression vectors. These corresponded to three predicted coiled-coil domains in the non-motor domain where KIF5C-(336-542) and KIF5C-(593-804) encompassed the KIF5C stalk region and KIF5C-(827-957) the cargo binding domain. All three constructs were used, as above, in both yeast two-hybrid interaction assays and immunoprecipitation experiments following the co-expression of GRIF-1 and KIF5C-(336-542) (37 kDa), KIF5C-(593-804) (31 kDa), or KIF5C-(827-957) (20 kDa) truncated constructs in HEK 293 cells. The results are summarized in Table 2 and Fig. 3. Both experimental paradigms demonstrate that it is only the KIF5C-(827-957) cargo binding domain that associates with GRIF-1. This domain associates with full-length GRIF-1 and the GRIF-1-(8-633) and GRIF-1-(124-283) fragments.


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  		TABLE 2
GRIF-1 interacts directly with the KIF5C cargo binding domain coil 3: demonstration by yeast two-hybrid interaction assays The yeast strain, L40, was co-transformed with bait and fish constructs and transformants grown on -2 SD, i.e. -W and -L and -3 SD, i.e. -W, -L, and -H. Colonies from -2 plates were re-streaked onto new plates and colony growth was determined as described under "Experimental Procedures."

 


Figure 4
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  		FIGURE 4.
Characterization of ECFP-GRIF-1 and EYFP-KIF5C: molecular size determination and co-association properties. In A and B, HEK 293 cells were transfected with either pCISGRIF-1, pECFP-GRIF-1, pcDNAHisMAXKIF5C, pEYFP-KIF5C, or pKIF5C-EYFP, cell homogenates were prepared 24 h post-transfection, and samples were analyzed by immunoblotting with antibody specificities as shown in the abscissae. A, lanes are: 1 and 4, untransfected cells; 2 and 5, cells transfected with pCISGRIF-1; 3 and 6, cells transfected with pECFP-GRIF-1. B, lanes are: 1 and 5, untransfected cells; 2 and 6, cells transfected with pcDNAHisMAXKIF5C; 3 and 7, cells transfected with pEYFP-KIF5C; and 4 and 8, cells transfected with pKIF5C-EYFP. In C-E, HEK 293 cells were co-transfected with either pECFP-GRIF-1 plus pcDNAHisMAXKIF5C (C), pECFP-GRIF-1 plus pEYFP-KIF5C (D), or pCISGRIF-1 plus pEYFP-KIF5C (E), homogenates were collected 24 h post-transfection, and immunoprecipitations were carried out using either sheep anti-GRIF-1874-889 antibodies or non-immune Ig and immune pellets analyzed by immunoblotting using antibody specificities as shown in the abscissae, all as described under "Experimental Procedures." The lane layout is identical with: lanes 1 and 4, detergent-soluble fraction; 2 and 5, immune pellet; and 3 and 6, control pellet. Arrows denote: A, GRIF-1, 115 kDa; B, KIF5C, 115 kDa; EYFP-KIF5C, 143 kDa; C, ECFP-GRIF-1, 143 kDa; KIF5C, 115 kDa; D, ECFP-GRIF-1, 143 kDa; EYFP-KIF5C, 143 kDa; and E, GRIF-1, 115 kDa; EYFP-KIF5C, 143 kDa. The positions of molecular mass standards (kDa) are shown on the right. The immunoblots are representative of at least n = 3 immunoprecipitations from n = 3 independent transfections.

 
Confocal Microscopy Studies of the Association between GRIF-1 and KIF5C
Characterization of ECFP-GRIF-1 and EYFP-KIF5C Constructs— The following in-frame constructs were generated by molecular cloning: full-length N- and C-terminally tagged KIF5C-EYFP, an N-terminal EYFP-tagged KIF5C-(1-335) motor domain, a C-terminal EYFP-tagged KIF5C-(336-957) non-motor domain, and an N-terminal ECFP-GRIF-1 (Fig. 1). Each was characterized with respect to molecular size by immunoblotting following expression in HEK 293 cells using anti-GFP antibodies and anti-GRIF-1 or anti-KIF5C antibodies. This was to ensure that the respective EYFP- or ECFP-tagged proteins did not undergo proteolytic digestion, thus the fluorescent moieties remained attached to the target proteins. The results for the full-length KIF5C constructs and GRIF-1 are shown in Fig. 4 and for the KIF5C-(1-335) motor domain and KIF5C-(336-957) non-motor domains in Fig. 5. In all cases, an increase in molecular weight for the tagged proteins was observed consistent with the addition of ECFP or EYFP. Further, a single band was found for all constructs in immunoblots using anti-GFP antibodies. The molecular masses of the tagged proteins were: GRIF-1, 115 kDa; ECFP-GRIF-1, 143 kDa; KIF5C, 115 kDa; EYFP-KIF5C, 143 kDa; c-Myc-tagged KIF5C-(1-335), 39 kDa; EYFP-KIF5C-(1-335), 66 kDa; c-Myc-tagged KIF5C-(336-957), 98 kDa; and EYFP-KIF5C-(336-957), 139 kDa.

