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The ribosome receptor, p180, interacts with kinesin heavy chain, KIF5B
BBRC Biochemical and Biophysical Research Communications 319 (2004) 987–992 www.elsevier.com/locate/ybbrc
The ribosome receptor, p180, interacts with kinesin heavy chain, KIF5B Russell J. Diefenbach,* Eve Diefenbach, Mark W. Douglas, and Anthony L. Cunningham Centre for Virus Research, Westmead Millennium Institute, The University of Sydney and Westmead Hospital, Westmead, NSW 2145, Australia Received 11 May 2004 Available online 28 May 2004
Abstract The conventional microtubule-dependent motor protein kinesin consists of heavy and light chains both of which have been documented to bind a variety of potential linker or cargo proteins. In this study we employed a yeast two-hybrid assay to identify additional binding partners of the kinesin heavy chain isoform KIF5B. A human brain cDNA library was screened with a bait corresponding to amino acid residues 814–963 of human KIF5B. This screen identified the ribosome receptor, p180, as a KIF5Bbinding protein. The sites of interaction are residues 1294–1413 of p180 and the C-terminal half of the cargo binding-domain of KIF5B (residues 867–907). The KIF5B-binding site in p180 is homologous to the previously determined KIF5B-binding site in kinectin. The interacting regions of p180 and KIF5B consist almost entirely of heptad repeats, suggesting the interaction is a coiledcoil. A role for the kinesin/p180 interaction may include mRNA localization and/or transport of endoplasmic reticulum-derived vesicles. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Kinesin; Ribosome receptor; Kinectin; Heptad repeats; Endoplasmic reticulum
The superfamily of kinesin molecular motor proteins together with cytoplasmic dynein is responsible for microtubule-dependent transport of cargo in eukaryotic cells [1]. Conventional kinesin, the first member of the kinesin superfamily (KIF) to be discovered [2], is a tetrameric protein consisting of two heavy chains (KIF5) and two light chains [3]. The mammalian genome contains three KIF5 genes (KIF5A, KIF5B, and KIF5C). KIF5B is ubiquitously expressed while KIF5A and KIF5C are expressed only in neuronal tissue [4–6]. Functions attributed to this class include transport of: mitochondria and lysosomes [7]; oligomeric tubulin [8]; neurofilament [9]; mRNA [10]; synaptic vesicle components [11]; and viruses [12,13]. Previous studies had implicated the C-terminal tail of KIF5 as having a direct role in binding of membranous cargo [14]. Several proteins have now been shown to directly bind to the KIF5 tail domain including kinectin [15], Ran-binding protein 2 [16], herpes simplex viral
*
Corresponding author. Fax: +61-2-9845-9103. E-mail address: [email protected] Diefenbach).
(R.J.
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.05.069
protein US11 [13], synaptosome-associated protein of 25 and 23 kDa (SNAP25 and SNAP23) [17], and glutamate receptor-interacting protein, GRIP1 [18]. In this study, we present evidence for another new KIF5B-interacting protein, the 180 kDa ribosome receptor, p180, identified using a yeast two-hybrid screen. The nature of the interaction is likely to be an a-helical coiled-coil mediated via heptad repeats.
Materials and methods Expression constructs. Fragments of human KIF5B and KLC1 inserted into the yeast LexA two-hybrid vectors displayBAIT and displayTARGET (Display Systems Biotech) or the bacterial expression vectors pET-28a (Novagen) and pGEX-2T have been previously described [17,19]. Full-length human SNAP25B inserted into pGEX-2T has been previously described [17]. Subunits of human dynein inserted into displayBAIT and displayTARGET have also been previously described [17]. Fragments of human ribosome receptor (RR or p180) were amplified either from: plasmid pEGFP-p100 [20] which was kindly provided by Dr. Tetsu Akiyama (Institute of Molecular and Cellular Biosciences, The University of Tokyo); plasmid pTSX3.8F (contains cDNA for p180 isoform with 24 tandem repeats) kindly provided by Dr K. Ogawa-Goto (National Institute of Infectious Diseases, Tokyo) [21]; or displayTARGET RR1127–1537 obtained in
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this study. Fragments were cloned into EcoRI and XhoI digested or XhoI digested displayTARGET or EcoRI and SalI digested pGEX5X-1 (Amersham Biosciences). A fragment corresponding to RR1127– 1537 was also excised from displayTARGET and inserted into EcoRI and XhoI digested displayBAIT. In addition, a fragment which encodes amino acids 1–84 of herpes simplex viral tegument protein US11 was amplified from full-length US11 in pET-28c [13] and inserted into BamHI digested pET-28c. Yeast two-hybrid assay. The use of the LexA-based two-hybrid assay to screen a human brain cDNA library with KIF5B heavy chain (residues 814–963) as bait has been previously described [17]. The yeast two-hybrid assay was also used to map the interacting domains of KIF5B heavy chain and p180 identified in the initial two-hybrid screen. The protocols for assessing interacting