Multiple Kinesin Family Members Expressed in Teleost Retina and RPE include a Novel C-terminal Kinesin

Exp. Eye Res. (1997) 64, 781–794

Multiple Kinesin Family Members Expressed in Teleost Retina and RPE include a Novel C-terminal Kinesin L A U R I E B O S T -U S I N G E R, R O B E R T J. C H E N, D A V I D H I L L M A N, H Y U N P A R K    B E T H B U R N S I DE* Department of Molecular and Cell Biology, 335 Life Sciences Addition, University of California, Berkeley, CA 94720-3200, U.S.A. (Received Seattle 3 September 1996 and accepted in revised form 10 December 1996) Kinesins comprise a large superfamily of microtubule-based motor proteins, individual members of which mediate specific types of motile processes. To identify kinesin family members (KIFs) that are critical to retinal function and thus to vision, a reverse transcriptase polymerase chain reaction (RT-PCR) cloning strategy was used to isolate putative KIFs expressed in the neural retina and retinal pigmented epithelium (RPE) of the striped bass, Morone saxatilus. Eleven fish KIFs (FKIFs) were isolated from neural retina and six of the same FKIFs were also isolated from RPE. One of the KIFs identified in this screen, FKIF2, was the most prevalent clone detected both in the retina (41 % of clones) and RPE (72 % of clones). Based on predicted amino acid sequence homology within the motor domain, seven of the FKIFs have been tentatively assigned to known kinesin families : the kinesin heavy chain family (FKIF1, 5 and 9), the unc104}KIF1 family (FKIF3 and 8), the KIF2 family (FKIF4), and the cKIF family (FKIF2). Northern blot analysis revealed that each detectable FKIF exhibited a unique tissue-specific expression pattern. Since FKIF2 was more highly expressed in retina than in any other tissue tested, including brain, and was the most abundant KIF message expressed in both retina and RPE, it was examined in more detail and the complete C 2±3 kb open reading frame for FKIF2 was cloned and sequenced. The predicted amino acid sequence indicates that FKIF2 has a C-terminal motor domain, and thus is a member of the cKIF family. FKIF2 is only 36±5 % identical at the amino acid level to the most closely related cKIF in the database, suggesting that FKIF2 may be a novel member of this family. Antibodies raised against a unique peptide specific to FKIF2 recognize an C 80 kd protein in homogenates of retina, RPE, brain and kidney. The pronounced expression of FKIF2 in retina and RPE suggests that FKIF2 may play an important role in microtubule-dependent motile events in these two tissues. # 1997 Academic Press Limited Key words : c-KIF ; C-terminal kinesin ; cytoskeleton ; kinesin ; microtubule motor ; retina ; retinal pigment epithelium.

1. Introduction Vertebrate vision depends upon the normal interaction and sustained vitality of retinal photoreceptors and retinal pigment epithelial (RPE) cells. Motile processes are required for photoreceptor and RPE morphogenesis as well as for maintenance of photopigment turnover throughout life (Besharse, 1986 ; Burnside and Dearry, 1986 ; Madreperla and Adler, 1989). Each day, photopigment-bearing membrane disks are generated at the base of the photoreceptor outer segment and old disks are shed from the distal tip (Besharse, 1986 ; Young and Bok, 1969). Shed packets of disks must be efficiently phagocytosed by adjacent RPE cells (Mullen and LaVail, 1976). If any of these motile processes are compromised in either photoreceptors or RPE cells, photoreceptor degeneration ensues, leading to blindness (Mullen and LaVail, 1976 ; Voaden, 1991 ; Wright, 1990). The defective genes have been identified for only about 20 % of the known forms of hereditary photoreceptor degeneration in humans (Wright, 1990 ; Rosenfeld et al., 1994). Since motile events are critical * For correspondence.

0014–4835}97}050781­14 $25.00}0}ey960271

to photoreceptor vitality, and since most motile events depend upon molecular motors (Schroer and Sheetz, 1991), we have sought to identify cytoskeletal motors critical to morphogenesis and maintenance of photoreceptors or RPE cells ; any such motor would be a reasonable candidate gene for causing one of the forms of photoreceptor degeneration whose underlying mechanism is not yet understood. That hereditary defects in specific cytoskeletal motors can produce photoreceptor degeneration has already been reported in flies and humans (Porter et al., 1992 ; Weil et al., 1995). Defects in the myosin III ninaC produce photoreceptor degeneration in Drosophila, and defects in myosin VIIA produce the human disease Usher Syndrome Type 1B, which is characterized by degeneration of photoreceptors and auditory hair cells. In this study we have set out to identify microtubuledependent motors of the kinesin superfamily that are expressed in vertebrate photoreceptors and RPE cells. Kinesins are mechanochemical motors which utilize the energy of ATP hydrolysis to walk along microtubules (Goldstein, 1993 ; Bloom and Endow, 1994). Ubiquitously expressed in all eukaryotic organisms, kinesin family members (KIFs) are defined by the extensive homology they share within a globular # 1997 Academic Press Limited