It was also requisite to demonstrate that the tagged proteins behaved as wild type. This was demonstrated here by the ability of the tagged protein to co-associate with the non-tagged or tagged binding partner. Thus the following pairwise combinations were co-expressed in HEK 293 cells, immunoprecipitations carried out with anti-GRIF-1874-889 antibodies, and immune pellets analyzed by anti-GRIF-1, anti-KIF5C, and anti-GFP antibodies: ECFP-GRIF-1 plus KIF5C; ECFP-GRIF-1 plus EYFP-KIF5C; GRIF-1 plus EYFP-KIF5C; ECFP-GRIF-1 plus KIF5C-EYFP; GRIF-1 plus KIF5C-EYFP; GRIF-1 plus EYFP-KIF5C-(1-335); ECFP-GRIF-1 plus EYFP-KIF5C-(1-335); GRIF-1 plus EYFP-KIF5C-(336-957); and ECFP-GRIF-1 plus EYFP-KIF5C-(336-957). The results are shown for full-length constructs in Fig. 4 (E-G) and for the KIF5C motor and non-motor domains in Fig. 5 (C-F). In all cases except as expected for the KIF5C-(1-335) and GRIF-1 double transfectants, a specific association was found between the tagged-untagged or tagged-tagged GRIF-1/KIF5C combinations. Note that the results for the immunoprecipitations for the C-terminal-tagged KIF5C constructs, i.e. KIF5C-EYFP, are not shown, because they gave identical results to those found for the immunoprecipitations of N-terminal-tagged KIF5C and GRIF-1/ECFP-GRIF-1.


Figure 5
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  		FIGURE 5.
Characterization of EYFP-KIF5C-(1-335) and EYFP-KIF5C-(336-957): molecular size determination and co-association properties. In A and B, HEK 293 cells were transfected with either pCMV-KIF5C-(1-335), pEYFP-KIF5C-(1-335), pCMV-KIF5C-(336-957), or pEYFP-KIF5C-(336-957), cell homogenates were prepared 24 h post-transfection, and samples were analyzed by immunoblotting with antibody specificities as shown in the abscissae. A, lanes are: 1 and 4, untransfected cells; 2 and 5, cells transfected with pCMV-KIF5C-(1-335); 3 and 6, cells transfected with pEYFP-KIF5C-(336-957). B, lanes are: 1 and 4, untransfected cells; 2 and 5, cells transfected with pCMV-KIF5C-(336-957); and 3 and 6, cells transfected with pEYFP-KIF5C-(336-957). In C-F, HEK 293 cells were co-transfected with either pCISGRIF-1 plus pEYFP-KIF5C-(1-335) (C), pECFP-GRIF-1 plus pEYFP-KIF5C-(1-335) (D), pCISGRIF-1 plus pEYFP-KIF5C-(336-957) (E), or pECFP-GRIF-1 plus pEYFPKIF5C-(336-957) (F), homogenates were collected 24 h post-transfection, and immunoprecipitations were carried out using either sheep anti-GRIF-1874-889 antibodies or non-immune Ig and immune pellets analyzed by immunoblotting using antibody specificities as shown in the abscissae all as described under "Experimental Procedures." The lane layout is identical with: 1 and 4, detergent-soluble fraction; 2 and 5, control pellet; and 3 and 6, immune pellet. Arrows denote: A, c-Myc-tagged KIF5C-(1-335), 39 kDa; EYFP-KIF5C-(1-335), 66 kDa; B, c-Myc-tagged KIF5C-(336-957), 98 kDa; EYFP-KIF5C-(336-957), 139 kDa; C, EYFP-KIF5C-(1-335), 66 kDa; ECFP-GRIF-1, 143 kDa; D, EYFP-KIF5C-(1-335), 66 kDa; ECFP-GRIF-1, 143 kDa; E, EYFP-KIF5C-(336-957), 139 kDa; GRIF-1, 115 kDa; and F, EYFP-KIF5C-(336-957), 139 kDa; ECFP-GRIF-1 143 kDa. The positions of molecular mass standards (kDa) are shown on the right. The immunoblots are representative of at least n = 3 immunoprecipitations from n = 3 independent transfections.

