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Human Molecular Genetics Advance Access originally published online on October 7, 2004
Human Molecular Genetics 2004 13(23):3017-3027; doi:10.1093/hmg/ddh321
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Human Molecular Genetics, Vol. 13, No. 23 © Oxford University Press 2004; all rights reserved

Interconnections of CLN3, Hook1 and Rab proteins link Batten disease to defects in the endocytic pathway

Kaisu Luiro, Kristiina Yliannala, Laura Ahtiainen, Heidi Maunu, Irma Järvelä, Aija Kyttälä and Anu Jalanko*

National Public Health Institute, Department of Molecular Medicine, Biomedicum Helsinki, FIN-00251 Helsinki, Finland

Received August 24, 2004; Accepted September 27, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The endosomal/lysosomal transmembrane protein CLN3 is mutated in the Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL). However, the molecular mechanism of JNCL pathogenesis and the exact function of the CLN3 protein have remained unclear. Previous studies have shown that deletion of BTN1, the yeast orthologue of CLN3, leads to increased expression of BTN2. BTN2 encodes Btn2p, a proposed homologue to a novel microtubule-binding protein Hook1, which regulates endocytosis in Drosophila. We analysed here the putative interconnection between CLN3 and Hook1 in the mammalian cells and discovered that overexpression of human CLN3 induces aggregation of Hook1 protein, potentially by mediating its dissociation from the microtubules. Using in vitro binding assay we were able to demonstrate a weak interaction between Hook1 and the cytoplasmic segments of CLN3. We also found receptor-mediated endocytosis to be defective in CLN3-deficient JNCL fibroblasts, connecting CLN3, Hook1 and endocytosis in the mammalian system. Moreover, co-immunoprecipitation experiments showed that Hook1 physically interacts with endocytic Rab7, Rab9 and Rab11, hence delineating a manifold role for mammalian Hook1 in membrane trafficking events. These novel interactions between the microtubule-binding Hook1 and the large family of Rab GTPases also suggest a link between CLN3 function, microtubule cytoskeleton and endocytic membrane trafficking.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Juvenile form of the neuronal ceroid lipofuscinoses (JNCL, Batten disease), the most common neurodegenerative disease of childhood, is caused by mutations in the CLN3 gene encoding a transmembrane CLN3 protein (1). JNCL belongs to a group of eight autosomal, recessively inherited neurodegenerative disorders, termed NCL diseases, which all share the histopathological finding of autofluorescent accumulation in various tissues and progressive death of cortical neurons (2). Six of the eight genes underlying NCL have been identified (1,3–8), but the underlying disease mechanisms have remained elusive.

We and others have previously shown that overexpressed CLN3 localizes to the late endosomal/lysosomal compartments and also in EEA1-positive early endosomes of neurons (9–13). The lysosomal targeting of CLN3 is facilitated by two different lysosomal sorting signals (13) and AP3 has been suggested to be involved in this sorting event (14). Endogenous Cln3 is also found in the pre-synaptic areas of mouse retinal neurons and in synaptosomal fractions of the brain (15). Empirical evidence on the topology of the hydrophobic CLN3 was first provided by Mao et al. (16) who utilized flag-tagged CLN3 and glycosylation mutagenesis and suggested that CLN3 has five membrane-spanning domains, an extracellular/intraluminal N-terminal region and a cytoplasmic C-terminal region (16). In contrast, selective cell permeabilization assays indicated a cytoplasmic localization for the N-terminal region and suggested that CLN3 is a type III transmembrane protein consisting of six membrane-spanning domains (13). The cytoplasmic orientation of the N-terminal region has also been reported by Ezaki et al. (17).

The exact function of the CLN3 protein is currently unknown, but the severe symptoms of the affected and the conservation of the protein among different species indicate a fundamental role in cell metabolism. Studies using the yeast knockout model (btn1-{Delta}) for the CLN3 homologue BTN1 showed an acidification of the yeast vacuole suggesting that CLN3 has a role in regulating the acidic lysosomal pH (18,19). In humans, the reverse was observed; while intracellular pH of JNCL fibroblasts was normal, lysosomal pH was elevated (20). The significance of this finding in terms of the pathogenesis is yet to be understood. Gene expression profiling of the yeast btn1-{Delta} strain showed an upregulation of a novel BTN2 gene encoding Btn2p (18). Btn2p has been shown to interact with the yeast Yif1 protein (21), which forms complexes with the yeast Rab1/Ypt1 required for the budding of ER-derived vesicles (22), suggesting that Btn2p is involved in membrane trafficking events.

Btn2p is a putative orthologue of the mammalian Hook1 protein, a member of a novel family of three microtubule-binding proteins, Hook1, Hook2 and Hook3, which share a common protein structure of a microtubule-binding domain at the N-terminal region which has the property to form coiled coils, and a C-terminal region which putatively interacts with membranes (23). Functions of the mammalian Hook1 proteins have not been dissected, but the Drosophila homologue of Hook1 regulates trafficking of internalized ligands to the late endosomes (24,25). Furthermore, it has been reported to function in the maturation or stabilization of multivesicular bodies (MVBs) of the endocytic pathway (26). Recent analysis have also implicated Hook1 participation in endosomal fusion events (27).