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motor domain which contains both microtubule- and ATP-binding sites (Bloom and Endow, 1994). Multiple members of the kinesin superfamily may be expressed in a single organism or even a single cell (Goldstein, 1993 ; Goodson, Kang and Endow, 1994) : for example, multiple KIF expression has been reported for Drosophila (Stewart et al., 1991 ; Endow and Hatsumi, 1991), mouse brain (Aizawa et al., 1992), Xenopus oocyte (Vernos, Heasman and Wylie, 1993), and yeast (Roof, Meluh and Rose, 1992). Kinesin motors have been implicated in several types of motile processes, including transport of mitochondrial or vesicular cargoes along microtubules, intermicrotubule sliding, and the assembly and motility of mitotic and meiotic spindles (Goldstein, 1993 ; Bloom and Endow, 1994). To date only two types of KIFs have been identified in vertebrate retina and RPE : members of the conventional kinesin heavy chain (KHC) family and of the heterotrimeric kinesin (KIF3) family. KHC isoforms have been detected in the rat retina, and the different isoforms appear to translocate different cargoes and travel at different rates of speed (Elluru, Bloom and Brady, 1995). Multiple KHC isoforms have also been identified in rabbit retina and RPE by immunological and molecular methods (Amaratunga et al., 1993 ; Elluru et al., 1995 ; King-Smith, Bost-Usinger and Burnside, 1995). Inhibition of KHC synthesis with antisense oligonucleotides blocked anterograde axonal transport in rabbit ganglion cells (Amaratunga et al., 1993). Two forms of KHC have been detected in fish RPE, but neither appear to be involved in pigment granule movement (King-Smith et al., 1995) ; however, conventional KHC is responsible for translocation of pigment granules in fish melanophores (Rodionov, Gyoeva and Gelfand, 1991). In contrast to KHC, very little is known about the expression pattern or functional roles of other KIFs in the retina or RPE. KIF3 family members have recently been shown to be associated with the connecting cilium of vertebrate rod photoreceptors (Beech et al., 1996). Since the connecting cilium is the only structural link between the inner segment and photosensitive outer segment, KIF3 or other kinesin family members may play a role in delivery of newly synthesized proteins to the photoreceptor outer segment. Abnormal axonemal microtubules and aberrant intraciliary membrane vesicles in the photoreceptor ciliary axoneme have been observed in retinas of people with Usher Syndrome Type II (Hunter et al., 1986 ; Barrong et al., 1992). Because of the multitude of microtubule-mediated motility events which occur in the vertebrate retina and RPE it was predicted that multiple kinesin family members might be expressed and needed in these tissues. Since photoreceptors and RPE cells interact so intimately in photoreceptor morphogenesis and maintenance, and since both photoreceptors and RPE cells have been shown to be loci of gene defects which lead to retinal degenerations, separate RT-PCR screens for

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kinesin proteins were carried out using both retina and RPE. These studies were performed in retina and RPE from the striped bass, Morone saxatilus, because it provides several advantages for identifying molecular motors preferentially expressed in photoreceptors and RPE. The striped bass retina is large and nonvascularized ; thus large amounts of tissue can be obtained and complications introduced by contaminating vascular elements can be avoided. Also, sheets of fish RPE can easily be isolated remarkably free of other cell types, permitting rapid preparation of mRNA directly from a differentiated, intact epithelium containing a single cell type. After using fish tissues to identify FKIFs expressed in photoreceptors and RPE, we can also look for mouse, chick, or human homologues for genetic mapping and}or further functional studies. PCR products encoding partial motor domains of 11 distinct putative FKIFs were identified and cloned. Seven of the 11 are expressed in bass retina or RPE at levels detectable by Northern blot analysis. Sequence analysis showed that all possess conserved sequences characteristic of kinesin family members. We have completely cloned and sequenced, and have begun to characterize FKIF2, a novel kinesin family member identified in this screen. FKIF2 is the predominant motor expressed in the RPE and retina, and belongs to the cKIF family of proteins. 2. Materials and Methods Animals and Tissue Preparation RPE sheets and retina tissue samples were isolated from dark-adapted striped bass, Morone saxatilus. Fish were dark-adapted 45 min during their light period and killed by spinal section and pithing. Eyes were removed, hemisected, and the neural retina was dissected away from the underlying RPE cell layer and immediately placed into liquid nitrogen. RPE sheets were dislodged by pipetting a gentle stream of Ringer (116±3 m NaCl, 5±4 m KCl, 1±8 m CaCl , 0±8 m # MgSO , 1±0 m Na HPO , 25±5 m glucose, 24 m % # % NaHCO , 3 m HEPES, 1±0 m ascorbic acid) which $ contained 0±1 units µl−" rRNasin (Promega, Madison, WI, U.S.A.) and 1 m DTT. The sheets and retinas were immediately quick-frozen in liquid nitrogen. Polymerase Chain Reaction The oligonucleotide primers used correspond to the I-F-A-Y-G-Q-T motif in the ATP binding site (PCR1), the G-K-T-Y}F}H-T-M motif adjacent to the ATP binding site (PCR2), and to sequences complementary to the V-D-L-A-G-S motif downstream of the ATP binding site (PCR3). Primer PCR1 was 5«-AT(C}A)TT(T}C)-GC(A}T}G}C)-TA(T}C)-GG(A}T}G}C)-CA(A} G)-AC-3« and is 256-fold degenerate. Primer PCR2 was 5«-GG(A}T}G}C)-AA(A}G)-AC(A}T}G}C)(T}C)A(T}C)-AC(A}T}G}C)-ATG-3« and was 512-fold

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F. 1. Alignment of partial motor domain sequences for FKIF1-6, 8 and 9 with the two closest related KIFs generated in a TFastA search (accession numbers can be found in the legend for Fig. 2). Regions corresponding to the degenerate oligonucleotide primers PCR1, PCR2 and PCR3 are indicated by arrows. Amino acid residues conserved among all of the family members are boxed, and amino acid residue numbers are indicated above the sequences. The alignment was generated using the UWGCG PileUp sequence analysis program (Devereux et al. 1984). FKIF sequence data are available from EMBL}GenBank}DDBJ under accession numbers MSU34653-MSU34660.

degenerate. Primer PCR3 was 5«-(T}A) (G}A}T)CC(A}T}G}C) GC-(G}C) A(G}A)-(G}A) TC-(C}A}G) AC-3« and was 576-fold degenerate. cDNA was synthesized from 5 µg of total RNA from RPE sheets or neural retina using the Stratascript RT-PCR kit (Stratagene,

La Jolla, CA, U.S.A.). Reaction mixtures (50 µl) contained approximately 50 ng cDNA, 60 m Tris– HCl (pH 8±5), 15 m NH SO , 1±5 m MgCl , 125 µ % % # dNTPs, 1 µ PCR1, 1 µ PCR3, and 2±5 units of Taq DNA polymerase. The primary PCR amplification