domains, quantification of each positive interaction using a b-galactosidase assay, and determination of fusion protein expression were as described previously [17]. In vitro binding studies. The glutathione-S-transferase (GST), oligohistidine (His), and untagged fusion constructs were expressed, harvested, and lysed as previously described [13]. The His-tagged proteins KIF5B amino acid fragments 771–963, 814–963, and US11,1– 84 were purified as previously described [17]. Prior to binding experiments, purified His-proteins were diluted tenfold with wash buffer (phosphate-buffered saline, pH 7.2, 0.1% (v/v) Triton X-100). GSTfusion proteins were immobilized on glutathione–Sepharose beads prior to incubation with either purified His-proteins or bacterial lysates containing untagged KLC4-569. Conditions for binding, wash, and elution were as previously described [17]. Analysis of protein complexes. Protein complexes were separated by SDS–PAGE using precast 4–20% gradient gels (Bio-Rad) and proteins were identified by immunoblotting as previously described [19]. Antibodies used included mouse monoclonal anti-KLC (L2; Chemicon Chemical), mouse monoclonal antibodies against fusion tags hemagglutinin (HA), LexA, and His (Santa Cruz Biotechnology).
Results Yeast two-hybrid analysis of the interaction of KIF5B with p180 We employed a yeast two-hybrid screen using a fragment (amino acids 814–963; Fig. 1A) of human KIF5B heavy chain as bait and a human brain cDNA library as target. As a result of this screen, two clones were identified from the screening of approximately 1.85 106 independent clones as fragments of the 180 kDa ribosome receptor, p180 (GenBank Accession No. NM_004587). These clones were in addition to the 13 clones previously identified as SNAP25 [17]. This p180 is an integral endoplasmic reticulum protein with a predicted N-terminal transmembrane domain spanning amino acids 8–33 (Fig. 1B) [22]. Isoforms of human p180 contain a 10 amino acid consensus motif, NQGKKAEGAQ, repeated in tandem up to 54 times [23]. These repeats contain the ribosome binding domain [24] and are inserted near the N-terminus of p180 while the remainder of the protein is composed predominantly of heptad repeats with unknown function (Fig. 1B). The positive clones identified in this study correspond to amino acids 1127–1537 and 1215–1537 of p180 and are located at the C-terminal heptad-repeat region (Fig. 1B).
Fig. 1. Diagram of proteins and fragments thereof expressed in this study. In each case fragments illustrated with the solid lines are positive for the identified KIF5B/p180 interaction. (A) Human KIF5B showing head, stalk, and tail domains [6]. (B) Human p180 [23] with the predicted transmembrane domain (TM; residues 8–33) indicated. The 54 tandem repeats (consensus NQGKKAEGAQ) of p180 that mediate ribosome binding are contained within residues 177–736 (gray box). The presence of heptad repeats (black boxes) and therefore the probability of forming coiled-coils was determined for each protein sequence using the Lupas algorithm [41]. GenBank accession numbers used included: NM_004521 for KIF5B; and NM_004587 together with AF007575 for p180. *Fragment 40–868 lacks residues 256–576 of fulllength p180 as it was generated from a p180 isoform containing 24 tandem repeats [21].
To further define the interaction between KIF5B and p180, fragments of both proteins were expressed in yeast two-hybrid vectors. The expression of all bait and target fusion proteins in the yeast two-hybrid assay was confirmed by immunoblotting (not shown). Positive interactions were initially assessed by activation of both reporter genes LEU2 and lacZ resulting in blue colonies on –Leu/X-gal plates. Quantitative b-galactosidase activity was also determined. The p180 fragment 1127– 1537 identified in the initial screen was tested along with fragments which span residues 40–1537 of p180 (Fig. 1B). Each fragment was tested against KIF5B555– 813 and KIF5B814–963. Results showed that RR1127– 1537, RR1215–1413, and RR1294–1413 all interacted with the original bait KIF5B814–963 (Fig 2A). Fragment RR40–868 which spans the ribosome binding site of p180 did not bind to KIF5B814–963 (Fig. 2A). Specificity was confirmed by the fact that none of these
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Fig. 2. Yeast two-hybrid analysis of the interaction of KIF5B with p180. Summary of binding data obtained with yeast co-transformed with various combinations of bait and target fusion constructs. (A) KIF5B and p180. (B) p180 and various motor protein subunits. Positive interactions were observed as blue colonies with + indicating the lightest blue and +++ the darkest blue. No growth ()) indicates a negative interaction. Positive interactions were then quantified using a liquid b-galactosidase assay. Given activity values are the average of measurements from at least three separate colonies. Negative interactions were not quantified and are designated ND (not determined). Background b-galactosidase activity (not shown) has a value typically less than 20, as previously reported [17].