In the present study we have analysed the interconnections of CLN3 and Hook1 proteins in the mammalian system. We show that CLN3 overexpression induces an aggregation of the microtubule-binding Hook1 protein in mammalian cells. We also demonstrate a defect in receptor-mediated endocytosis in CLN3-deficient fibroblasts. Most importantly, we discovered novel interactions between the mammalian Hook1 and several endocytic Rab GTPases. These findings implicate a role for CLN3 in the complex cellular machinery connecting cytoskeletal functions to endocytic membrane trafficking.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Overexpression of CLN3 causes aggregation of Hook1 protein
To examine the putative interaction between CLN3 and the microtubule-binding Hook1 protein in mammalian cells, HeLa cells were transiently transfected with Hook1, GFP-CLN3 or both and analysed by double immunostaining and confocal microscopy. Localization of the newly generated GFP–CLN3 was similar to that reported in the previous studies with overexpressed non-tagged CLN3; substantial co-localization was observed with the lysosomal Lamp1 marker (Fig. 1A–C). Transfected Hook1 localized uniformly throughout the cytoplasm in small punctuate subcellular structures of unknown origin (Fig. 1G). As described previously (23), no co-localization was observed with Hook1 and variety of immunological markers for diverse subcellular compartments including ER–Golgi intermediate compartment (ERGIC), Golgi (TGN46), early endosomes (EEA1), late endosomes (LBPA) and lysosomes (Lamp1) (data not shown). However, a dramatic change in the immunofluorescence staining of Hook1 was observed when co-expressed with GFP–CLN3; CLN3 caused Hook1 to appear in larger dot-like structures (Fig. 1D–F). These Hook1-positive subcellular structures were also observed upon co-expression of native CLN3 and Hook1 in both HeLa and COS-1 cells (data not shown). To test whether the observed phenomenon was specific for CLN3, we co-expressed another lysosomal membrane protein GFP-sialin (28) with Hook1 but no alteration in terms of Hook1 localization was observed (Fig. 1G–I). We then proceeded to explore the molecular link between the Hook1 and CLN3 proteins by pulse-chase analysis and immunoprecipitation. COS-1 cells were transiently transfected either with Hook1 or Hook1 and CLN3 together, the cells were then metabolically labelled, chased for 0–6 h, and subsequently immunoprecipitated with Hook1 antibodies. Hook1 co-transfected with an empty vector (Fig. 2A, lanes 2 and 3) or with sialin (Fig. 2B) immunoprecipitated as a ~85 kDa protein as predicted. However, when CLN3 was overexpressed simultaneously, Hook1 was no longer immunoprecipitable (Fig. 2A, lanes 4 and 5). The same phenomenon was observed when Hook1 was co-expressed with the deletion mutant form of the CLN3 (CLN3{Delta}ex7–8) or with a point mutant form causing milder phenotype, CLN3E295K (Fig. 2B). This was observed with three different Hook1 antibodies and it suggests, that upon co-expression with CLN3, Hook1 is found in aggregates. It has been shown that Hook1 binds microtubules with its conserved amino terminal domain (23). We were able to demonstrate that microtubule-depolymerizing agent nocodazole induced similar dot-like Hook1 aggregates as did CLN3, suggesting that CLN3 overexpression detaches Hook1 from the microtubules (Fig. 2C).



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  		Figure 1. Confocal immunofluorescence analysis of the co-expression of CLN3 and Hook1 in HeLa cells. HeLa cells immunostained with anti-Lamp1 in red (A) and transfected with GFP-CLN3 (green) (B) show substantial co-localization of GFP-CLN3 and the late endosomal/lysosomal Lamp1 (yellow, C). Co-expression of Hook1 (red, D) and GFP-CLN3 (green, E) induces a dramatic change in the subcellular localization of Hook1 making it appear in aggregate-like structures. The red cytoplasmic Hook1 aggregates and GFP–CLN3 do not co-localize (F). Transfected Hook1 (G) is localized diffusely in the cytoplasm, and co-expression with another lysosomal membrane protein, GFP-sialin (H), induces no apparent change in the subcellular localisation of Hook1; merged image shown in (I). Hook1 immunostaining was performed with polyclonal 9005/Hook1 antibody. Scale bar: (A–F) 14 µm, (G–I) 10 µm.