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consisted of 35 cycles of 1 min at 95°C, 2 min at 45°C, and 3 min at 72°C. The final extension step was performed at 72°C for 10 min. Approximately 20 ng of the primary PCR product was reamplified under identical cycle conditions using primers PCR2 and PCR3. Isolation and Sequencing of PCR Products The RPE and retina PCR products were treated with the Klenow fragment of DNA polymerase 1 and then phosphorylated with T4 polynucleotide kinase (Boehringer Mannheim, Indianapolis, IN, U.S.A.). The PCR-amplified DNA bands of approximately 450 bp were fractionated in a 1 % agarose gel, excised, and gel purified using the QIAEX (Qiagen, Chatsworth, CA, U.S.A.) DNA purification kit. The products were then cloned into the EcoRV site of pBluescript SK+ (Stratagene, La Jolla, CA, U.S.A.) which had been dephosphorylated with calf intestinal alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN, U.S.A.). Individual clones containing inserts were picked at random and sequenced using the Sequenase 2.0 kit (US Biochemical, Cleveland, OH, U.S.A.). The sequences were analysed by Genetics Computer Group Wisconsin Sequence Analysis package (UWGCG). Analysis of RNA Expression Frozen tissues were transferred to a 10-fold volume of RNA-statB (Tel-TestB, Inc., Friendsworth, TX, U.S.A.) homogenization buffer, homogenized for 30 sec, and total RNA was isolated according to the manufacturer’s instructions. A 15 µg sample of total RNA was electrophoresed in formaldehyde-agarose gels, transferred to a nylon membrane and hybridized according to standard procedures (Sambrook, Fritsch and Maniatis, 1989). Northern blots were sequentially probed with FKIF cDNA probes and a tubulin cDNA probe (Hieber, Agranoff and Goldman, 1992) which were radiolabelled with [$#P]dCTP using a random priming labeling kit (BRL, Gaithesburg, MD, U.S.A.). Blots were prehybridized in 50 % formamide hybridization buffer, washed and exposed according to standard procedures. Hybridization signals were quantified using a phosphorimager and ImageQuant software (Molecular Dynamics). Northern Blots were normalized by removing cDNA probes and rehybridizing with a 28 s ribosomal RNA cDNA probe (Gonzalez et al., 1985). Southern Blotting and Colony Hybridization For Southern blot analysis, 3 ng of the FKIF generated fragments were electrophoresed in a 1 % agarose gel. The gel was denatured and neutralized and the DNA was then transferred to a 0±45 µ Hybond-N membrane according to standard procedures (Sambrook et al., 1989). For colony hybridizations, insert-containing bacterial colonies

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were lifted onto sterilized nitrocellulose filters and placed in denaturing solution for 3 min, followed by two 5 min neutralizations. The filters were dried at 37°C, fixed by UV cross-linking, and rehydrated in 2¬ SSC. The filters were prewashed in 5¬ SSC, 0±5 % SDS and 1 m EDTA, pH 8±0 and excess bacterial debris was removed. The filters and Southern blots were then prehybridized and hybridized with the appropriate FKIF cDNA probes exactly as described above for Northern blots.

Cloning of the FKIF2 cDNA The complete FKIF2 cDNA sequence was generated using a combination of PCR amplification, λ Zap cDNA library screening, and 5« RACE amplification. The upstream motor domain sequence (1351 to 1816 bp) was generated by PCR amplification using primers PCR2 and PCR3 and was described in detail above. The upstream FKIF2 motor domain fragment was radiolabelled with [$#P]dCTP using a random priming labeling kit (BRL, Gaithesburg, MD, U.S.A.), and used as a probe to screen a λ Zap striped bass RPE-retina cDNA library using standard plaque hybridization techniques (Sambrook et al., 1989). The downstream motor domain sequence and partial 3« untranslated region (UTR) sequence was obtained from the library screening (mRNA size from Northern blot analysis suggests a cDNA clone did not contain the full length UTR). The FKIF2 tail domain sequence was obtained using 5« RACE amplification. Bass polyA RNA, purified using the polyA tract purification kit (Promega, Madison, WI, U.S.A.), was used to construct RACE ready cDNA using the Marathon RACE amplification system (Clonetech, Mountain View, CA, U.S.A.). RACE reactions using the Advantage amplification kit (Clonetech, Mountain View, CA, U.S.A.), were performed on approximately 50 ng cDNA, according to the manufacturer’s instructions. The 5« RACE amplification consisted of 35 cycles of 1 min at 96°C, 3 min at 72°C, with a final extension step performed at 72°C for 10 min. Ten percent of the RACE product was electrophoresed on a 1 % agarose gel, transferred to a nylon membrane, and analysed by Southern blot analysis using 1351 to 1750 bp of the motor domain region as a probe. An C 2±2 kb Southern positive band was excised, purified using the QIAEX gel purification kit (Qiagen, Chatsworth, CA, U.S.A.), and subcloned into the pGEM-T (Invitrogen, San Diego, CA, U.S.A.) cloning vector. All sequence information shown in Fig. 4 was obtained from both cDNA strands and generated using manual didedoxy chain termination sequencing (USB Sequenase kit, Cleveland, OH, U.S.A.) or by automated cycle sequencing using the Perkin Elmer Terminator Ready Reaction mix (Perkin-Elmer, Norwalk, CT, U.S.A.) and the products were run on an ABI 373 Stretch Apparatus (Applied Biosystems, Foster City, CA, U.S.A.).