p180 fragments interact with the KIF5B stalk fragment 555–813 (Fig. 2A). An interaction above background was also observed between RR822–1214 (which does not interact with KIF5B814–963) and KIF5B555–813, suggesting this is an additional, though probably minor,
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contact site between KIF5B and p180 (Fig. 2A). Overall this indicates that the minimal binding site for KIF5B maps to region 1294–1413 of p180. This region corresponds to a heptad-repeat region near the C-terminus of p180 (Fig. 1B). Of note is that this region is also homologous to the previously identified KIF5B-binding site in kinectin [15] but has no obvious homology with the heptad-repeat KIF5B-binding site in SNAP25 [17] (not shown). Kinectin and p180, though functionally distinct, have been identified as belonging to a related family of proteins with homology between heptadrepeat regions in each protein [25]. Mapping of the p180-binding site in KIF5B showed that RR1215–1413 binds to KIF5B814–907 and 867– 963 but not to 908–963 or 555–866 (Fig. 2A). Lack of auto-activation by bait/KIF5B814–907 and 867–963 has been previously demonstrated [17]. The KIF5B region 814–907 corresponds to a predicted cargo-binding site in KIF5B [26] and maps to the heptad-repeat region in the tail domain of KIF5B (Fig. 1A). We have reported a similar observation for the binding of SNAP25 and SNAP23 to KIF5B [17]. In contrast to the SNAP25 and SNAP23 interaction, p180 still bound to region 867– 963, suggesting that the minimal binding site in KIF5B for p180 maps to region 867–907 (Fig. 1A). A similar interaction has been previously described for herpes simplex viral protein US11 and KIF5B [13]. The interaction of p180 and KIF5B was also confirmed in the reverse orientation in the yeast two-hybrid assay (Fig. 2B). Lack of auto-activation by target/KIF5B814– 963 was also demonstrated (not shown). This served as a positive control for assessing possible interactions between RR1127–1537 and either KLC or subunits of the retrograde molecular motor dynein. Analysis with bait/ RR1127–1537 was necessary as KLC and dynein subunits LC8, RP3, and TcTex1 autoactivate as bait fusion constructs [17]. KLC, in the absence of KIF5B, was found not to bind directly to the KIF5B-binding fragment RR1127– 1537. Fragments of KLC tested included the heptad-repeat region 4–199, which contains the KIF5B-binding site [17,27,28], and the TPR repeat region 200–569, which contains a cargo-binding domain [29] (Fig. 2B). Furthermore, RR1127–1537 did not interact with subunits of dynein. The dynein subunits tested included dynein intermediate chain (DIC) and the dynein light chains LC8, RP3, and TcTex1 (Fig. 2B). Similar results were obtained previously with SNAP25 and SNAP23 [17]. In vitro analysis of the interaction of KIF5B and KLC with p180 The interaction of KIF5B and KLC with p180 was also analyzed using an in vitro pull-down assay. Purified His-tagged fragments KIF5B814–963 and KIF5B771– 963 were analyzed for interaction with GST-tagged RR1215–1413. In agreement with the yeast two-hybrid
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Fig. 3. In vitro pull-down assay to assess the interaction of KIF5B and KLC with p180. His-US11, His-KIF5B or untagged KLC was added to GST-tagged proteins on glutathione–Sepharose beads. Protein complexes were subsequently eluted and separated by SDS–PAGE (4– 20%). (A) Immunoblot with anti-His tag antibody confirms the presence of His-KIF5B co-eluting preferentially with GST-RR1215–1413. No binding to p180 was observed with His-US11. (B) Immunoblot with anti-KLC antibody confirms the presence of untagged KLC4–569 co-eluting only with GST-KIF5B771–963.
