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Human Molecular Genetics Pages 523-531  

The Batten disease gene product (CLN3p) is a Golgi integral membrane protein
Introduction
Results
   Transient expression of CLN3p protein chimeras
   CLN3p/GFP expression in stable transfectants
   Localization of CLN3p in fractionated subcellular compartments of stable CLN3p/GFP transfectants
   Localization of CLN3p in endosomes and lysosomes
Discussion
Materials And Methods
   DNA constructs
   RT-PCR and FISH
   Cell culture and transfections
   Fluorescence/immunofluorescence
   Subcellular fractionation and western blot analysis
Abbreviations
Acknowledgements
References

The Batten disease gene product (CLN3p) is a Golgi integral membrane protein

The Batten disease gene product (CLN3p) is a Golgi integral membrane protein

Gabriel Kremmidiotis*, Ingrid L. Lensink, Rebecca L. Bilton, Erica Woollatt, Timothy K. Chataway1, Grant R. Sutherland and David F. Callen

Centre for Medical Genetics, Department of Cytogenetics and Molecular Genetics and 1Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women’s and Children’s Hospital, 72 King William Road, Adelaide, SA 5006, Australia

Received November 11, 1998; Revised and Accepted December 21, 1998

Batten disease (juvenile neuronal ceroid lipofuscinosis) is a recessive neurodegenerative disorder of childhood. The gene, CLN3, was recently identified and found to encode a novel 438 amino acid protein of unknown function. In order to gain insight into the function of the Batten disease protein (CLN3p), we investigated its subcellular localization. Protein constructs incorporating CLN3p fused to the green fluorescence protein or an eight amino acid peptide tag were transiently expressed in fibroblasts, HeLa and COS-7 cells. A juxtanuclear, asymmetric localization pattern was observed that correlated with the Golgi apparatus in all three cell types. However, a proportion of transiently transfected cells exhibited a punctate vesicular distribution throughout the cytoplasm in addition to or without the Golgi localization. In order to account for localization patterns arising from intracellular protein transport disruption due to exaggerated overexpression in transiently transfected cells, we isolated a stably transfected cell line expressing only one copy of the CLN3-GFP DNA construct. Fluorescence and biochemical analyses using this cell line demonstrated that CLN3p is an integral membrane protein that localizes primarily in the Golgi apparatus. The functional implications of this finding are discussed.

INTRODUCTION

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigments in neurons and other cell types (1). At least five types of NCL are recognized based on age of onset and clinicopathological features. Batten disease, the juvenile onset form of NCL, is the most common neurodegenerative disorder of childhood. Its incidence is estimated at up to 1/25 000 births (2), with an increased prevalence in the North European population. Clinical onset begins with visual failure between the ages of 5 and 10 years. Seizures and mental deterioration ensue with decline to death usually in the second or third decade. Diagnostic criteria include the presence of intracellular inclusions which appear as ‘fingerprint profiles’ on electron microscopy (3). The Batten disease gene (CLN3) contains 15 exons over 15 kb of genomic sequence (4,5) that encode a 1689 bp transcript which is translated into a 438 amino acid protein. The CLN3 transcript exhibits high conservation in other species with the yeast protein homologue (BTN1) exhibting 39% identity and 59% similarity (6).

The function of the Batten gene protein (CLN3p) is currently unknown. The accumulation of mitochondrial ATP synthase subunit c (7) in patient lysosomes has implied CLN3p involvement in the degradation pathway of this compound. Elleder et al. (8) suggested that the accumulation of this compound is a non-specific phenomenon common to all types of NCL. In addition, Pearce and Sherman (6) demonstrated that the yeast CLN3p homologue is not essential and does not play a direct role in the degradation of mitochondrial ATP synthase subunit c.

The subcellular localization of CLN3p may provide clues in evaluating the potential function of this protein. Katz et al. (9) detected CLN3p in the mitochondria of mouse retina photoreceptor cells using polyclonal antibodies. Jarvela et al. (10) on the other hand reported CLN3p localization in the lysosomes of HeLa cells transiently transfected with CLN3 cDNA. Against this background we studied the subcellular localization of CLN3p in transiently and stably transfected cells. Recombinant CLN3p tagged with green fluorescent protein (GFP) or the FLAG peptide was studied using detailed fluorescence microscopy and biochemical analyses. Our studies suggest that CLN3p is an integral Golgi membrane protein.