 


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  		Figure 2. Immunoprecipitation analysis of Hook1 and CLN3. Hook1 was transiently co-transfected with empty pCMV5-vector, CLN3, CLN3E295K, CLN3{Delta}ex7–8 or sialin, radioactively labelled for 1 h, and immunoprecipitated with Hook1 antibody after 0 or 6 h chase. (A) No endogenous Hook1 was immunoprecipitated in the untransfected control (lane 1). Hook1 immunoprecipitates as a ~85 kDa protein when co-transfected with the empty pCMV vector (lanes 2 and 3). When Hook1 is co-transfected with wild-type CLN3, it is no longer immunoprecipitable (lanes 4 and 5). (B) Co-transfection with the mutant forms of CLN3, CLN3E295K and CLN3{Delta}ex7–8, also make Hook1 unimmunoprecipitable (lanes 3–6). No Hook1 is immunoprecipitated in the untransfected control (lane 7). In contrast, Hook1 immunoprecipitates as a ~85 kDa protein when co-transfected with another lysosomal membrane protein sialin (lanes 1 and 2, lower panel). (C) HeLa cells were transiently transfected with Hook1 and treated with nocodazole to depolymerize the microtubules. Hook1 was detected in similar aggregates as previously described with co-expression with CLN3. Hook1 immunostaining was performed with polyclonal 9005/Hook1 antibody. Scale bar 14 µm.

 
JNCL fibroblasts exhibit defective endocytosis
Due to the dramatic influence of CLN3 overexpression on the Hook1 protein and the proposed function of Drosophila hook in endocytosis, we investigated whether CLN3 deficiency results in defects in endocytosis in the mammalian cells. We first investigated the uptake and recycling of transferrin in JNCL and wild-type (WT) human fibroblasts using biotinylated transferrin and a solid phase assay. For the uptake studies, the biotin–transferrin was bound to the cell surfaces on ice and the internalization at 37°C at indicated time points was analysed. No difference in terms of the transferrin endocytosis between the JNCL cells and the WT cells was observed (Fig. 3A). For the recycling assay, the endosomes were loaded with the biotin–transferrin at 17°C and, subsequently, recycling at 37°C at indicated time points was measured. Results from three independent experiments showed that the recycling of biotinylated transferrin was increased in the JNCL cells (Fig. 3B).



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  		Figure 3. Effect of CLN3 deficiency on the transferrin cycle. (A) Transferrin endocytosis. Fibroblasts from Batten patients (JNCL) and wild-type (WT) controls were incubated with biotinylated transferrin at 0°C (surface binding) and the uptake at various time points at 37°C was measured as described in Materials and Methods. Values were calculated as an average of two independent experiments performed in triplicate. No difference in terms of the transferrin endocytosis was observed between these two cell lines. (B) Transferrin recycling. JNCL and WT cells were incubated with biotinylated transferrin for 2 h at 17°C (endosome loading) and the recycling at 37°C was measured as described in Materials and Methods. Values were calculated as an average of three independent experiments (only one experiment in triplicate at time point 20 min) performed in triplicate. For each curve, the absorbance value (A490 nm) at the 0 time point was set to 100. It was observed that compared with the wild-type cells, transferrin recycling was increased in the CLN3-defective JNCL cells.

 
We further examined receptor-mediated endocytosis by analysing the trafficking of fluorescent low-density lipoprotein (BODIPY FL-LDL) on the degradative endocytic pathway. JNCL and WT fibroblasts were starved in lipoprotein-free medium overnight in order to up-regulate the LDL receptor expression on the cell surface. After lipoprotein starvation the cells were allowed to internalize BODIPY FL-LDL for 30 min and either fixed immediately or chased for 40 min or 2 h and analysed by confocal microscopy. In the WT cells, LDL had passed the EEA1-positive early endosomes (Fig. 4A), and predominantly co-localized with the late endosomal (LE)/lysosomal marker LBPA immediately after the uptake period (Fig. 4B). In comparison, in the JNCL cells LDL was found exclusively in the EEA1-positive early endosomes at this time point and no co-localization of the LDL and LBPA was detected (Fig. 4C and D), indicating a delay in the routing through the endocytic pathway. However, after 2 h of chase, BODIPY FL-LDL has partially reached the LE/lysosomes also in the JNCL cells (Fig. 4E and F).



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  		Figure 4. Delay in the receptor-mediated endocytosis of BODIPY FL-LDL in JNCL cells. Fibroblasts from Batten patients (JNCL) and wild-type (WT) controls were starved overnight in lipoprotein-free medium and subsequently incubated with BODIPY FL-LDL (green) for 30 min and then either fixed directly (A–D) or chased for 2 h followed by fixation (E and F). Cells were then immunostained either with monoclonal EEA1 or LBPA antibodies (red). (A) Partial co-localization of the BODIPY FL-LDL with the early endosomal marker EEA1 is detected in the WT cells directly after the internalization. (B) Major portion of the BODIPY FL-LDL co-localizes with the LE/lysosomal marker LBPA in WT cells at this time point. (C) BODIPY FL-LDL co-localizes well with the early endosomal EEA1 in the JNCL cells after the uptake period. (D) There is no co-localization with the BODIPY FL-LDL and the LE/lysosomal marker LBPA in the JNCL cells. (E) After 2 h of chase some BODIPY FL-LDL is still present in the early endosomes in the JNCL cells, as demonstrated by the co-localization with the EEA1; however (F), it has partially reached the LEs and lysosomes and co-localizes with the LBPA. Scale bar 20 µm.