analysis an interaction was confirmed between both KIF5B814–963 and KIF5B771–963, both of which contain the p180-binding region 814–907 and RR1215– 1413, which contains the KIF5B-binding site 1294–1413 (Fig. 3A). No interaction was observed between RR1215–1413 and the known KIF5B-binding protein US11 [13]. An in vitro pull-down assay was used to confirm that KLC, in the absence of KIF5B, does not directly bind to the region of p180 that contains the KIF5B-binding site (residues 1294–1413). Bacterial lysates containing untagged KLC4–569 were added to various GST-tagged proteins. In agreement with the yeast two-hybrid analysis no interaction was observed between KLC and GST-RR1215–1413 nor, as previously documented, with a GST fusion of full-length SNAP25, another KIF5B-binding protein [17] (Fig. 3B). An interaction was confirmed for the positive control, in this case GSTKIF5B771–963 which contains the known KLC-binding site (residues 789–813) [17]. For each pull-down assay, Coomassie blue staining of protein gels was performed to confirm the presence of GST proteins eluted from glutathione–Sepharose beads (not shown).
Discussion The precise role of the mammalian ribosome receptor, p180, in cells has not been determined. Studies in
yeast, which lack p180, have shown that expression of p180 results in proliferation of rough membranes coupled with an increased secretory activity [30]. Induction of secretory pathway components in yeast was found to be subsequently associated with increased stability of their mRNA [31]. These functions are dependent on the ribosome binding activity of p180 which resides in the N-terminus of the protein [30,31]. The role of the heptad repeat-containing C-terminal domain in these processes is not clear and prior to this study no ligand for this domain had been identified. The predicted extended stalk-like nature of this domain may facilitate the maintenance of regular intermembrane spacing, the regulation of ribosome binding, and/or arrangement of p180 on the cytoplasmic surface of the rough endoplasmic reticulum [30]. Overall it is likely that p180 is involved in the terminal differentiation of mammalian secretory tissue [31]. A recent finding has shown a direct link between hDLG, a mammalian homolog of Drosophila discs large suppressor, and p180 [20]. Binding was shown to require the PDZ domain of DLG, while it did not require the 10 amino acid repeat ribosome-binding domain of p180. Binding of p180 to hDLG was predicted to occur at the C-terminus of p180 since it contains a consensus PDZbinding domain (consensus XT/SXV/I/L; GTSV in p180) [20]. The conclusion was made that this interaction may play a role in regulation of secretion. This is because DLG (which contains three PDZ domains, one SH3 domain, and one guanylate kinase-like domain) functions as a scaffolding protein to recruit components of various signaling pathways. In addition, like kinectin, p180 may function as a RhoA GTPase effector since it contains a potential RhoA-binding motif [20]. In this study we have identified an interaction between the kinesin motor, KIF5B, and p180. This interaction depends on the heptad-repeat containing regions in the C-terminal domain of p180 and the tail domain of KIF5B (Fig. 1). The biological significance of this interaction is not clear. Like the related kinesin receptor, kinectin, p180 is an endoplasmic reticulum-integral membrane protein. However, unlike kinectin, p180 has the ability to bind ribosomes. The potential of p180 to bind ribosomes and kinesin simultaneously through separate domains suggests it may be important for cytoplasmic mRNA localization. The role of cytoplasmic mRNA localization is to restrict the synthesis of proteins to the sites of their functions in eukaryotic cells [32]. This is most evident in the localization and translation of mRNA in dendrites (reviewed in [33]). The mRNA is typically transported in the form of RNA granules which consist of several components including mRNAs and RNA-binding proteins (often Staufen) [32,34]. The role of the cytoskeleton, and particularly kinesin, in the transport of these RNA granules has been documented [35–37]. These
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RNA granules may bind directly to kinesin or via kinesin linkers, with both certainly a possibility. Distinct RNA granule transport intermediates have been observed in neurons. These intermediates were all found to contain Staufen and kinesin, KIF5, but could be distinguished by the presence or absence of ribosomes and endoplasmic reticulum [38,39]. Presumably only those RNA granules which contain both endoplasmic reticulum and ribosomes could be transported by a kinesin/p180 complex. Indeed, organelle-free but kinesin-dependent transport of myelin basic protein mRNA-containing granules has been reported in oligodendrocytes [35]. Further determination of the composition of RNA transport granules will elucidate what are the various kinesin linkers. Additionally, as initially proposed for kinectin [40], p180 may function as a receptor for kinesin-driven microtubule-dependent transport of vesicles derived from the endoplasmic reticulum. Interestingly, a structural protein encoded by the UL48 gene from human cytomegalovirus has been reported to interact with p180 [21]. The interaction did not require the C-terminal heptad-repeat domain of p180. The implication being that the virus exploits the p180/kinesin vesicle transport system to facilitate movement of the viral capsid through the cytoplasm.