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Antibody Preparation and Affinity Purification A 14 amino acid stretch of peptide unique to FKIF2 (residues 66–79, Fig. 1 ; DIKMNPDGSGQLYV) was chosen to generate FKIF2 specific antibodies. The peptide was synthesized, conjugated to KLH, and injected into rabbits for the production of polyclonal antisera (all phases of peptide and antibody production was performed by Genosys, The Woodlands, TX, U.S.A.). The peptide antisera was affinity purified against a bacterially expressed histadine-tagged FKIF2 protein (His-FKIF2). The FKIF2 motor domain (amino acids 1–145, Fig. 1) was subcloned into the pQE30 histadine-tag vector (Qiagen, Chatsworth, CA, U.S.A.). Purified His-FKIF2 was conjugated to CNBr-activated Sepharose 4B (Pharmacia, Piscataway, NJ, U.S.A.) according to manufacturer’s instructions. A 3 ml sample of FKIF2 peptide antisera was batch adsorbed onto the motor column by rocking overnight at 4°C. The column was washed with 10 column volumes of 50 m Tris, 500 m NaCl, 0±01 % Triton-X100, pH 7±5. FKIF2 peptide antibodies were eluted using 4  urea in 100 m glycine–HCl, pH 2±5, containing 1 mg ml−" BSA. Fractions were immediately dialysed against 50 m Tris–HCl, pH 7±6, 150 m NaCl, 0±02 % NaN and tested for titers using dot-blot $ analysis against dilutions of His-FKIF2. Peak fractions were pooled and stored in aliquots at ®80°C. Immunoblotting Tissue samples (bass RPE, sunfish RPE, chick RPEretina) and cell pellets (human and rat RPE) were homogenized in ice-cold hypotonic lysis buffer composed of 50 m Tris, pH 7±2, 5 m EDTA, 2 % SDS, 0±5 m PMSF, 157 mg ml−" benzamidine, 10 µg ml−" leupeptin, 10 µg ml−" aprotinin, N α-p-tosyl--arginine methyl ester (TAME) and soybean trypsin inhibitor. Samples were transferred to 1±5 ml tubes and centrifuged for 5 min at 800 g to pellet pigment granules and}or large pieces of connective tissue. The tissue supernatants were removed and protein concentration was determined using the DC-Bradford dyebinding assay (BioRad, Richmond, CA, U.S.A.). Twenty micrograms of total protein was loaded in each lane and proteins were separated on 8 % SDS–PAGE gels using standard procedures (Laemmli, 1970). Proteins were electro-transferred to nitrocellulose in 25 m Tris, 192 m glycine, 20 % MeOH, pH 8±3 using a BioRad transblot apparatus. Blots were blocked overnight at 4°C with 10 % non-fat dry milk (NFDM) in TTBS (10 m Tris, 150 m NaCl, 0±05 % Tween-20). FKIF2 peptide antibodies were diluted in 3 % NFDM in TTBS (1 : 500 dilution, final concentration of 0±038 µg ml−"). Blots were exposed to primary antibody for 3 hr at room temperature, washed in TTBS, and incubated in secondary antibodies conjugated to HRP (1 : 5000). Blots were developed using ECL chemiluminescence reagents

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(Amersham, Boston, MA, U.S.A.) according to manufacturer’s instructions.

3. Results Identification of Genes Encoding Kinesin Family Members in Fish RPE-Retina To identify genes encoding kinesin family members in RPE and retina, separate RT-PCR procedures were performed on RNA and RPE sheets and neural retinas of the striped bass Morone saxatilus. For the PCR amplification, three degenerate oligonucleotide primers corresponding to three highly conserved amino acid sequences in the motor domains of most kinesin proteins (Fig. 1) were synthesized. These primers were based on fish codon usage ascertained from sequencing the bass kinesin heavy chain (KingSmith et al., 1995). Primers PCR1 and PCR2 reside within the consensus ATP binding domain (Hirokawa et al., 1989), while PCR3 resides downstream in what is thought to be the microtubule binding domain (Fig. 1) (Yang, Layman and Goldstein, 1989). Amplification of RPE and retinal cDNA with PCR1 and PCR3 produced a major product of C 450 bp, the size expected for members of the kinesin family. To increase the number of authentic kinesin family members in the primary PCR product, the 450-bp fragment was re-amplified with PCR2 and PCR3, generating a major product of C 430 bp, which was then subcloned into pBluescript. Following purification, independent colonies were either sequenced directly or analysed by colony hybridization for identity to previously sequenced FKIFs. This nested primer approach produced a high percentage of PCR products encoding kinesin family members. Of 96 RPE-derived clones examined, 81 were found to encode six distinct putative fish kinesin family members (FKIF1-6) (Table I), identified as such by the presence of highly conserved amino acid sequence motifs characteristic of kinesins (shown boxed in Fig. 1). Of 80 retina-derived clones examined, 61 were found to encode 11 distinct FKIFs (Table I). The retina-derived FKIF clone included the same six FKIFs cloned from RPE (FKIF1-6), although the relative detection frequency of each FKIF clone differed in the two tissues (Table I). An additional five FKIFs, designated FKIF7-11, were each detected in only one copy in the retinal PCR screen. Three of these retinal FKIF PCR products were only partially sequenced (C 150 bp) and identified as FKIFs based upon predicted amino acid sequence homology ; however, they were found to be below the level of detection in Northern blots of retina or RPE, and were not analysed further at this time (FKIF7, 10 and 11 ; data not shown). The six most frequently encountered FKIFs from the PCR screen (FKIF1-6) and the two additional FKIFs expressed at levels detectable in retinal Northern blots (FKIF8 and 9) were analysed in more detail.

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T I Relative abundance of FKIF PCR clones Family member FKIF1 FKIF2 FKIF3 FKIF4 FKIF5 FKIF6 FKIF7 FKIF8 FKIF9 FKIF10 FKIF11

Clones (No.) RPE

Clones (No.) retina

% of total RPE

5 58 9 3 4 2

9 25 4 4 11 3 1 1 1 1 1 61*

6±2 71±6 11±1 3±7 4±9 2±5

81*

100

% of total retina 14±8 40±9 6±6 6±6 18±0 4±9 1±6 1±6 1±6 1±6 1±6 100

* These numbers were generated from sequence analysis of the C 430 bp PCR2-PCR3 amplified PCR products. The inserts from 81 RPE clones and 61 retina clones were sequenced. The relative numbers and percentages of each FKIF found in both RPE and retina are given in the table.