RESULTS

Transient expression of CLN3p protein chimeras

The GFP gene was cloned 3[prime] to the open reading frame coding for CLN3p (Fig. 1). The resulting DNA construct was used to transiently transfect HeLa cells, COS-7 cells and fibroblasts. The GFP fluorescence signal was examined 28, 48 and 72 h post-transfection. The CLN3p protein concentrated in an asymmetric, paranuclear pattern that is typical of localization to the Golgi apparatus (Fig. 2). There was no apparent localization in the nucleus.


Figure 1. (A) Diagramatic representation of the tagged CLN3 DNA constructs used in this report. The names of the corresponding cloning vectors and expected protein products are shown. (B) Diagram showing the positions of primers used for the amplification of CLN3p/GFP (carboxy) mRNA from the stably transfected cell line CLN3p/GFP-HeLa.8.


Figure 2. Localization of GFP-tagged CLN3p by fluorescence microscopy. Cells transiently transfected with the GFP vector (control) exhibit diffuse green fluorescence in both the nucleus and the cytoplasm. Cells transfected with the CLN3p/GFP (amino) DNA construct show paranuclear, asymetric, Golgi-like fluorescence. There is no CLN3p/GFP (amino) signal in the DAPI stained nuclei (blue).

The incorporation of GFP in the C-terminus of the CLN3p protein may be contributing to misfolding and mislocalization of the corresponding protein chimera. This issue was addressed by studying the subcellular localization of three different CLN3p constructs (Fig. 1). A construct containing the GFP tag in the N-terminus of CLN3p gave the same localization pattern as the construct containing GFP in the CLN3p C-terminus (Fig. 3). This demonstrates that the GFP cloned on either end of CLN3p does not affect the localization pattern observed. In addition, when CLN3p was instead tagged with the eight amino acid peptide FLAG (Fig. 1) the localization pattern observed was the same as that seen with the GFP tag (Fig. 3). This shows that the GFP tag does not interfere with the subcellular localization of CLN3p.


Figure 3. Expression of CLN3p constructs in transiently transfected COS 7 (A-C) and HeLa (D-F) cells. CLN3p was studied tagged with GFP in the C-terminus (A and D) or with GFP in the N-terminus (B and E) or tagged with the FLAG peptide (C and F). In all cases the localization pattern observed was the same. GFP-tagged CLN3p constructs were studied in live cells. The FLAG-tagged CLN3p construct was studied by immunohistochemistry using an anti-FLAG monoclonal antibody.

Correlation of the CLN3p/GFP localization with subcellular organelle-specific dyes indicated co-localization with the Golgi apparatus. There was only minor localization in the endoplasmic reticulum and lysosomes (data not shown). There was no apparent CLN3p localization on the cell surface membrane visualized by staining with wheatgerm agglutinin (WGA)/Texas Red in non-permeabilized transient transfectants (data not shown).

CLN3p/GFP expression in stable transfectants

A number of transiently transfected cells exhibited punctate vesicular distribution of CLN3p throughout the cytoplasm in addition to or without the Golgi-like localization (Fig. 4). This vesicular localization pattern was mainly observed in transiently transfected HeLa cells. The relative proportion of cells exhibiting these patterns differed from experiment to experiment and was not found to be related to the transfection lipid used (Superfect, Lipofectamine or Lipofectamine plus) or the post-transfection time. Only a small proportion of these vesicles stained with the lysosome-specific dye Lysotracker (data not shown). This cytoplasmic vesicular localization may be a true reflection of CLN3p expression or it may be the result of protein transport disruption due to exaggerated overexpression in transiently transfected cells. In order to resolve this problem we isolated a stably transfected cell line expressing only one copy of a CLN3-GFP DNA construct.


Figure 4. Live HeLa cell transiently transfected with CLN3p/GFP (amino) DNA construct. This cell is an example of the small proportion of HeLa cells expressing CLN3p/GFP in a vesicular punctate pattern distributed throughout the cytoplasm.

The CLN3-GFP (carboxy) DNA construct was used to stably transfect HeLa cells. Cells expressing the fluorescent CLN3p/GFP protein were isolated by culture in G418. The number of CLN3p/GFP (carboxy) copies incorporated into the genome of each isolated clone was assessed by fluorescence in situ hybridization (FISH). Clone CLN3p/GFP-HeLa.8 indicated stable incorporation of one copy of the CLN3-GFP (carboxy) construct (Fig. 5A). The expression of CLN3-GFP (carboxy) RNA in this stable line was confirmed by RT-PCR analysis using primers spanning the CLN3-GFP DNA fusion junctions (Figs 1B and 5B). Sequence analysis of the CLN3-GFP RT-PCR product indicated that the open reading frame of the construct had not been compromised. Fluorescence activated cell sorter analysis indicated that the CLN3p/GFP-HeLa.8 line is comprised in its entirety of cells expressing the CLN3p/GFP (carboxy) protein (Fig. 5C).