 
Co-localization Studies—In transfected COS-7 cells, ECFP-GRIF-1 was found to be distributed throughout the cell cytoplasm with some areas showing notable enrichment at sites close to the nucleus (Fig. 6, B and C). This localization pattern is similar to that reported for the expression of GRIF-1 in HEK 293 cells (1) where the enriched areas were shown to co-localize with aggregated mitochondria (5). EYFP-KIF5C and KIF5C-EYFP expressed alone in COS-7 cells showed the same distribution profile with fluorescence visible throughout the cell cytoplasm, and, when they are present, EYFP-KIF5C was concentrated at the ends of cellular extensions (Fig. 6E). When ECFP-GRIF-1 plus EYFP-KIF5C or KIF5C-EYFP was co-expressed, the distribution of ECFP-GRIF-1 was changed. In all cells imaged (n = 35), the majority of ECFP-GRIF-1 was recruited to KIF5C-enriched areas, which were particularly apparent at the end of cellular extensions. In cells where no processes were evident, co-localization was seen adjacent to the cell membrane (supplemental Fig. S1). In some co-transfected cells (19 out of 35 cells; 54%), some ECFP-GRIF-1 was still found in the cell cytoplasm where it was not associated with EYFP-KIF5C (Fig. 6, G and H). This may be a result of overexpression of ECFP-GRIF-1 in those particular cells. Fig. 6 also shows the distribution of mitochondria in COS-7 cells transfected with either ECFP-GRIF-1 (L-P), ECFP-KIF5C (Q-U), or ECFP-GRIF-1 plus EYFP-KIF5C (V-AA). Aggregated mitochondria are only seen in cells when ECFP-GRIF-1 was overexpressed; i.e. compare Fig. 6N (ECFP-GRIF-1 transfectant) with Fig. 6S (EYFP-KIF5C transfectant).

EYFP-KIF5C-(1-335) expressed alone showed a diffuse distribution throughout the whole cell, including in 55% of transfected cells (16 of 29 cells), and the cell nucleus (Fig. 7B). In addition, in ~24% of transfected cells (7 of 29 cells), KIF5C-(1-335) was associated with filamentous structures thought to be the microtubules, consistent with the existence of a microtubule binding site within the KIF5C-(1-335) (supplemental Fig. S2). This distribution pattern was not changed by co-expression of EYFP-KIF5C-(1-335) with ECFP-GRIF-1 (Fig. 7, E-G). EYFP-KIF5C-(336-957) expressed alone was localized as filamentous structures in the cell cytoplasm (Fig. 7J). This distribution was similar to that reported by Navone et al. (18) following overexpression in CV-1 monkey kidney endothelial cells of a vesicular stomatitis virus-tagged KHC construct that contained the KHC C-terminal portion of the {alpha}-helical coiled-coil rod and the C-terminal tail. The filamentous labeling found by Navone et al. (18) co-localized with microtubules and a microtubule binding site within the non-motor domain was identified (18). In the presence of ECFP-GRIF-1 the distribution pattern of EYFP-KIF5C-(336-957) was changed. It was always co-localized with ECFP-GRIF-1-rich regions close to the nucleus (Fig. 7, M and O). This pattern is reminiscent of the localization of GRIF-1 with aggregated mitochondria as reported for GRIF-1-transfected HEK 293 cells (5). Indeed, when COS-7 cells were transfected with pECFP-GRIF-1, pEYFP-KIF5C-(336-957), and pDsRed1Mito to label mitochondria, all three fluorescent moieties were co-localized close to the cell nucleus (Fig. 7, Q-V).