 
Interactions of Hook1 with CLN3 and the Rab proteins
Potential interactions of the CLN3, Hook1 and the endocytic machinery were further elucidated by detailed biochemical assays. To examine the putative interaction between CLN3 and microtubule-binding Hook1, we performed an in vitro binding assay for the cytoplasmic Hook1 and two of the cytoplasmic domains of the CLN3 (amino acids 1–33, 232–280; Fig. 5A) (13). Lumenal domain of CLN3 (amino acids 56–97) and bare GST protein were used as negative controls. CLN3 domains were produced as GST fusion proteins, affinity purified by glutathione Sepharose beads and incubated with radioactively labelled Hook1-protein produced in vitro. Resulting immunocomplexes were analysed by autoradiography. Weak interaction between the cytoplasmic domains of 1–33 and 232–280 and Hook1 was observed (Fig. 5A). Densitometric analysis of three separate autoradiograms showed that when compared with the GST vector alone, the binding of the cytoplasmic domains to Hook1 was significant for both the N-terminal region (amino acids 1–33; P=0.03) and the cytoplasmic loop (amino acids 232–280; P=0.03) (Fig. 5B). We were unable to confirm the interaction vice versa, i.e. using in vitro translated CLN3 and Hook1 as GST fusion protein, since GST–Hook1 remained insoluble after production.



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  		Figure 5. Weak interaction of the cytoplasmic domains of CLN3 and Hook1 demonstrated by the GST pull down assay. (A) Schematic diagram of the membrane topology of CLN3 showing the cytoplasmic domains 1–33 and 232–280 and the lumenal domain 56–97. In vitro translated Hook1 binds to both of the cytoplasmic domains (lanes 3 and 5, left). Lumenal domain (lane 4, left), bare GST beads and the GST vector were used as negative controls (lanes 1 and 2, left). (B) Densitometric analysis shows a significant increase in the binding of the CLN3 cytoplasmic domains to Hook1 (*P=0.03, n=3; **P=0.03, n=3) compared to the GST vector alone.

 
As recent work with the Btn2p, the Hook1 homologue in yeast, has demonstrated a link to the Ypt/Rab proteins (21), we explored putative interactions of Hook1 and CLN3 with the major Rab proteins of the endocytic pathway in mammalian cells. In particular, we concentrated on investigating the potential interaction between Hook1 and CLN3 with Rab7, Rab9 and Rab11. The non-endocytic Rab24 was used as a negative control. Hook1 or CLN3 was co-expressed with the individual GFP-tagged Rab proteins in COS-1 cells, which were then subjected to immunoprecipitation with a monoclonal GFP antibody conjugated to agarose beads, and subsequently analysed by western blotting with an antibody specific for Hook1 or CLN3. Hook1 was found to specifically interact with Rab7, Rab9 and Rab11 but not with Rab24 (Fig. 6A and B). In contrast, we were not able to demonstrate interactions between CLN3 and the Rab proteins using this method (data not shown).



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  		Figure 6. Interaction of the endocytic Rab proteins and Hook1 demonstrated by co-immunoprecipitation assay. COS-1 cells were co-transfected with Hook1 and GFP–Rab constructs and the lysate was co-immunoprecipitated with anti-GFP agarose beads followed by western blotting with hHK1/Hook1 antibody. (A) Hook1 interacts with Rab7, Rab9 and Rab11 but not with Rab24 (lanes 1–4, left). GFP vector and untransfected control cells were used as negative controls (lanes 5 and 6). (B) Transfection efficiency was confirmed by western blotting with monoclonal GFP antibody prior to the co-immunoprecipitation.

 
To visualize and compare the subcellular distribution of Hook1 and the interacting Rab proteins, we performed a confocal immunofluorescence microscopy analysis of the co-transfected proteins in HeLa cells. Compared to the transfected Rab7 alone (Fig. 7A, left), co-expression of Hook1 with Rab7 markedly affected the distribution of Rab7 (Fig. 7B, left). In addition, the distribution of Hook1 appeared to be altered and it was seen to co-localize with Rab7 in the same intracellular structures. In contrast, when co-expressed with Rab9, Hook1 appeared to retain its normal cytoplasmic localization (Fig. 7B, right). In addition, when compared with the transfected Rab9 alone (Fig. 7A, right), Rab9 distribution appears unaltered when co-expressed with Hook1. Similarly, there was no change in the subcellular distribution of Hook1 and Rab11 when expressed together (data not shown).