Figure 5. Characterization of the stably transfected cell line CLN3p/GFP-HeLa.8. (A) FISH of the GFP gene DNA to metaphases of the CLN3p/GFP (carboxy) stably transfected HeLa cell line indicates the presence of a single copy of the CLN3p/GFP (carboxy) DNA integrated in the genome. The hybridization site is indicated (arrow). The FISH signal and DAPI banding pattern were merged for figure preparation. (B) RT-PCR analysis of RNA extracted from the CLN3p/GFP-HeLa.8 line demonstrates expression of the CLN3p/GFP (carboxy) mRNA transcript. Lane 1, GFPC-f and CLN3p.880-861; lane 2, CLN3.862-881 and GFPC-r; lane 3, no reverse transcriptase negative control using primers GFPC-f and GFPC-r; lane 4, GFPC-f and GFPC-r. (C) Flow cytometry analysis of the fluorescence distribution displayed by the CLN3p/GFP-HeLa.8 cell line demonstrates that all the cells express the CLN3p/GFP (carboxy) protein. The fluorescence distributions are displayed as histograms of fluorescence intensity on a log scale (GFPlog PMT2) versus cell count (Count). The fluorescence distribution of untransfected HeLa cells is shown as a negative control reference. The mean fluorescence intensity exhibited by each cell population is shown.

Fluorescent microscopy of live CLN3p/GFP-HeLa.8 cells indicated paranuclear, asymmetric, Golgi-like localization of the CLN3p/GFP (carboxy) chimeric protein similar to that obtained with the transient transfectants (Fig. 6). However, the cytoplasmic punctate vesicular distribution exhibited by a number of transient cell transfectants was not apparent. Correlation of the CLN3p/GFP (carboxy) signal with organelle-specific stains indicated localization primarily in the Golgi apparatus. In some cells, the CLN3p/GFP (carboxy) protein appeared to localize in some lysosomes located close to the Golgi apparatus. The nuclei, surface membrane and mitochondria were totally devoid of CLN3p/GFP (carboxy) fluorescence (Fig. 6).


Figure 6. Expression of the CLN3p/GFP (carboxy) protein in the stably transfected CLN3p/GFP-HeLa.8 cell line. The green fluorescence signal obtained with the CLN3p/GFP (carboxy) protein was correlated with lysosomes, Golgi, nuclei, surface membrane, mitochondria and endoplasmic reticulum. Live CLN3p/GFP-HeLa.8 cells expressing CLN3p/GFP (carboxy) (A) were stained with the lysosomal dye Lysotracker (B). There is only a minor overlap of the green CLN3p/GFP signal with the red lysosomal marker (C). CLN3p/GFP-HeLa.8 cells expressing CLN3p/GFP (carboxy) (D) were fixed/permeabilized and their Golgi stained with Texas Red/WGA (E). There seems to be a complete overlap between the green CLN3p/GFP signal and the red Golgi apparatus signal (F), whereas the DAPI (blue) stained nuclei are devoid of green fluorescence. CLN3p/GFP-HeLa.8 cells expressing CLN3p/GFP (carboxy) (G) were fixed and their surface membrane stained with Texas Red/WGA (H). There is no expression of CLN3p/GFP on the cell membrane (I). Live CLN3p/GFP-HeLa.8 cells expressing CLN3p/GFP (carboxy) (J) were stained with the mitochondrial dye Mitotracker (K). There is no overlap between the green CLN3p/GFP signal and the red mitochondrial marker (L). Live CLN3p/GFP-HeLa.8 cells expressing CLN3p/GFP (carboxy) (M) were stained with the endoplasmic reticulum-specific dye ERtracker (N). There is a minor overlap between the blue endoplasmic reticulum fluorescence and the green CLN3p/GFP signal (O).


Localization of CLN3p in fractionated subcellular compartments of stable CLN3p/GFP transfectants

The CLN3p/GFP-HeLa.8 cell line was used in biochemical subcellular fractionation experiments (Fig. 7). Western blot analysis using an anti-GFP antibody revealed the presence of an ~70 kDa band in the microsomal pellet fraction of the HeLa stable transfectants. This band is of the expected size (considering that CLN3p is ~40-45 kDa and GFP is 27 kDa) and it is not seen in corresponding fractions obtained from non-transfected controls (Fig. 7A). As would be predicted for a membrane-associated protein, the CLN3p/GFP (carboxy) construct was completely solubilized by treatment with 1% Triton X-100 (Fig. 7B). However, detergent solubilization does not distinguish integral membrane proteins that are embedded in the membrane bilayer from proteins that are held in association with the membrane by electrostatic interactions. In order to distinguish between these two possibilities microsomal pellets were extracted with sodium carbonate at high pH (11.5). This treatment has been shown to release peripheral proteins while not significantly disturbing the membrane bilayer or its embedded integral proteins (11). Following extraction with sodium carbonate at pH 11.5, the CLN3p protein construct remained in the insoluble pellet (Fig. 7B). This indicates that CLN3p is an integral and not a peripheral membrane protein.