Figure 6
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  		FIGURE 6.
Confocal microscopy imaging of ECFP-GRIF-1 and EYFP-KIF5C expressed in COS-7 cells. ECFP-GRIF-1 and/or EYFP-KIF5C and/or DsRed1-Mito were co-expressed in COS-7 cells, cells were fixed 24-40 h post-transfection and imaged by confocal microscopy all as described under "Experimental Procedures." A-C, ECFP-GRIF-1; D-F, EYFP-KIF5C and G-K, co-expression of ECFP-GRIF-1 plus KIF5C-EYFP; L-P, ECFP-GRIF-1 plus DsRed1-Mito; Q-U, ECFP-KIF5C plus DsRed1-Mito; V-AA, ECFP-GRIF-1 plus EYFP-KIF5C plus DsRed1-Mito. A, D, G, L, Q, and V are images with saturated fluorescence intensity to show the complete cell outline; B, E, H, I, J, M, N, O, R, S, T, W, X, Y, and Z are a single confocal section of a selected transfected cell; C, F, K, P, U, and AA are pixel intensity profiles of the line scans shown in B, E, J, O, T, and Z, respectively; J, O, T, and Z are the merged images of (H plus I), (M plus N), (R plus S), and (W plus X plus Y), respectively, to show the co-localization. Note that in G-J and L-O, the transfected cell is multinucleated. It has been previously reported that there is a higher percentage of multinucleated cells when cells transfected with KHC clones are compared with untransfected cells (18). However, multinucleate and uninucleate transfected cells showed the same distribution of KIF5C in single transfections and co-distribution of KIF5C and GRIF-1 in co-transfected cells. Images are representative of n = 35 cells from at least n = 3 independent transfections. Scale bars are 10 µm.

 


Figure 7
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  		FIGURE 7.
Confocal microscopy imaging of ECFP-GRIF-1 and EYFP-tagged KIF5C-(1-335) and KIF5C-(336-957) expressed in COS-7 cells. ECFP-GRIF-1 and/or EYFP-KIF5C-(1-335), EYFP-KIF5C-(336-957), and/or DsRed1-Mito were co-expressed in COS-7 cells, cells were fixed 24-40 h post-transfection and imaged by confocal microscopy all as described under "Experimental Procedures." A-C, EYFP-KIF5C-(1-335); D-H, EYFP-KIF5C-(1-335) plus ECFP-GRIF-1; I-K, EYFP-KIF5C-(336-957); L-P, EYFP-KIF5C-(336-957) plus ECFP-GRIF-1; and Q-V, ECFP-GRIF-1 plus EYFP-KIF5C-(336-957) plus DsRed1-Mito. A, D, I, L, and Q are images with saturated fluorescence intensity to show the complete cell outline; B, E, F, G, J, M, N, O, R, S, T, and U are a single confocal section of a selected transfected cell; C, H, K, P, and V, pixel intensity profile of the line scans shown in B, G, J, O, and U, respectively; G, merged E and F; O, merged M and N; and U is merged R, S, and T to show areas of co-localization. Images are representative of at least n = 25 cells from n = 3 independent transfections. Scale bars are 10 µm.

 
FRET Measurements—To test whether ECFP-GRIF-1 associated directly with fluorescently tagged KIF5C, FRET studies were carried out in transfected HEK 293 cells. FRET efficiencies were measured by acceptor photobleaching. It was necessary to carry out these measurements in HEK 293 cells rather than COS-7 cells, because in the latter, the expression level of the fluorescent constructs was relatively low thus exposure to the appropriate laser for cell imaging resulted in the photobleaching of either EYFP or ECFP. In HEK 293 cells, the expression of the constructs was higher due to both a higher transfection efficiency and greater protein expression thus circumventing problems due to the photobleaching. Note that similar subcellular distribution profiles were found for the expression of single and pairwise GRIF-1/KIF5C combinations for both COS-7 cells and HEK 293 cells, i.e. GRIF-1/KIF5C and GRIF-1/KIF5C-(336-957) both co-localized.

In initial studies, optimum conditions were established for the measurement of FRET efficiency by acceptor photobleaching using positive and negative ECFP/EYFP controls (Fig. 8). A FRET efficiency of 24.6% ± 2.0 (n = 22 cells) was determined for the positive control (ECFP-EYFP tandem construct) and a FRET efficiency of 3.8% ± 0.5 (n = 18 cells) was measured for the negative control (pECFP and pEYFP). The percentages of FRET efficiency for the GRIF-1/KIF5C constructs were: 5.6% ± 0.5 for ECFP-GRIF-1/N-EYFP-KIF5C (n = 15 cells); 10.3% ± 0.7 for ECFP-GRIF-1/KIF5C-EYFP (n = 20 cells); 4.0% ± 0.4 for ECFP-GRIF-1/EYFP-KIF5C-(1-335) (n = 11 cells); and 12.5% ± 0.5 for ECFP-GRIF-1/EYFP-KIF5C-(336-957) (n = 16 cells). Thus, a significant FRET value was only found for the ECFP-GRIF-1/KIF5C-EYFP and ECFP-GRIF-1/EYFP-KIF5C-(336-957) pairs showing a direct association between GRIF-1 and the C-terminal, non-motor domain of the kinesin-1 molecule, KIF5C.