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  		Figure 7. Confocal immunofluorescence analysis of Hook1 with Rab7 and Rab9 in HeLa cells. Cells were transfected with GFP–Rab constructs alone (A) or co-transfected with a GFP–Rab constructs and Hook1 and immunostained with 9005/Hook1 antibody (B). (A) Subcellular localization of GFP–Rab7 (left) and GFP–Rab9 (right). (B) In the left panels, co-expression of GFP–Rab7 (green) and Hook1 (red) leads to an altered subcellular distribution of Rab7. A partial co-localization (yellow) is also observed. In the right panels, Rab9 partial co-localization of GFP–Rab9 (green) and Hook1 (red) is also observed, but Rab9 localization seems unaffected. Scale bar: (A) 5 µm, (B) 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present study, we demonstrate that CLN3 overexpression has profound effects on the microtubule-binding Hook1 by affecting its localization and ability to be immunoprecipitated. Utilizing the recently solved membrane topology of CLN3 (13) we were also able to demonstrate a weak interaction of the cytoplasmic N-terminal region and the cytoplasmic loop domain of CLN3 with in vitro translated Hook1. Our findings suggest that overexpression of CLN3 causes conformational changes or aggregation of Hook1, and it could be hypothesized that this phenomenon is due to a competition of both CLN3 and Hook1 for binding with the same third molecule, in which case excess CLN3 would saturate the binding sites, therefore inhibiting Hook1 binding and making it unstable. Mechanistically, this action of CLN3 may be mediated through Hook1 dissociation from the microtubules, as Hook1 alone forms similar aggregates also after microtubule depolymerization by nocodazole. Although the molecular basis of the interaction between CLN3 and Hook1 remains mostly unclear, the data presented here constitutes a novel link between CLN3, Batten disease and cellular functions involving the microtubule cytoskeleton.

Impaired membrane trafficking and dysfunctional endocytosis form the foundation to numerous pathological processes and have been recognized as underlying molecular bases for several human genetic disorders (29–31). For example, an increased rate of cargo transport into the lysosomes has been demonstrated in mucolipidosis type IV (32), proposing a defect in the late steps of endocytosis. Endocytic defects have also been connected to elevated endosomal pH, as illustrated by recent studies with chloride channels ClC-4 and ClC-5, which share a dual role as a structural component of the endocytic apparatus and in the acidification of endosomes (33,34). Previous studies have suggested a functional role also for CLN3 in the regulation of intraorganellar pH (18–20). This is in part supported by the present study as we demonstrate an impairment of receptor-mediated endocytic pathway in the CLN3-deficient JNCL fibroblasts. We show a delay in the trafficking of LDL along the endocytic pathway and, conversely, an increase in the recycling of transferrin. These endocytic defects seen in the JNCL cells may be directly attributed to the CLN3 deficiency, although they may alternatively be secondary effects resulting, for instance, from interference with the Hook1 function. It would be of interest to examine whether the expression levels of Hook1 are altered in JNCL cells, similarly to the yeast model. However, due to the extremely low expression level of Hook1 in these primary fibroblasts, assessed by real-time PCR analysis, no reliable comparison of the relative mRNA levels of Hook1 could be made.

Drosophila Hook1 has been shown to have a role in endocytosis (24,25), in particular in the maturation of the MVBs and in the negative regulation of cargo delivery from mature MVBs to late endosomes and lysosomes (26). Genetic analyses in yeast have demonstrated interaction of Btn2p (proposed Hook1 homologue) and the Golgi associated Yif1p protein (21), which binds several RabGTPases (35), thus supporting the role of Btn2p in membrane trafficking. Membrane fusion events and microtubule-binding Hook1 have also been connected in a recent study demonstrating Hook1 interaction with mammalian Vps18, a component of the mammalian HOPS complex involved in membrane tethering and fusion (27). In this study, we demonstrate novel interactions of mammalian Hook1 with the endocytic Rab GTPases Rab7, Rab9 and Rab11. In contrast, no interaction was found with Rab24, a non-endocytic member of the Rab GTPase family (36), confirming the specificity of this interaction to the endocytic Rabs. The interaction of Rab7 and Hook1 is strongly supported by the immunofluorescence findings showing extensive co-localization of the two proteins when co-expressed. However, this was not observed for Hook1 and Rab9 or Rab11, suggesting that the interaction detected in vitro may be weak in vivo, being perhaps mediated by other molecules. Rab7 and Rab9 localize to the late endocytic organelles, where they occupy distinct domains (37). Specific interaction of Hook1 with these Rab proteins asserts the function of Hook1 in the late endocytic organelles and is consistent with earlier studies (26,27). In contrast, Rab11 is known to mediate the membrane fusion events between the endocytic recycling compartment and the TGN (38). Our finding of Hook1 interaction with Rab11 as well as the previously reported interaction with the HOPS complex (27) suggest a multi-level role for Hook1 along the endocytic pathway. Studies with other species have emphasized the microtubule-binding properties of Hook1; in mice, Hook1 function has been reported to be necessary for the correct positioning of microtubular structures within the haploid germ cell (39) and Hook1 homologue in Caenorhabditis elegans, named ZYG-12, is found to be essential for the attachment between the centrosome and nucleus (40). Rab proteins have also been found to interact with cytoskeletal elements, exemplified by the interaction of Rab6 with a kinesin-like protein Rabkinesin-6 involved in cytokinesis (41) and the regulation by Rab5 on attachment and movement of early endosomes along microtubules (42). The interactions between the Rab GTPase family members and the microtubule-binding Hook1 thus reinforces the idea of more diverse functions of the Rab proteins besides tethering/docking of vesicles at their target membranes (43).