FKIF2 was by far the most predominant kinesin family member detected in both screens, representing C 72 % of all RPE-derived clones and C 41 % of all retinaderived clones. Sequence Comparison of FKIFs The amino acid sequences of the PCR products derived from FKIF1-6, 8 and 9 were aligned with the two most closely related KIF sequences from the database search using the UWGCG PileUp program (Devereux, Haeberli and Smithies, 1984) (Fig. 1). Amino acids corresponding to conserved regions found throughout the kinesin superfamily are boxed, indicating that all eight putative FKIFs are bona fide kinesin family members. The extent of identity among the FKIFs themselves was evaluated using the UWGCG BestFit program (Devereux et al., 1984) (Table II). FKIF1, 5 and 9 were more than 80 % identical in amino acid sequence within the cloned 430 bp portion of the motor domain. FKIF3 and 8 were also more than 82 % identical. All other combinations of FKIFs showed less than 45 % identity with one another, indicating that they are no more closely related than members of different kinesin subfamilies. In order to classify each FKIF according to placement within the kinesin superfamily, a GCG TFastA search was performed by comparing the FKIF motor domain sequences with other sequences in the GenBank database. The top three matches of the TFastA search were used to generate a dendogram which graphically depicts how the FKIF members fit into the kinesin superfamily (Fig. 2). The closest match for each FKIF from the TFastA search was used in a Bestfit analysis, and these data are summarized in Table III. Three of the FKIFs (1, 5 and 9) are more than 75 % identical to members of the KHC family ; two FKIFs (3 and 8) are more than 81 % identical to

members of the unc104}KIF1 subfamily, and FKIF4 is more than 63 % identical to a member of the KIF2 central motor family (Bloom and Endow, 1994 ; Goodson et al., 1994 ; Sekine et al., 1994). FKIF2 and FKIF6 are less than 42 % identical to any known kinesin and thus appear to represent novel family members. Tissue Expression Patterns of FKIFs Northern blot analysis was performed to determine transcript size and tissue expression pattern for each FKIF. Total RNA was prepared from a variety of bass tissues, transferred to a membrane, and sequentially probed with each FKIF. For qualitative comparison, the blot was also probed with a cDNA encoding the αtubulin gene (Hieber et al., 1992). Transcripts for FKIF4, 7, 10 and 11 were not abundant enough to be detected by Northern blot as performed (data not shown). The expression patterns for the remaining FKIFs differed in transcript size or tissue distribution or both, even including the closely related FKIF1 and 9 (Fig. 3). The three KHC isoform (FKIF1, 5 and 9) tissue-specific expression patterns are seen in the first column of Fig. 3. The FKIF1 transcript is 6±5 kb and is strongly expressed in the retina, brain, and kidney, and less so in muscle and liver. FKIF5 is expressed as a single 6±6 kb transcript in retina and brain and a 1±9 kb transcript in muscle. FKIF9 is expressed strongly as 7±5, 6±4 and 5±4 kb transcripts in the brain, and much weaker as 6±4 and 5±4 kb transcripts in the retina, and as a single 6±4 kb transcript in the kidney. The two unc104 family members are also differentially expressed and are seen in the third column of Fig. 3. FKIF3 is expressed as a single 9±3 kb transcript in retina and brain and a 1±9 kb transcript in muscle. FKIF8 is expressed as a 9±0 kb transcript in retina and brain.

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T II Percent identity between FKIFs FKIF1 FKIF1 FKIF2 FKIF3 FKIF4 FKIF5 FKIF6 FKIF8 FKIF9

FKIF2

FKIF3

FKIF4

FKIF5

FKIF6

FKIF8

FKIF9

39±3

40±8 33±6

42±9 36±0 38±6

80±6 37±6 44±4 42±3

38±3 43±0 36±1 36±4 35±9

46±2 38±3 82±1 39±4 46±5 33±8

85±5 40±8 43±8 41±5 86±2 38±0 44±8

FKIF1 FKIF2 FKIF3 FKIF4 FKIF5 FKIF6 FKIF8 FKIF9

Comparisons were performed using the bestfit UWGCG sequence analysis program (Devereux et al., 1984). Values " 80 % homology are italicized.

The novel kinesin family member, FKIF2, was the most abundantly expressed kinesin in the retina and in the RPE. FKIF2 is strongly expressed as 5±8 and 4±1 kb transcripts in the retina. Both transcripts are expressed in the brain, but at much lower levels. Only the 4±1 kb transcript is detectable in RPE by Northern blot analysis. Normalization to 28s rRNA levels showed that brain RNA levels are approximately twofold greater than RPE and retina is approximately 1±5 fold greater than RPE. Southern blot analysis showed no cross-reactivity among FKIF clones except for barely detectable cross-reactivity between the two most closely related FKIF clones (FKIF1 and 9), which nonetheless had very different Northern blot expression patterns (data not shown). cDNA Cloning and Sequencing of FKIF2 The combined approaches of PCR amplification, cDNA library screening, and RACE amplification were used to generate the complete cDNA open reading frame (ORF) sequence for FKIF2 (Fig. 4). The upstream motor domain sequence (1351 to 1816 bp) was generated by PCR amplification using the GKTY}F} HTM and VDLAG primers. The PCR-generated fragment was used to screen a bass RPE-retina cDNA library. Multiple copies of a 0±9 kb clone resulted from this screen, and all encoded the downstream motor domain sequence (1747 to 2220 bp) and a short stretch of 3« UTR. Rescreening this library with the downstream motor domain failed to produce additional FKIF2 sequence. 5« RACE amplification was used to generate the 5« tail region and complete the FKIF2 cloning. The FKIF2 ORF is 2320 bp and is predicted to encode a protein of approximately 87 kd. An amino acid composition profile showed that the FKIF2 protein is very rich in glutamic acid residues (14 % of all amino acids). Moreover, FKIF2 is predicted to be an acidic protein, having a pI of 6±1. According to secondary structure predictions using the ChouFasman and Robson-Garnier methods (Chou and Fasman, 1974 ; Garnier, Osguthorpe and Robson,