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Acknowledgments This work was supported by Grants 107374 and 253617 from the Australian National Health and Medical Research Council (to A.L.C. and R.J.D.).
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The ribosome receptor, p180, interacts with kinesin heavy chain, KIF5B Russell J. Diefenbach,* Eve Diefenbach, Mark W. Douglas, and Anthony L. Cunningham Centre for Virus Research, Westmead Millennium Institute, The University of Sydney and Westmead Hospital, Westmead, NSW 2145, Australia Received 11 May 2004 Available online 28 May 2004
Abstract The conventional microtubule-dependent motor protein kinesin consists of heavy and light chains both of which have been documented to bind a variety of potential linker or cargo proteins. In this study we employed a yeast two-hybrid assay to identify additional binding partners of the kinesin heavy chain isoform KIF5B. A human brain cDNA library was screened with a bait corresponding to amino acid residues 814–963 of human KIF5B. This screen identified the ribosome receptor, p180, as a KIF5Bbinding protein. The sites of interaction are residues 1294–1413 of p180 and the C-terminal half of the cargo binding-domain of KIF5B (residues 867–907). The KIF5B-binding site in p180 is homologous to the previously determined KIF5B-binding site in kinectin. The interacting regions of p180 and KIF5B consist almost entirely of heptad repeats, suggesting the interaction is a coiledcoil. A role for the kinesin/p180 interaction may include mRNA localization and/or transport of endoplasmic reticulum-derived vesicles. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Kinesin; Ribosome receptor; Kinectin; Heptad repeats; Endoplasmic reticulum
The superfamily of kinesin molecular motor proteins together with cytoplasmic dynein is responsible for microtubule-dependent transport of cargo in eukaryotic cells [1]. Conventional kinesin, the first member of the kinesin superfamily (KIF) to be discovered [2], is a tetrameric protein consisting of two heavy chains (KIF5) and two light chains [3]. The mammalian genome contains three KIF5 genes (KIF5A, KIF5B, and KIF5C). KIF5B is ubiquitously expressed while KIF5A and KIF5C are expressed only in neuronal tissue [4–6]. Functions attributed to this class include transport of: mitochondria and lysosomes [7]; oligomeric tubulin [8]; neurofilament [9]; mRNA [10]; synaptic vesicle components [11]; and viruses [12,13]. Previous studies had implicated the C-terminal tail of KIF5 as having a direct role in binding of membranous cargo [14]. Several proteins have now been shown to directly bind to the KIF5 tail domain including kinectin [15], Ran-binding protein 2 [16], herpes simplex viral
*
Corresponding author. Fax: +61-2-9845-9103. E-mail address: [email protected] Diefenbach).
(R.J.
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.05.069
protein US11 [13], synaptosome-associated protein of 25 and 23 kDa (SNAP25 and SNAP23) [17], and glutamate receptor-interacting protein, GRIP1 [18]. In this study, we present evidence for another new KIF5B-interacting protein, the 180 kDa ribosome receptor, p180, identified using a yeast two-hybrid screen. The nature of the interaction is likely to be an a-helical coiled-coil mediated via heptad repeats.
Materials and methods Expression constructs. Fragments of human KIF5B and KLC1 inserted into the yeast LexA two-hybrid vectors displayBAIT and displayTARGET (Display Systems Biotech) or the bacterial expression vectors pET-28a (Novagen) and pGEX-2T have been previously described [17,19]. Full-length human SNAP25B inserted into pGEX-2T has been previously described [17]. Subunits of human dynein inserted into displayBAIT and displayTARGET have also been previously described [17]. Fragments of human ribosome receptor (RR or p180) were amplified either from: plasmid pEGFP-p100 [20] which was kindly provided by Dr. Tetsu Akiyama (Institute of Molecular and Cellular Biosciences, The University of Tokyo); plasmid pTSX3.8F (contains cDNA for p180 isoform with 24 tandem repeats) kindly provided by Dr K. Ogawa-Goto (National Institute of Infectious Diseases, Tokyo) [21]; or displayTARGET RR1127–1537 obtained in
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this study. Fragments were cloned into EcoRI and XhoI digested or XhoI digested displayTARGET or EcoRI and SalI digested pGEX5X-1 (Amersham Biosciences). A fragment corresponding to RR1127– 1537 was also excised from displayTARGET and inserted into EcoRI and XhoI digested displayBAIT. In addition, a fragment which encodes amino acids 1–84 of herpes simplex viral tegument protein US11 was amplified from full-length US11 in pET-28c [13] and inserted into BamHI digested pET-28c. Yeast two-hybrid assay. The use of the LexA-based two-hybrid assay to screen a human brain cDNA library with KIF5B heavy chain (residues 814–963) as bait has been previously described [17]. The yeast two-hybrid assay was also used to map the interacting domains of KIF5B heavy chain and p180 identified in the initial two-hybrid screen. The protocols for assessing