What is the function of the CLN3 protein in the light of this novel data? CLN3 has been reported to localize to various intracellular vesicles (reviewed in 44) but previous studies in non-neuronal cells have mostly supported the lysosomal localization of overexpressed CLN3. It is, nonetheless, known from our previous analyses that the endogenous Cln3 in the mouse brain is localized and fractionated into small vesicular presynaptic compartment (15). In addition, transfected CLN3 has been shown to localize not only to lysosomes but also to EEA1-positive early endosomes in cultured rat hippocampal neurons (13). These data indicated that CLN3 might possess functions outside the lysosomes. As a multispan membrane protein, CLN3 could participate in transport of solutes or ions across the membranes. Recent studies in yeast have indeed implicated that the CLN3 orthologue Btn1p has a role in vacuolar arginine transport (45). A distant but potentially biologically significant relationship between CLN3 and a equilibrative nucleoside transporter family SLC29 has also been illustrated (46). In addition, there are observed phenotypic similarities between Cln3–/– mice (47) and the knockout mice lacking the chloride CLC-3 channel function on the endosomal membranes and synaptic vesicles (48,49). Therefore, the loss of CLN3 could directly influence the ion content of endocytic organelles and result in defective endocytosis per se. However, the novel link between CLN3, Hook1 and the Rab proteins suggests that the role of CLN3 in the vesicular trafficking events may be more complex. JNCL is severely manifested in the central nervous system, and accordingly, it will be of great interest to analyse the fate of both Hook1 and the Rab GTPases in the Cln3-deficient neuronal cells. In conclusion, the data presented here provide important new insights into the functional roles of CLN3, and also support the viewpoint that several proteins that control cytoskeletal functions are also involved in endocytosis and membrane fusion events (reviewed in 50). It will be crucial to unravel the molecular basis behind these interconnections, and the data presented here will provide new tools for this analysis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
JNCL cells
Primary fibroblasts were obtained from skin biopsies of three JNCL patients homozygous for the major deletion removing nucleotides 461–677 from the CLN3 coding region and three normal controls. Cells were maintained in DMEM medium (Gibco BRL, Life Techologies Inc.) supplemented with 10% fetal calf serum (FCS), 0.5 mm L-glutamine, 50 mg/ml streptomycin and 100 IU/ml penicillin at 37°C, 5% CO2. Experiments were performed on monolayer cultures grown to 50–90% confluency on 10 mm coverslips for microscopy.

Antibodies
Two novel Hook1 antibodies 9005 and 9019 were produced against a synthetic MAP-peptide (Invitrogen) corresponding human Hook1 amino acid sequence 130–147. Two rabbits were immunized with 500 µg of peptide emulsified in complete Freund's adjuvant and injected intradermally at multiple sites. Injections were performed at 3 week intervals for total of 9 weeks. The antisera were IgG purified and tested in immunofluorescence staining of Hook1-transfected HeLa cells (dilution 1 : 500). In addition, immunoprecipitation after in vitro translation was performed confirming specificity. Antibody for human Hook1 (hHK1, 1 : 2000) was a generous gift from H. Krämer (University of Texas Southwestern Medical Centre, Dallas, TX, USA) and described earlier (23). Production of the polyclonal 385/CLN3 antibody was described earlier (9). The LBPA antibody (51) was kindly provided by J. Gruenberg (University of Geneva, Switzerland). Other primary antibodies used were mouse anti-human EEA1 (1 : 50, Transduction Laboratories) and mouse anti-human LAMP1 (1 : 100, August JT, Hildreth JEK, Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA). Secondary antibodies used in immunofluorescence stainings were Rhodamine Red X-conjugated AffiPure goat anti-rabbit IgG (H+L) and Rhodamine (TRITC)-conjugated F(ab')2 fragment-specific goat anti-mouse IgG+IgM from Jackson ImmunoResearch Laboratories (dilution 1 : 200).