1978), the N-terminal tail region of FKIF2 (amino acids 1–440) is rich in α-helices. The motor domain (amino acids 450–773) is located at the C-terminus. A TFastA search was performed on the complete motor domain sequence (amino acids 450–773) and on the entire coding sequence. The results of the search showed that the FKIF2 motor domain is most closely related to KatA (52±3 % identity), an Arabidopsis kinesin which belongs to the cKIF family (Mitsui et al., 1993) and the entire protein is most closely related to the human neuronal KHC (38±9 % identity ; Navone et al., 1992). Based upon this average level of identity within the motor domain and low level of identity throughout the entire protein, FKIF2 is believed to be a novel, previously uncharacterized kinesin family member. A gene closely related to FKIF2 has recently been cloned in the mouse (mouse clone zyb3) ; this mouse gene shows C 70 % identity in the motor domain and C 60 % identity overall (L. Goldstein, personal communication). The full length amino acid sequences of FKIF2 and three members from each KIF superfamily were used to generate a dendogram (Fig. 5), which illustrates the placement of FKIF2 into the C-terminal family of kinesins. Protein Expression Pattern of FKIF2 In order to examine the protein expression pattern of FKIF2, antibodies were raised to a stretch of amino acids within the motor domain that was unique to FKIF2 [amino acids 521–534 ; DIKMNPDGSGQLYV, shown boxed in Fig. 4(A)]. Polyclonal rabbit antibodies were raised against the conjugated peptide and affinity purified against a peptide affinity column and against a bacterially expressed His-FKIF2 protein. Antisera from both purifications recognized a common band of C 80 kd in Western blots of total retina protein. The data presented were generated from the His-FKIF2 purification column. Total cell lysates were prepared from the same panel of tissues analysed by Northern blot analysis (RPE, retina, muscle, kidney, brain, liver). The antibodies recognized an C 80 kd protein in RPE, retina, kidney, brain, and an C 79}82 kd

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comigrates with FKIF2 was also detected in human RPE. 4. Discussion

FKIF4 MCAK bimC KIP1 EG5 FKIF2 ncd FKIF6 KIF1B FKIF3 FKIF8 unc104 HUMORFW HKHC(u) MKHC(b) FKIF1 FKIF9 MKHC(a) HKHC(n) FKIF5 DKHC SKHC unc116 KIF3 FLA10

F. 2. Dendogram analysis of the relationships of FKIFs to one another and to the three most closely related KIFs identified by a TFastA search. FKIF sequences are marked by arrowheads. The comparison was based on 430 bp partial motor domain sequence. The amino acid residues between primer sequences GKTH}YFM and VDLAG were analysed by the UWGCG PileUp sequence analysis program (Devereux et al. 1984), which maximizes the amino acid alignment among compared sequences. Similarity is inversely proportional to the distance along the horizontal axis. The sequences followed by their GenBank accession No’s are as follows : KAR3 M31719 ; KLPA X64603 ; KIF2 D12644 ; MCAK U11790 ; bimC M32075 ; KIP1 Z11962 ; Eg5 X54002 ; ncd M33932 ; KIF1B D17577 ; unc104 M58582 ; HUMORFW D26361 ; HKHC(u) X65873 ; MKHC(b) L27153 ; MKHC(a) X61435 ; HKHC(n) U06698 ; DKHC M24441 ; SKHC J05258 ; unc116 L19120 ; KIF3 D12645 ; FLA10 L33697 ; KIF4 D12646 ; PIR u : XKLP1 A44835.

doublet in liver (Fig. 6). This apparent molecular weight is in close agreement to the predicted size of C 87 kd encoded by the FKIF2 cDNA sequence. Although mRNA levels were much greater in retina and brain as compared to RPE, the protein levels appear to be equal, or greater in the RPE as compared to retina and brain. In order to determine whether a homologue of FKIF2 exists in mammals and other vertebrates, a panel of tissue and cell culture lysates from sunfish, rat, chick and human RPE was screened. In addition to the C 80 kd protein seen in the closely related bass and sunfish RPE lysates, an C 80 kd protein which

Using RT-PCR of RNA from neural retina and RPE of the striped bass, Morone saxatilus, PCR products representing 11 distinct members of the kinesin superfamily were obtained. All 11 were amplified from retina, and a subset of six was amplified from RPE. These PCR products were identified as putative KIFs based on their possession of highly conserved characteristic sequences in the ATP- and microtubulebinding regions of kinesin motor domains (Fig. 1) (Goldstein, 1993). Eight of the FKIF PCR products were fully sequenced (C 430 bp of motor domain) and compared to previously identified KIFs in the GenBank database. Based on sequence comparisons, the eight FKIFs have been tentatively assigned to known classes of the kinesin superfamily (Bloom and Endow, 1994 ; Goodson et al., 1994 ; Sekine et al., 1994) (Fig. 2). FKIF2 and FKIF6 showed a low level of identity to previously identified KIFs and appear to represent novel genes. Because of its overwhelming prevalence in the PCR screen and its preferential expression in the retina and RPE, the FKIF2 gene was characterized in more detail. The cDNA structure of FKIF2 shows that FKIF2 is a member of the cKIF family, members of which possess C-terminal motors (Hirokawa, 1996 ; Moore and Endow, 1996). Protein secondary structure predictions suggest that the amino-terminal tail of FKIF2 is very rich in α-helices and could be predicted to form a coiled-coil type structure. Tail domains of other kinesin family members are also very α-helical, and coiled-coil domains are predicted to be involved in dimer or oligimer formation (Cole et al., 1993 ; Yang et al., 1989). FKIF2 is strongly expressed as an C 80 kd protein in RPE, retina, and kidney, with much lower protein levels found in brain and liver. A protein with a similar molecular weight was also detected in cultured human RPE, suggesting that human RPE expresses a related homologue of FKIF2 [Fig. 6(B)]. A comigrating band was not detected in chick or rat tissues ; the absence of an C 80 kd band in chick or rat could mean that these species do not express detectable levels of an FKIF2 homologue, or that the epitopes recognized by the peptide antibody are not present in chick or rat protein. The protein expression pattern seen in the panel of fish tissues [Fig. 6(A)] was quite different from the mRNA expression pattern, where message was highly expressed in retina, less so in brain and RPE, and undetectable in kidney. This discrepancy in protein and mRNA levels suggests that protein turnover and}or message stability of FKIF2 may be different in the different tissues. There are other reported examples where KIF mRNA and protein expression patterns are not parallel. Abundant levels of KHC were detected in fish RPE by Western blot using several different KHC antibodies (King-Smith et