interacting domains, quantification of each positive interaction using a b-galactosidase assay, and determination of fusion protein expression were as described previously [17]. In vitro binding studies. The glutathione-S-transferase (GST), oligohistidine (His), and untagged fusion constructs were expressed, harvested, and lysed as previously described [13]. The His-tagged proteins KIF5B amino acid fragments 771–963, 814–963, and US11,1– 84 were purified as previously described [17]. Prior to binding experiments, purified His-proteins were diluted tenfold with wash buffer (phosphate-buffered saline, pH 7.2, 0.1% (v/v) Triton X-100). GSTfusion proteins were immobilized on glutathione–Sepharose beads prior to incubation with either purified His-proteins or bacterial lysates containing untagged KLC4-569. Conditions for binding, wash, and elution were as previously described [17]. Analysis of protein complexes. Protein complexes were separated by SDS–PAGE using precast 4–20% gradient gels (Bio-Rad) and proteins were identified by immunoblotting as previously described [19]. Antibodies used included mouse monoclonal anti-KLC (L2; Chemicon Chemical), mouse monoclonal antibodies against fusion tags hemagglutinin (HA), LexA, and His (Santa Cruz Biotechnology).
Results Yeast two-hybrid analysis of the interaction of KIF5B with p180 We employed a yeast two-hybrid screen using a fragment (amino acids 814–963; Fig. 1A) of human KIF5B heavy chain as bait and a human brain cDNA library as target. As a result of this screen, two clones were identified from the screening of approximately 1.85 106 independent clones as fragments of the 180 kDa ribosome receptor, p180 (GenBank Accession No. NM_004587). These clones were in addition to the 13 clones previously identified as SNAP25 [17]. This p180 is an integral endoplasmic reticulum protein with a predicted N-terminal transmembrane domain spanning amino acids 8–33 (Fig. 1B) [22]. Isoforms of human p180 contain a 10 amino acid consensus motif, NQGKKAEGAQ, repeated in tandem up to 54 times [23]. These repeats contain the ribosome binding domain [24] and are inserted near the N-terminus of p180 while the remainder of the protein is composed predominantly of heptad repeats with unknown function (Fig. 1B). The positive clones identified in this study correspond to amino acids 1127–1537 and 1215–1537 of p180 and are located at the C-terminal heptad-repeat region (Fig. 1B).
Fig. 1. Diagram of proteins and fragments thereof expressed in this study. In each case fragments illustrated with the solid lines are positive for the identified KIF5B/p180 interaction. (A) Human KIF5B showing head, stalk, and tail domains [6]. (B) Human p180 [23] with the predicted transmembrane domain (TM; residues 8–33) indicated. The 54 tandem repeats (consensus NQGKKAEGAQ) of p180 that mediate ribosome binding are contained within residues 177–736 (gray box). The presence of heptad repeats (black boxes) and therefore the probability of forming coiled-coils was determined for each protein sequence using the Lupas algorithm [41]. GenBank accession numbers used included: NM_004521 for KIF5B; and NM_004587 together with AF007575 for p180. *Fragment 40–868 lacks residues 256–576 of fulllength p180 as it was generated from a p180 isoform containing 24 tandem repeats [21].
To further define the interaction between KIF5B and p180, fragments of both proteins were expressed in yeast two-hybrid vectors. The expression of all bait and target fusion proteins in the yeast two-hybrid assay was confirmed by immunoblotting (not shown). Positive interactions were initially assessed by activation of both reporter genes LEU2 and lacZ resulting in blue colonies on –Leu/X-gal plates. Quantitative b-galactosidase activity was also determined. The p180 fragment 1127– 1537 identified in the initial screen was tested along with fragments which span residues 40–1537 of p180 (Fig. 1B). Each fragment was tested against KIF5B555– 813 and KIF5B814–963. Results showed that RR1127– 1537, RR1215–1413, and RR1294–1413 all interacted with the original bait KIF5B814–963 (Fig 2A). Fragment RR40–868 which spans the ribosome binding site of p180 did not bind to KIF5B814–963 (Fig. 2A). Specificity was confirmed by the fact that none of these
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Fig. 2. Yeast two-hybrid analysis of the interaction of KIF5B with p180. Summary of binding data obtained with yeast co-transformed with various combinations of bait and target fusion constructs. (A) KIF5B and p180. (B) p180 and various motor protein subunits. Positive interactions were observed as blue colonies with + indicating the lightest blue and +++ the darkest blue. No growth ()) indicates a negative interaction. Positive interactions were then quantified using a liquid b-galactosidase assay. Given activity values are the average of measurements from at least three separate colonies. Negative interactions were not quantified and are designated ND (not determined). Background b-galactosidase activity (not shown) has a value typically less than 20, as previously reported [17].