GRIF-1, KIF5C, and KLC Interactions
To investigate whether GRIF-1 associates with the assembled heavy chain plus light chain tetrameric kinesin complex, immunoprecipitation and confocal imaging experiments were carried out on cells co-transfected with GRIF-1, KIF5C, and KLC clones. The results are shown in Fig. 9.

Co-immunoprecipitation Studies—In the following experiments, it was necessary to use the EYFP-tagged form of KIF5C, because pcDNAHisMaxKIF5C generates a His-tagged protein that is recognized by anti-GRIF-18-633 antibodies (anti-GRIF-18-633 antibodies were raised to poly-His tagged GRIF-18-633 (5)). In GRIF-1/EYFP-KIF5C double transfectants, GRIF-1 and EYFP-KIF5C co-immunoprecipitated (Fig. 4E); in KIF5C and KLC double transfectants, KIF5C and KLC co-immunoprecipitated (Fig. 9B), but in GRIF-1 and KLC double transfectants, no association between the two proteins was detected (Figs. 1E, 1F, and 9A). When HEK 293 cells were transfected with GRIF-1 plus EYFP-KIF5C plus KLC clones, detergent extracts were prepared and samples were immunoprecipitated with anti-GRIF-18-633 antibodies, and GRIF-1, KIF5C, and KLC immunoreactivities were then all found in the test but not control pellets (Fig. 9C). Because KLC did not associate with GRIF-1 in the absence of KIF5C, this must mean that GRIF-1 associates with the KHC, KLC tetrameric complex.

Co-localization Studies—The same combinations of clones as described for the co-immunoprecipitation studies were used for the transfection of COS-7 cells. Representative confocal microscopy images are shown in Fig. 9. Points to note are: (i) overexpression of GRIF-1 did not change the distribution pattern of KLC and no areas of co-localization were found (Fig. 9, D-H); (ii) EYFP-KIF5C and KLC mostly co-localized within the cell cytoplasm (Fig. 9, I-M); (iii) it was noted that in the majority of the EYFP-KIF5C plus KLC double transfectants, no fluorescence was detected at the tips of cell extensions such as seen in Fig. 6E; (iv) in the triple transfections, co-localization of EYFP-KIF5C, KLC, and ECFP-GRIF-1 was seen both in the cell cytoplasm and later, as for both EYFP-KIF5C alone and EYFP-KIF5C plus ECFP-GRIF-1, concentrated at focal points at the tips of cell processes (Fig. 9, N-S).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have previously shown that GRIF-1 associates with the molecular motor, kinesin (5). In this report, we have used four experimental approaches to map the GRIF-1 binding domain of the prototypic kinesin-1, KIF5C. Immunoprecipitations and yeast two-hybrid studies both mapped the GRIF-1 binding region of KIF5C to the C3 cargo binding domain. Further, in the yeast two-hybrid interaction assays it was found that the C3 cargo binding domain specifically associated with GRIF-1-(124-283) in agreement with the previous mapping of the kinesin binding domain of GRIF-1 (5). Overexpression of EYFP- and ECFP-tagged KIF5C and GRIF-1, respectively, in either HEK 293 cells or COS-7 cells showed that GRIF-1 co-localized with KIF5C (Fig. 6, H-J) and in refined studies, with the KIF5C non-motor domain, KIF5C-(336-957). FRET experiments established that the interaction between the two proteins was direct. The fact that a significant FRET value was found between the C-terminal-tagged KIF5C and ECFP-GRIF-1, the C-terminal-tagged KIF5C-(336-957) and GRIF-1, but not between the N-terminal-tagged KIF5C nor the motor domain, EYFP-KIF5C-(1-335), confirmed the direct association between the two proteins at the KIF5C C-terminal and GRIF-1 N-terminal regions. Finally, GRIF-1 was shown to form a ternary complex with KHC and KLC. These findings substantiate a role for GRIF-1 as an adaptor protein linking kinesin to its cargo in the anterograde trafficking processes in neurons. A schematic model summarizing the possible interactions between GRIF-1 and kinesin is shown in Fig. 10.