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789

T III Percent identity between FKIFs and representative kinesin family members HKHC(u)

HKHC(n)

DKHC

MKHC

KIFIB

KIF2

KIF3

NCD

BIMC

95±2 39±9 42±8 39±6 84±1 37±8 40±5 88±3

87±6 41±2 43±4 40±3 86±2 37±8 39±5 91±0

79±3 41±9 40±8 36±9 75±9 39±9 38±8 77±2

86±9 40±5 43±4 41±6 86±9 36±5 38±8 94±5

39±7 32±2 90±1 38±7 42±1 33±1 81±5 39±1

38±3 30±3 39±4 63±7 37±6 31±8 37±2 38±3

38±9 39±7 41±4 31±4 41±6 35±8 40±8 42±3

34±5 39±7 35±4 29±3 33±1 41±5 33±6 33±8

38±6 34±2 36±2 31±2 36±9 37±4 33±5 33±8

FKIF1 FKIF2 FKIF3 FKIF4 FKIF5 FKIF6 FKIF8 FKIF9

FKIF1 FKIF2 FKIF3 FKIF4 FKIF5 FKIF6 FKIF8 FKIF9

Comparisons were performed using the bestfit UWGCG sequence analysis program (Devereux et al., 1984). Values " 63 % homology are italicized.

re tin a m us cl e ki dn ey br ai n liv er

R PE

FKIF3

re tin a m us cl e ki dn ey br ai n liv er

R PE

FKIF2

re tin a m us cl e ki dn ey br ai n liv er

R PE

FKIF1

28 s 18 s

n

er liv

ai

ey

br

cl e

dn

ki

a m

us

tin re

PE R

er

n ai

liv

ey

FKIF8

br

cl e

dn

us m

ki

a tin re

PE R

n

er liv

ai

ey

FKIF6

br

dn

ki

m

us

tin re

R

PE

a

cl e

FKIF5

28 s 18 s

liv er

ai n

ey

br

cl e

dn

ki

a

us m

tin re

PE R

ai n liv er

ey

28 s

br

dn

ki

cl e

m

us

a tin re

PE R

liv er

ai n

ey

Tubulin

br

dn

ki

us m

tin re

R

PE

a

cl e

FKIF9

28 s 18 s

F. 3. Tissue-specific expression patterns of FKIFs. Northern blot analysis was performed using the cloned FKIF PCR fragments and a tubulin cDNA probe. Samples of total RNA (15 µg) from RPE, retina, muscle, kidney, brain, and liver were electrophoresed and transferred to a nylon membrane. The blot was stripped and probed with a cDNA encoding 28s ribosomal RNA to quantitate the amount of RNA loaded.

al., 1995) ; however transcripts of the KHC isoforms (FKIF1, 5 and 9) were not detectable in RPE by Northern blot in the present study. Similarly, the expression of mouse KIF3 transcripts (as detected by Northern blot) did not correlate with detection of protein in Western blots of the same tissues (Aizawa et al., 1992 ; Kondo et al., 1994).

FKIF1, 5 and 9 showed a high level of sequence identity (87–95 %) to previously identified members of the KHC family, suggesting that all three are KHC isoforms (Navone et al., 1992 ; Kato, 1990). This is the first report of three KHC isoforms expressed in one species. Only two KHC isoforms were detected in human tissues : one detected by both Northern and

790

L. B O S T -U S I N G E R E T A L. (A)

(B)

0

1

2

2.3 kb

Motor

cDNA clone 5' RACE clone PCR fragment

F. 4. For legend see opposite.

N O V E L C -T E R M I N A L K I N E S I N I N R E T I N A A N D R P E FKIF2 Zyb3 KatA KatB KAR3 KLPA ncd MCAK XKCM1 KIF2 HKHC(u) HKHC(n) DKHC KIF3A KRP85 FLA10 bimC Eg5 KLP1 KIF4 unc104 KIF1B HUMORFW

F. 5. Dendogram analysis of the relationship of FKIF2 to representative KIFs. FKIF2 sequence is marked by an arrowhead. The complete amino acid sequences were analysed by the UWGCG PileUp sequence analysis program (Devereux et al., 1984), which maximizes the amino acid alignment among compared sequences. Similarity is inversely proportional to the distance along the horizontal axis. The sequences followed by their GenBank accession No’s are as follows (see legend of Fig. 2 for additional accession numbers) : XKCM1 U36485 ; KRP85 L16993 ; KatA D11371 ; KatB D21137.

Western blot in many tissues and thus termed ubiquitous (KHC(u)], plus another detected exclusively in neuronal tissues and termed neuronal [KHC(n)] (Niclas et al., 1994). Two different KHCs have also been identified in mouse (Aizawa et al., 1992 ; Kato, 1990). Only one KHC has been reported for Drosophila (Yang et al., 1989), squid (Kosik et al., 1990), or sea urchin (Wright et al., 1991). The three putative fish KHC isoforms exhibited different tissue specific expression patterns by Northern blot analysis, although all three showed strong hybridization to a transcript in the 6±5–6±6 kb size range. The major transcripts reported for both ubiquitous and neuronal KHCs in