Figure 7. (A) Western blot analysis of subcellular fractions derived from stably transfected CLN3p/GFP-HeLa.8 cells (+) and untransfected HeLa cells (-) using a monoclonal antibody specific for GFP. Differential centrifugation was performed to obtain the nuclei pellet (P1), the mitochondrial/microsomal pellet (P2), the microsomal pellet (P3) and the cytosolic fraction supernatant (S3). An ~70 kDa band corresponding to the CLN3p/GFP (carboxy) protein is present in the CLN3p/GFP-HeLa.8 cell extracts but not in the untransfected cell extracts. This band is present in the microsome-containing pellet fractions (P2 and P3) but not in the nuclear (P1) and the cytosolic (S3) fractions. A smaller ~48 kDa band is present in both transfected and untransfected cell fractions suggesting that it is a protein that the GFP antibody cross-reacts with. (B) Western blot analysis of the pellet fraction containing the CLN3p/GFP (carboxy) band, treated with ionic detergent or alkaline sodium carbonate. Following treatment, samples were separated at 200 000 g into soluble (Sn) and insoluble membrane pellet (P) fractions. The CLN3p/GFP (carboxy) protein was found in the microsomal pellet (P) in untreated fractions (I); it was released into the soluble fraction (Sn) upon treatment with 1% Triton X-100 (II) and remained in the pellet (P) when extracted with 100 mM sodium carbonate (III).


Localization of CLN3p in endosomes and lysosomes

The fluorescence microscopy results indicated predominant localization of the CLN3p/GFP protein in the Golgi of the stably transfected HeLa cell line. There was some localization evident in a small number of vesicles staining with the lysosomal dye Lysotracker. The presence of the CLN3p/GFP (carboxy) protein construct in lysosomes was also tested using a biochemical approach. Refined fractionation of the microsomal fraction derived from CLN3p/GFP-HeLa.8 cells was performed using a Percoll density gradient. The presence of the CLN3p/GFP protein construct in the different density fractions was detected by western blot analysis using anti-GFP antibody and was correlated with the activities of enzyme markers for lysosomes [N-acetyl-[beta]-d-glucosaminidase ([beta]-Hex)], Golgi (galactosyl transferase) and mitochondria ([alpha]-ketoglutarate dehydrogenase) (Fig. 8). The 70 kDa band corresponding to the CLN3p/GFP (carboxy) protein was predominantly detected in the fractions exhibiting high activity for the Golgi-specific enzyme galactosyl transferase. There were negligible amounts of the CLN3p/GFP (carboxy) protein in the fractions exhibiting activity for the mitochondrial enzyme [alpha]-ketoglutarate dehydrogenase.


Figure 8. Percoll density gradient separation of the microsomal fractions derived from the HeLa cell line stably expressing CLN3p/GFP (carboxy). Fractions were assayed for enzyme activities of [beta]-Hex (lysosomal marker), galactosyl transferase (Golgi marker) and [alpha]-ketoglutarate dehydrogenase (mitochondrial marker). [beta]-Hex enzyme activity is expressed in relative fluorescence units (RFU), galactosyltransferase in 3H c.p.m. and [alpha]-ketoglutarate dehydrogenase in 14C c.p.m. Fractions 9-22 were assayed for the presence of the CLN3p/GFP protein using western blot analysis (insert). The CLN3p/GFP (carboxy) protein was found predominantly in the fractions exhibiting activity for the Golgi marker enzyme.

The activity of the lysosomal enzyme [beta]-Hex exhibited a bimodal distribution over the Percoll fractions examined. A high activity peak was detected in the high density fractions 19-22 (lysosomal fraction, density 1.10-1.15 g/cm3; 12). The 70 kDa CLN3p/GFP (carboxy) band was very faint in these fractions, suggesting that lysosomes contain very low amounts of the GFP/CLN3p protein construct. A secondary [beta]-Hex activity peak was detected in the low density fractions 11-17 (density 1.03-1.08 g/cm3; 12). These fractions contain the CLN3p/GFP (carboxy) protein and exhibit high activity for the Golgi enzyme galactosyl transferase. This is likely to be an endosomal peak as [beta]-Hex is trafficked to lysosomes through endosomes. Re-assay of the gradient for the endosomal marker acid phosphatase revealed that the endosomal peak is coincident with the secondary [beta]-Hex peak (data not shown).