p180 fragments interact with the KIF5B stalk fragment 555–813 (Fig. 2A). An interaction above background was also observed between RR822–1214 (which does not interact with KIF5B814–963) and KIF5B555–813, suggesting this is an additional, though probably minor,
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contact site between KIF5B and p180 (Fig. 2A). Overall this indicates that the minimal binding site for KIF5B maps to region 1294–1413 of p180. This region corresponds to a heptad-repeat region near the C-terminus of p180 (Fig. 1B). Of note is that this region is also homologous to the previously identified KIF5B-binding site in kinectin [15] but has no obvious homology with the heptad-repeat KIF5B-binding site in SNAP25 [17] (not shown). Kinectin and p180, though functionally distinct, have been identified as belonging to a related family of proteins with homology between heptadrepeat regions in each protein [25]. Mapping of the p180-binding site in KIF5B showed that RR1215–1413 binds to KIF5B814–907 and 867– 963 but not to 908–963 or 555–866 (Fig. 2A). Lack of auto-activation by bait/KIF5B814–907 and 867–963 has been previously demonstrated [17]. The KIF5B region 814–907 corresponds to a predicted cargo-binding site in KIF5B [26] and maps to the heptad-repeat region in the tail domain of KIF5B (Fig. 1A). We have reported a similar observation for the binding of SNAP25 and SNAP23 to KIF5B [17]. In contrast to the SNAP25 and SNAP23 interaction, p180 still bound to region 867– 963, suggesting that the minimal binding site in KIF5B for p180 maps to region 867–907 (Fig. 1A). A similar interaction has been previously described for herpes simplex viral protein US11 and KIF5B [13]. The interaction of p180 and KIF5B was also confirmed in the reverse orientation in the yeast two-hybrid assay (Fig. 2B). Lack of auto-activation by target/KIF5B814– 963 was also demonstrated (not shown). This served as a positive control for assessing possible interactions between RR1127–1537 and either KLC or subunits of the retrograde molecular motor dynein. Analysis with bait/ RR1127–1537 was necessary as KLC and dynein subunits LC8, RP3, and TcTex1 autoactivate as bait fusion constructs [17]. KLC, in the absence of KIF5B, was found not to bind directly to the KIF5B-binding fragment RR1127– 1537. Fragments of KLC tested included the heptad-repeat region 4–199, which contains the KIF5B-binding site [17,27,28], and the TPR repeat region 200–569, which contains a cargo-binding domain [29] (Fig. 2B). Furthermore, RR1127–1537 did not interact with subunits of dynein. The dynein subunits tested included dynein intermediate chain (DIC) and the dynein light chains LC8, RP3, and TcTex1 (Fig. 2B). Similar results were obtained previously with SNAP25 and SNAP23 [17]. In vitro analysis of the interaction of KIF5B and KLC with p180 The interaction of KIF5B and KLC with p180 was also analyzed using an in vitro pull-down assay. Purified His-tagged fragments KIF5B814–963 and KIF5B771– 963 were analyzed for interaction with GST-tagged RR1215–1413. In agreement with the yeast two-hybrid
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Fig. 3. In vitro pull-down assay to assess the interaction of KIF5B and KLC with p180. His-US11, His-KIF5B or untagged KLC was added to GST-tagged proteins on glutathione–Sepharose beads. Protein complexes were subsequently eluted and separated by SDS–PAGE (4– 20%). (A) Immunoblot with anti-His tag antibody confirms the presence of His-KIF5B co-eluting preferentially with GST-RR1215–1413. No binding to p180 was observed with His-US11. (B) Immunoblot with anti-KLC antibody confirms the presence of untagged KLC4–569 co-eluting only with GST-KIF5B771–963.
analysis an interaction was confirmed between both KIF5B814–963 and KIF5B771–963, both of which contain the p180-binding region 814–907 and RR1215– 1413, which contains the KIF5B-binding site 1294–1413 (Fig. 3A). No interaction was observed between RR1215–1413 and the known KIF5B-binding protein US11 [13]. An in vitro pull-down assay was used to confirm that KLC, in the absence of KIF5B, does not directly bind to the region of p180 that contains the KIF5B-binding site (residues 1294–1413). Bacterial lysates containing untagged KLC4–569 were added to various GST-tagged proteins. In agreement with the yeast two-hybrid analysis no interaction was observed between KLC and GST-RR1215–1413 nor, as previously documented, with a GST fusion of full-length SNAP25, another KIF5B-binding protein [17] (Fig. 3B). An interaction was confirmed for the positive control, in this case GSTKIF5B771–963 which contains the known KLC-binding site (residues 789–813) [17]. For each pull-down assay, Coomassie blue staining of protein gels was performed to confirm the presence of GST proteins eluted from glutathione–Sepharose beads (not shown).