791

human were of a similar size (6±2 kb) (Niclas et al., 1994). FKIF1 was detectable as a single 6±5 kb transcript in all tissues examined (except RPE), suggesting that it might represent the fish homologue of the ubiquitous KHC isoform (King-Smith et al., 1995). Consistent with this suggestion is the finding that the predicted FKIF1 amino acid sequence shares a higher percent identity with human KHC(u) (95 %) than with KHC(n) (87 %). Additional sequence information outside of the conserved motor domain would be needed to verify that FKIF1 is a bone fide KHC isoform. FKIF5 was detected exclusively in retina and brain, suggesting it might be the fish neuronal isoform of KHC. FKIF5 shared the highest percent identity with human KHC(n) and with KHC identified from mouse brain (Table III). There was also a 1±9 kb FKIF5 transcript barely detectable in muscle. In human brain, both the KHC(u) and the KHC(n) probes hybridized to a 1±7 kb transcript in addition to the 6±2 and other intermediate-sized transcripts (Niclas et al., 1994). The significance of this smaller transcript is not understood. FKIF9 appears to be preferentially expressed in neuronal tissues (brain and retina), but also shows expression in kidney ; thus FKIF9 falls in between FKIF1 and FKIF5 in terms of the different types of tissues which express this gene. FKIF3 and 8 were 82 and 90 % identical to mouse KIF1B which was previously identified as a member of the unc104}KIF1 family (Nangaku et al., 1994 ; Sekine et al., 1994 ; Wright et al., 1991). Unc104 and KIF1A have been implicated in anterograde translocation of synaptic vesicle precursors in neuronal axons (Otsuka et al., 1991 ; Okada et al., 1995), while KIF1B has been reported to mediate anterograde axonal transport of mitochondria in mouse brain (Nangaku et al., 1994). Since both FKIF3 and 8 show higher sequence identity to mouse KIF1B than to any other KIF in the database, they may be fish homologues mediating similar functions in retina or RPE. Although we identified numerous KIFs by our PCR screens, we know that our search did not exhaustively identify all kinesins expressed in the RPE and retina. The lack of detection of a KIF3 family member was somewhat surprising since members of this family have been identified in brain (Kondo et al., 1994) and sensory receptors (Tabish et al., 1995). Furthermore, recent studies have shown that antibodies to KIF3A bind to bands of appropriate molecular weight in western blots of fish retina and also to the basal body and connecting cilium of fish photoreceptors, in addition to binding to other microtubules in fish photoreceptors (Beech et al., 1996). Thus a KIF3

F. 4. Nucleotide and predicted amino acid sequence of FKIF2. (A) The nucleotide and corresponding amino acids are numbered as shown. Primers used in PCR and RACE amplifications are shown as arrows. The peptide used for antibody construction is boxed. (B) Schematic representation of FKIF2 cDNA structure. The regions cloned by RACE amplification, PCR amplification, and cDNA library screening are noted.

L. B O S T -U S I N G E R E T A L.

(B)

ba

ss

re tin a m us cl e ki dn ey br ai n liv er

Bass tissues

R PE

(A)

R PE su nf is ch h R PE ic k R PE ra -r tR et in PE a hu (c m ul an tu r R PE ed) (c ul tu r

ed )

792

200 200 116 97

116 97

66

66

45

45

F. 6. Tissue-specific expression pattern of FKIF2 protein. The tissue and species distribution of FKIF2 was analysed by Western blot analysis using the anti-peptide FKIF2 antibody. Equal amounts (20 µg) of protein isolated (A) from a panel of bass tissues (RPE, retina, muscle, kidney, brain and liver) and (B) from RPE or RPE-retina lysates prepared from different species (bass, sunfish, chick, rat, and human) were loaded in each lane.

homologue appears to be expressed in fish retina but nonetheless was not detected by RT-PCR in our screen. It is possible that KIF3 message is present in retina in low copy number, perhaps because retinal KIF3 has a low protein turnover in relatively few retinal cell types. No representatives from the KIF3} osm3, the KIF4}XKLP1 or the bimC}Eg5 families of kinesins were detected in the PCR screen (Bloom and Endow, 1994 ; Goodson et al., 1994 ; Sekine et al., 1994). Since many of these latter two families contain kinesins primarily associated with mitotic or meiotic processes (Goldstein, 1993), their absence from the post-mitotic retina and RPE is not surprising. The presence of multiple kinesin family members in the retina and RPE may arise because different motile processes are mediated by distinct motors. FKIF2 is the predominant member expressed in both the retina and RPE and is a member of the C-terminal family of kinesins. The C-terminal motor ncd has been characterized by motility assays and shown to function as a minus-end directed motor (Moore and Endow, 1996). If FKIF2 similarly translocates towards microtubule minus-ends, it would mediate transport toward the apical surface in RPE cells (Burnside and Bost-Usinger, 1997). Photoreceptor microtubules are oriented in such a way that minus-end directed transport would be toward the connecting cilium in both the inner and outer segments (Troutt and Burnside, 1988). For example, the preferential localization of mitochondria in distalmost regions of the inner segments of differentiated photoreceptors is consistent with transport along inner segment microtubules by a minusend directed motor (Madreperla and Adler, 1989). We should note that in vitro motility assays performed on native FKIF2 protein or on in vitro expressed protein are needed to definitively show that FKIF2 has microtubule-dependent motility.

The multiplicity of kinesin family members expressed in retina and RPE suggests that microtubule motors are highly specialized for particular transport functions. Expression of multiple motors in the retina may ensure by redundancy that more than one motor is capable of mediating certain functions. Numerous motile processes in retinal and RPE cells have been shown to be microtubule-dependent. Among them are axonal transport in retinal ganglion cells (Amaratunga et al., 1993 ; Elluru et al., 1995), lysosome and phagosome transport in RPE cells (Beauchemin and Leuenberger, 1977 ; Besharse and Dunis, 1982 ; Herman and Steinberg, 1982) and morphogenesis and maintenance of photoreceptor morphology (Besharse and Dunis, 1982 ; Madreperla and Adler, 1989). The identification of FKIFs reported here and the initial characterization of the novel kinesin FKIF2 provide groundwork for future efforts to understand the roles of specific kinesins in photoreceptors and RPE. First steps toward understanding the function of FKIF2 including its immunolocalization, which is currently being investigated. Acknowledgements We would like to thank S. Endow for helpful advice throughout this project, A. Pidoux, Z. Cande, K. Pagh-Roehl, L. Wong, E. Hoang, and C. King-Smith for their helpful discussions and critical reading of the manuscript, and Kris Okumu for excellent technical assistance. We would like to thank J. Korenbrot for the generous gift of the striped bass RPE-retina cDNA library. This work was supported by NIH grants EY03575 and EY6534-02.

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