Figure 8
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  		FIGURE 8.
Measurement of FRET efficiencies between ECFP-GRIF-1 and EYFP-tagged KIF5C constructs by acceptor photobleaching. ECFP-GRIF-1 and either EYFP-KIF5C, KIF5C-EYFP, EYFP-KIF5C-(1-335), EYFP-KIF5C-(336-957) combinations were co-expressed in HEK 293 cells and FRET efficiencies by acceptor photobleaching were measured exactly as described under "Experimental Procedures." A, the FRET efficiencies for the negative control, i.e. HEK 293 cells transfected with pECFP plus pEYFP and the positive control, i.e. the pECFP-EYFP tandem construct. B, the FRET efficiencies for the negative control and each GRIF-1/KIF5C combination as labeled. The results are the means for at least n = 8 individual cells from n = 3 independent transfections for each pairwise combination. Each FRET efficiency was compared with the FRET efficiency obtained for the negative control using an unpaired Student t test. *, p < 0.025; **, p < 0.0005.

 


Figure 9
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  		FIGURE 9.
GRIF-1 associates with the KHC/KLC tetrameric complex: demonstration by co-immunoprecipitation studies and confocal microscopy imaging. I, immunoprecipitation experiments where HEK 293 cells were transfected with either pCISGRIF-1 plus pCMVKLC (A); pEYFP-KIF5C plus pCMVKLC (B), or pCISGRIF-1 plus pEYFP-KIF5C plus pCMVKLC (C), detergent extracts of cell homogenates were prepared 48 h post-transfection, immunoprecipitation assays were carried out using either affinity-purified anti-GRIF-18-633 (A and C), anti-KIF5C938-957 antibodies (B), or non-immune Ig (A-C), and immune pellets were analyzed by immunoblotting using antibodies as shown in the abscissae, all as described under "Experimental Procedures." The gel layout has the same format for each immunoprecipitation as follows: lanes 1, 4, and 7, detergent-solubilized transfected HEK 293 cell homogenate; lanes 2, 5, and 8, non-immune pellet; and lanes 3, 6, and 9, immune pellet. Note that 10% of the immune pellet was analyzed for the detection of the immunoprecipitating antibody protein, whereas 90% of the immune pellets were analyzed for the detection of co-associating proteins. The positions of molecular mass standards (kDa) are shown on the right. The immunoblots are representative of at least n = 3 immunoprecipitations from n = 3 independent transfections. II, confocal microscopy imaging experiments. COS-7 cells were transfected with either A-C, pCMVKLC; D-H, pCMVKLC plus pECFP-GRIF-1; I-M, pCMVKLC plus pEYFP-KIF5C; and N-S, pCMVKLC plus pECFP-GRIF-1 plus pEYFP-KIF5C. Cells were fixed 24-40 h post-transfection, stained with anti-c-Myc antibodies, and imaged by confocal microscopy all as described under "Experimental Procedures." A, D, I, and N are images with saturated fluorescence intensity to show the complete cell outline; B, E, J, and O, are each a single confocal section of a selected transfected cell showing the distribution of c-Myc-tagged KLC; F and P, are each a single confocal section of a selected transfected cell showing the distribution of ECFP-GRIF-1; K and Q, are each a single confocal section of a selected transfected cell showing the distribution of EYFP-KIF5C; G, L, and R, are merged to show the co-localization; H, M, and S, pixel intensity profile of the line scans shown in G, L, and R, respectively. Images are representative of at least n = 10 cells from n = 3 independent transfections for all combinations. Scale bars are 10 µm.

 
The concept of the selective transport of proteins and/or organelles via adaptor proteins linking kinesin motor proteins to their cargoes is an emerging feature of trafficking mechanisms in neurons. As described in the introduction, there are now several examples of such kinesin-associated adaptors and their cargoes, i.e. mitochondria and syntaxin-1-containing vesicles are attached to the KHC cargo binding domain by the adaptor protein, syntabulin, for their transport to synapses (9); JIP-3, a JNK signaling pathway protein, binds to a six tetratricopeptide motif in KLCs to transport the cargo, APP (10); KIF17 forms a complex with mLin10 in the transportation of N-methyl-D-aspartate receptor NR2B subunits to the synapse (11, 12); and GRIP1 is an adaptor protein linking the AMPA subtype of glutamate receptor-containing vesicles to KIF5 (13). In all these examples, the KHC or the KLC were studied in isolation, whereas, here it is clearly shown that GRIF-1 binds to the KHC when assembled with the KLC. Further, it was noted that the distribution pattern of EYFP-KIF5C is affected by co-expression of KLCs. This observation agrees with that reported by Verhey et al. (19). Here it was shown that association of KHCs with KLCs inhibits the binding of KHC to microtubules by inducing the formation of a folded KHC conformation. In the presence of GRIF-1, KHCs and KLCs are co-associated with GRIF-1 in the cell cytoplasm and at the tips of transfected COS-7 cell processes as observed for cells transfected with EYFP-KIF5C alone but not for the majority of cells for EYFP-KIF5C plus KLC transfectants. This suggests that, perhaps, GRIF-1 mediates conformational changes of KHC/KLC complexes such as unfolding. This then permits binding of the complex to microtubules, and active motor transport can now occur as has been shown in kinesin motility assays in vitro (20, 21).