Discussion The precise role of the mammalian ribosome receptor, p180, in cells has not been determined. Studies in
yeast, which lack p180, have shown that expression of p180 results in proliferation of rough membranes coupled with an increased secretory activity [30]. Induction of secretory pathway components in yeast was found to be subsequently associated with increased stability of their mRNA [31]. These functions are dependent on the ribosome binding activity of p180 which resides in the N-terminus of the protein [30,31]. The role of the heptad repeat-containing C-terminal domain in these processes is not clear and prior to this study no ligand for this domain had been identified. The predicted extended stalk-like nature of this domain may facilitate the maintenance of regular intermembrane spacing, the regulation of ribosome binding, and/or arrangement of p180 on the cytoplasmic surface of the rough endoplasmic reticulum [30]. Overall it is likely that p180 is involved in the terminal differentiation of mammalian secretory tissue [31]. A recent finding has shown a direct link between hDLG, a mammalian homolog of Drosophila discs large suppressor, and p180 [20]. Binding was shown to require the PDZ domain of DLG, while it did not require the 10 amino acid repeat ribosome-binding domain of p180. Binding of p180 to hDLG was predicted to occur at the C-terminus of p180 since it contains a consensus PDZbinding domain (consensus XT/SXV/I/L; GTSV in p180) [20]. The conclusion was made that this interaction may play a role in regulation of secretion. This is because DLG (which contains three PDZ domains, one SH3 domain, and one guanylate kinase-like domain) functions as a scaffolding protein to recruit components of various signaling pathways. In addition, like kinectin, p180 may function as a RhoA GTPase effector since it contains a potential RhoA-binding motif [20]. In this study we have identified an interaction between the kinesin motor, KIF5B, and p180. This interaction depends on the heptad-repeat containing regions in the C-terminal domain of p180 and the tail domain of KIF5B (Fig. 1). The biological significance of this interaction is not clear. Like the related kinesin receptor, kinectin, p180 is an endoplasmic reticulum-integral membrane protein. However, unlike kinectin, p180 has the ability to bind ribosomes. The potential of p180 to bind ribosomes and kinesin simultaneously through separate domains suggests it may be important for cytoplasmic mRNA localization. The role of cytoplasmic mRNA localization is to restrict the synthesis of proteins to the sites of their functions in eukaryotic cells [32]. This is most evident in the localization and translation of mRNA in dendrites (reviewed in [33]). The mRNA is typically transported in the form of RNA granules which consist of several components including mRNAs and RNA-binding proteins (often Staufen) [32,34]. The role of the cytoskeleton, and particularly kinesin, in the transport of these RNA granules has been documented [35–37]. These
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RNA granules may bind directly to kinesin or via kinesin linkers, with both certainly a possibility. Distinct RNA granule transport intermediates have been observed in neurons. These intermediates were all found to contain Staufen and kinesin, KIF5, but could be distinguished by the presence or absence of ribosomes and endoplasmic reticulum [38,39]. Presumably only those RNA granules which contain both endoplasmic reticulum and ribosomes could be transported by a kinesin/p180 complex. Indeed, organelle-free but kinesin-dependent transport of myelin basic protein mRNA-containing granules has been reported in oligodendrocytes [35]. Further determination of the composition of RNA transport granules will elucidate what are the various kinesin linkers. Additionally, as initially proposed for kinectin [40], p180 may function as a receptor for kinesin-driven microtubule-dependent transport of vesicles derived from the endoplasmic reticulum. Interestingly, a structural protein encoded by the UL48 gene from human cytomegalovirus has been reported to interact with p180 [21]. The interaction did not require the C-terminal heptad-repeat domain of p180. The implication being that the virus exploits the p180/kinesin vesicle transport system to facilitate movement of the viral capsid through the cytoplasm.
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Acknowledgments This work was supported by Grants 107374 and 253617 from the Australian National Health and Medical Research Council (to A.L.C. and R.J.D.).
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