Cloning of cDNAs
The construction of pCMV5–CLN3 was described earlier (9). For expression in mammalian cells, cDNA encoding the wild-type CLN3 from the pCMV–CLN3 construct was inserted in frame between EcoRI and BamHI sites of pEGFP-C2 (Clontech) producing a fluorescent fusion protein with an N-terminal EGFP-tag. For the GST pull-down experiments, PCR-generated cDNAs encoding different segments of the CLN3 were cloned in frame into pGEX–4T-1 (Pharmacia Biotech) between the EcoRI and XhoI sites to produce GST fusion proteins of peptides 232–280 and 384–438 and into pGEX–4T-3 (Pharmacia Biotech) between the BamH1 and SalI sites to produce GST fusion proteins of peptides 1–33 and 56–97. For the in vitro translation assay, the cDNA of the HOOK1 gene was obtained from an IMAGE Consortium clone (592403) (52) in pBluescript (Stratagene). For the expression studies, HOOK1 cDNA was cut off with SmaI–KpnI and cloned into the SmaI site in pCMV5 (53). Construction of pSVpoly-sialin and pEGFP-sialin was described elsewhere (28). Fluorescent Rab constructs were generously provided by M. Zerial (pEGFP-Rab11, Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany), A. Wandinger-Ness (pEGFP-Rab7, University of New Mexico, Albuquerque, NM, USA), E. Ikonen (pEGFP-Rab9, National Public Health Institute, Helsinki, Finland) and V. Olkkonen (pEGFP-Rab24, National Public Health Institute).

Transfections, immunofluorescence staining and confocal microscopy
HeLa cells were transiently transfected with Fugene (Roche) according to the manufacturer's instructions. Where specified, after 24 h of transfection, cells were treated with 33 µM nocodazole (Sigma) in DMEM with 10% FCS for 30 min followed by fixation. Cells were then fixed with fresh 4% paraformaldehyde in PBS for 15 min at RT, washed with PBS and permeabilized with 0.1% Triton X-100 (Sigma) in 0.5% bovine serum albumin (BSA, Sigma)/PBS for 15 min. Non-specific binding was blocked with 0.5% BSA in PBS for 30 min. For double immunostaining, the cells were incubated with the primary antibodies diluted in the blocking buffer for 1 h at RT. Subsequently, the cells were incubated with the appropriate secondary antibodies diluted in the blocking buffer for 40 min. The coverslips were mounted in GelMount (Biomeda Corp.) and viewed with Leica DMR confocal microscope with TCS NT software (Leica Microscope and Scientific Instruments Group). The confocal images were completed with Adobe Illustrator 8.0 software.

Immunoprecipitation assay
COS-1 cells were transfected with the calcium-phosphate method for 2 days (54) in the following combinations: pCMV5–Hook1 alone, pCMV5–Hook1 and pCMV5–CLN3, pCMV5–Hook1 and pCMV5–CLN3E295K, pCMV5–Hook1 and pCMV5–CLN3{Delta}ex7–8 or pCMV5–Hook1 and pSVpoly-sialin. Untransfected cells were used as a negative control. Cells were then metabolically labelled for 1 h with L-[35S]cysteine (Amersham Pharmacia Biotech) and then chased for 0 or 6 h and lysed in 1% Triton X-100/PBS. Immunoprecipitation was carried out with fixed Staphylococcus aureus cells (Calbiochem) using Hook1-specific antibodies (9005, 9019 and hHK1). The labelled proteins were separated by 14% SDS–PAGE and visualized by autoradiography. Transfection efficiency was checked with western blotting using antibodies for Hook1, CLN3 or sialin.

Transferrin endocytosis and recycling
To study the endocytosis of transferrin, both JNCL and wild-type fibroblasts, plated onto 1.9 cm2 wells, were washed three times with PBS and pre-incubated in serum-free medium (DMEMB=AIR MEM, 10 mM HEPES, 0.1% BSA) for 1 h at 37°C. The cells were then transferred on ice, washed with cold DMEMB and incubated with biotinylated holotransferrin (Sigma) for 1 h (surface binding). Cells were then washed thoroughly with ice-cold 200 µg/ml holotransferrin in DMEMB and PBS to remove unbound biotin–holotransferrin. The biotinylated holotransferrin was internalized at 37°C and the cells were collected at indicated time points. At each time point, non-endocytosed transferrin was washed away with 200 µg/ml holotransferrin in DMEMB (four times) and PBS, and lysed with ST buffer (1 mM EDTA, 50 mM NaCl, 0.1% SDS, 1% Triton X-100, 10 mM Tris–HCl, pH 7.5). The amount of biotin–holotransferrin was measured from the lysates with a solid phase assay. ImmunoMaxiSorp 96-well plates (Nalge Nunc Int.) were coated with goat anti-human transferrin antisera (1 : 100, Sigma) in 50 mM NaHCO3 pH 9.6 overnight. To block unspecific binding, the wells were incubated with 0.2% BSA in ST buffer for 4 h. Fifty microlitres of the cell lysates were added to the wells followed by an overnight incubation at 4°C. The wells were washed twice with PBS, blocked with 0.2% BSA in ST buffer for 5 min and incubated with horseradish peroxidase-conjugated streptavidin (Immunopure Str-HRP, 1 : 5000, Pierce) for 1 h. The wells were washed twice with PBS and the HRP substrate was added (0.5 mg/ml phenyldiamine, 50 mM Na2HPO4, 30 mM Na-citrate pH 5, 0.03% H2O2) and incubated for 30 min. The reaction was stopped with 50 µl of 2N H2SO4 and the absorbance ({lambda}=490 nm) was detected. The recycling assay was performed similarly, with the exception of loading the early endosomes with biotin–holotransferrin at 17°C for 2 h.