A further example of kinesin adaptor proteins are the {alpha} and beta isoforms of dystrobrevin (22). Like GRIP1 (13) and now, GRIF-1, beta-dystrobrevin was shown to bind to the C-terminal KHC cargo binding domain (23). Both GRIP-1 and {alpha}- and beta-dystrobrevins bind to the cargo binding domain with similar affinities as determined by surface plasmon resonance analysis, i.e. GRIP-1, KD = 1.9 x 10-8 M (13); beta-dystrobrevin, KD = 4 x 10-8 M (23). Moreover and interestingly, Ceccarini et al. (23) showed that two distinct regions of beta-dystrobrevin contribute to the binding to KHC and that in vitro phosphorylation of a glutathione-beta-dystrobrevin construct resulted in a decreased binding to KHC (23). This suggests that both tertiary structure and post-translational modification may modulate beta-dystrophin-KHC or, more generally, adaptorkinesin interactions.

The one or more cargoes transported by GRIF-1 (and the homologous protein, OIP106) have yet to be ascertained. The first description of GRIF-1 identified it as a GABAA receptor beta2 subunit-associated protein (1). This suggested that the cargo may be assembled, beta2 subunit-containing GABAA receptors, but in vivo evidence to support this function is still lacking. However, Gilbert et al. (6) recently showed that OIP106 (TRAK1) co-associated with GABAA receptor {alpha}1 subunits, thus corroborating a role for the GRIF-1/OIP106 family in transporting GABAA receptors. Additionally, both GRIF-1 and OIP106, like the Drosophila orthologue Milton, have been shown to aggregate mitochondria following their respective overexpression in mammalian cells (3, 5). Further, without Milton, Drosophila are blind and mitochondria accumulated in neuronal cell bodies, whereas synaptic terminals and axons were depleted of mitochondria (3). However, if the cargo is a mitochondrion, it is unclear with which mitochondrial protein GRIF-1 or OIP106 associates. It is unlikely that GABAA receptors are expressed in mitochondria. GRIF-1 and OIP106 have, however, both been shown to associate with the enzyme, OGT (2). There are two forms of OGT, a mitochondrial (m) and a nucleocytoplasmic (nc) variant. The GRIF-1 or OIP106 binding domain of OGT is conserved between both forms mapping to mOGT-(51-100) and ncOGT-(167-283) (2, 5).6 mOGT has been reported to localize within the mitochondrial inner membrane (24) and would not therefore be accessible for binding to soluble GRIF-1-KHC or OIP106-KHC complexes. It is thus more likely that GRIF-1 and OIP106 associate with ncOGT.


Figure 10
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  		FIGURE 10.
A schematic diagram depicting putative GRIF-1-kinesin-cargo interactions for the anterograde trafficking of defined cargoes to synapses. Kinesin is depicted as a tetramer with two copies of the KHC and two copies of the KLC. For the heavy chain, the motor domain is shown associated with microtubules and contains, additionally, the stalk and cargo binding regions. GRIF-1 is depicted as a dimer (G. Ojla, M. Beck, K. Brickley, and F. A. Stephenson, manuscript in preparation) forming a ternary complex with the cargo binding domain of the KHC and the cargo. Yeast two-hybrid interaction assays using GRIF-1 deletion constructs previously showed that GABAA receptor beta2 subunits and KIF5C share the same coiled-coil binding domain of GRIF-1, i.e. GRIF-1-(124-283) (1 and 5) that is depicted in the figure by the lighter shading. A is a model where the KHC and the cargo bind separately to each of the GRIF-1 polypeptides; B is an alternative model whereby the KHC, GRIF-1, and the cargo associate with the same GRIF-1 subunit.

 
Other candidate proteins that mediate the association between GRIF-1 and mitochondria include syntabulin (25) and the mitochondrial GTPase, Miro (26). Both have both been implicated in anterograde mitochondrial trafficking in neurons. Indeed, it was recently demonstrated that Miro forms complexes with both GRIF-1 and OIP106 (27) and in Drosophila, Milton with Miro (28).

GRIF-1 is found within the brain in both neurons and glial cells.7 Furthermore, it is also expressed in the heart and at lower levels in skeletal muscle (1). It is possible that the cargo transported by GRIF-1-KHC complexes may be cell type-specific depending on the repertoire of KHCs expressed by the host cell and that the formation of such complexes may possibly be regulated by post-translational modifications such as that recently described for {alpha}- and beta-dystrobrevin. The enzyme, OGT, with which GRIF-1 and OIP106 are known to be associated, catalyzes the addition of N-acetylglucosamine onto serine and threonine residues of protein substrates. This O-glycosylation post-translational modification occurs in the cell cytoplasm, and it is thought to regulate protein function and to have a reciprocal relationship with post-translational modification and regulation via phosphorylation, the so-called "YinOYang" relationship (29). The YingOYang neural network predictions for O-beta-GlcNAc attachment sites in eukaryotic protein sequences (www.cbs.dtu.dk/services/YinOYang) predicts several sites for O-glycosylation within the non-kinesin binding GRIF-1 C-terminal domain. It is known that GRIF-1 and OIP106 are O-glycosylated in vivo (2), although the residues that are actually O-glycosylated have not been identified. Thus perhaps the association of GRIF-1 and OIP106 with OGT is to regulate GRIF-1-KHC interactions rather than OGT per se being a cargo.

In summary, the work described herein maps the GRIF-1 binding domain of KIF5C and shows that GRIF-1 forms a ternary complex with KIF5C and KLC that results in the aggregation of mitochondria. These findings consolidate the role of GRIF-1 as an adaptor protein involved in motor-dependent anterograde transport. Defects in these transport mechanisms, especially in the transport of mitochondria and GABAA receptors, purported to be GRIF-1 cargoes, may contribute to the pathology of neurodegenerative diseases such as Alzheimer disease, amyotrophic lateral sclerosis, hypertonia, and hereditary spastic paraplegia (30).


   FOOTNOTES
 
* This work was funded by the Biotechnology and Biological Sciences Research Council, United Kingdom. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1 and S2. Back

1 Both authors contributed equally to this work. Back

2 Current address: Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115. Back

3 To whom correspondence should be addressed: Tel.: 44-207-753-5877; Fax: 44-207-753-5964; E-mail: anne.stephenson{at}pharmacy.ac.uk.

4 The abbreviations used are: GABA, {gamma}-aminobutyric acid; AMPA, {alpha}-amino-3-hydroxy-5-methylisoxazole-4-propionate; APP, amyloid precursor protein; cfu, colony forming units; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; GRIF-1, GABAA receptor interacting factor-1; GRIP1, glutamate-receptor-interacting protein 1; HAP-1, huntingtin-associated protein; HEK, human embryonic kidney; KHC, kinesin heavy chain; IL, intracellular loop; KIF, kinesin superfamily protein; KLC, kinesin light chain; N-methyl-D-aspartate, N-methyl-D-aspartate; OGT, O-GlcNAc transferase; OIP, OGT interacting protein; JNK, c-Jun N-terminal kinase; CMV, cytomegalovirus; AD, activation domain; SD, synthetic defined media. Back

5 K. Pozo, K. Brickley, M. Beck, and F. A. Stephenson, unpublished observations. Back

6 M. Beck, K. Pozo, and F. A. Stephenson, unpublished results. Back

7 K. Brickley, G. S. Ojla, M. J. Smith, and F. A. Stephenson, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Dr. T. Nagase and colleagues (Kazusa DNA Research Institute, Chiba, Japan) for the gift of the clone KIAA0531 (KIF5C) and Dr. L. B. Lachman (M.D. Andersen Cancer Center, Houston, TX) for the gift of the kinesin light chain clone, Dr. J. G. McNally and Dr. L. He (Johns Hopkins University) for the gift of pEYFP-ECFP, Dr. J. McIlhinney (University of Oxford) for initial help with the FRET studies, Ana Salgueiro (Universidade do Algarve, Portugal) for the preparation of pECFP-GRIF-1 and pEYFP-KIF5C, and Dr. R. J. Harvey (School of Pharmacy, University of London) for the gift of pDsRed1-Mito.



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 EXPERIMENTAL PROCEDURES
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 DISCUSSION
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