Receptor-mediated endocytosis
For LDL uptake studies, JNCL and normal control fibroblasts were starved overnight in DMEM with lipoprotein-free serum. Cells were washed briefly with PBS and incubated with BODIPY FL-AcLDL (20 µg/ml, Molecular Probes) in serum-free DMEM for 30 min at 37°C. Then the cells were washed thoroughly with PBS and either fixed immediately with fresh 4% paraformaldehyde in PBS for 15 min at RT or chased for 40 min or 2 h in maintaining medium at 37°C followed by fixation.

Co-immunoprecipitation assay
For co-immunoprecipitation assay, African Green Monkey kidney cells (COS-1, ATCC CRL-1650) cells were co-transfected by calcium phosphate method (54) with pCMV5–Hook1 or pCMV–CLN3 and pEGFP–Rab7, pEGFP–Rab9, pEGFP–Rab11 or pEGFP–Rab24. The pEGFP–C2 vector and untransfected cells were used as negative controls. After 2 days, cells were lysed with lysis buffer A (50 mM Tris, pH 7.4, 300 mM NaCl, 1% Triton X-100 and 0.1% BSA) for 30 min at 4°C with shaking. Lysate was centrifuged and the supernatant was immunoprecipitated with monoclonal GFP antibody conjugated to agarose beads (Santa Cruz Biotechnology). Prior to the immunoprecipitation, equal expression levels of each construct were confirmed by western blotting with monoclonal GFP antibody (Santa Cruz Biotechnology), 385/CLN3 antibody or hHK1/Hook1 antibody. Immunocomplexes were analysed by western blotting with hHK1/Hook1 or 385/CLN3 antibodies.

In vitro binding assay
For the in vitro binding assay, the cytoplasmic domains of CLN3 protein (1–33, 232–280) and one lumenal domain 56–97 were produced as GST fusion proteins in Escherichia coli (we were unable to produce the C-terminal domain 384–438), and combined with glutathione Sepharose 4B beads (Amersham Pharmacia Biotech) that were washed three times with 0.5% Triton X-100 in PBS, and incubated at 4°C overnight. Hook1 in pBluescript was in vitro translated using the TNT T7 Coupled Reticulocyte lysate systen (Promega) and labelled with L-[35S]cysteine (Amersham Pharmacia Biotech) according to the instructions of the manufacturer. The resulting protein samples were analysed on 14% SDS–PAGE gel followed by autoradiography. Thereafter, 5 µl of the radioactively labelled Hook1 was incubated with equal amounts of GST–CLN3 fusion proteins bound to the GST beads in the binding buffer (0.5 mM EDTA, 50 mM Tris–HCl, pH 7.8, 150 mM KCl, 5% glycerol, 0.3% Nonidet P-40, 0.3% Triton X-100, 5 mM MgCl2, 0.1% BSA, protease inhibitor cocktail from Boehringer Mannheim) overnight at 4°C. The beads were washed five times with the binding buffer and analysed on 14% SDS–PAGE gel followed by autoradiography. Densitometric analysis of the autoradiograms was performed by Scion Image Beta 4.02 software. Student's t-test was used for the statistical analyses.


   ACKNOWLEDGEMENTS
 
We thank Ms Kaija Antila, Ms Anne Nyberg and Ms Marja Weckström for their superb technical assistance. Dr Maria Halonen and Dr Jouni Vesa are thanked for their valuable advice with the interaction studies and Dr Vesa Olkkonen is thanked for critical reading of the manuscript. Dr Maria Kauppi is also acknowledged for her essential advice with the transferrin experiments. This study was financially supported by the Academy of Finland (Center of Excellence in Disease Genetics, grant 44870), EC Grant LSHM-CT-2003-503051, The Sigrid Juselius Foundation, Rinnekoti Research Foundation, Arvo and Lea Ylppö Foundation, De Blindas Vänner Foundation and Helsinki Biomedical Graduate School (MD/PhD program).


   FOOTNOTES
 
* To whom correspondence should be addressed at: National Public Health Institute, Department of Molecular Medicine, PO Box 104 (Haarmaninkatu 8), FIN-00251 Helsinki, Finland. Tel: +358 947448392; Fax: +358 947448480; Email: anu.jalanko{at}ktl.fi


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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