DISCUSSION

The function of the protein coded by the Batten disease gene remains unknown. The accumulation of F1F0-ATP synthase subunit c in the cells of Batten disease patients suggested the involvement of CLN3p in the degradative pathway of this mitochondrial protein (13). Analysis of the predicted CLN3p amino acid sequence has revealed the presence of a possible mitochondrial targeting site spanning residues 11-19 (14), suggesting that CLN3p is a mitochondrial protein. In agreement with this prediction are the findings of Katz et al. (9) who reported exclusive localization of CLN3p in the mitochondria of mouse retina photoreceptor cells. In that study CLN3p was not detected in the mitochondria of other retina cell types, suggesting that it may be only indirectly correlated with the F1F0-ATP synthase subunit c degradation pathway and that its expression may be confined to mitochondria only in certain cell types. However, the findings of Katz et al. (9) should be interpreted with caution. These workers detected CLN3p in the mitochondria of mouse retina cells using an anti-CLN3p serum that was not affinity purified or tested on transfectants for its specificity. This serum exhibited high cross-reactivity on human tissue homogenates.

Mitochondrial proteins encoded by nuclear genes are synthesized on cytosolic ribosomes and are directly imported into various mitochondrial compartments by virtue of specific import presequences (15). However, Jarvela et al. (10) reported that CLN3p is post-translationally translocated into the endoplasmic reticulum where it undergoes heterogeneous N-glycosylation and is subsequently targeted to the lysosomes. These workers investigated the expression of CLN3p in transiently transfected HeLa cells. Their immunofluorescence data showed a vesicular punctate subcellular distribution pattern that partially overlapped with the localization of the lysosomal protein Lamp1.

The high cross-species conservation and the hydrophobic nature of CLN3p make it a very poor immunogen. Consequently, the production of good CLN3p-specific polyclonal antisera or monoclonal antibodies have proven difficult (G. Kremmidiotis, unpublished data). As a result, the detection of endogenous CLN3p has not been possible. There have been no reports describing the detection of endogenous human CLN3p. Papers published to date have relied on the use of transient transfectants to study this protein (10). We studied the subcelluar localization of CLN3p in transiently transfected cells and a stably transfected cell line.

We observed two patterns of CLN3p expression in transiently transfected fibroblasts, HeLa and COS-7 cells. Paranuclear asymmetric Golgi-like concentration was the predominant pattern seen in all cell types examined. Punctate vescicular subcellular distribution was observed in some cells. The latter pattern was mainly seen in HeLa cell preparations. Organelle-specific fluorochrome dyes demonstrated correlation of CLN3p expression with the Golgi apparatus and partial overlaps with the endoplasmic reticulum and a small proportion of lysosomes. These observations are partially consistent with the findings of Jarvela et al. (10); however, the localization in the Golgi is a novel finding.

Some transiently transfected cells may be expressing more than one episomal copy of the transfected DNA leading to an exaggerated overexpression of the corresponding protein. Subsequently some of the intracellular localization distribution patterns may be the result of protein transport disruption due to overexpression rather than a reflection of the true subcellular targeting of the protein under study. In order to account for the possibility of such an experimental artifact we studied the expression of CLN3p in a stably transfected HeLa cell line that contained only one copy of the transfected CLN3p-coding DNA integrated into its genome. The intracellular location of CLN3p in these cells was clearly the Golgi apparatus, with some overlap in the endoplasmic reticulum. Only a very small number of vesicles located close to the Golgi bodies appeared to contain CLN3p. These vesicles stained with the acidotropic dye Lysotracker.

The acidotropic dye Lysotracker used in our fluorescence microscopy experiments stains acidic organelles of the endosomal/lysosomal subcellular compartment. The observation of the CLN3p construct in a small number of vesicles staining with the Lysotracker dye in our stable transfectants suggested some minor localization of this protein construct in the endosomal/lysosomal compartment. We investigated this further by detailed biochemical studies. Biochemical fractionation and western blot analyses revealed the CLN3p construct to be present in fractions containing high levels of the Golgi enzyme marker galactosyl transferase. Very low levels of the CLN3p construct were found in fractions containing high levels of the lysosomal enzyme marker [beta]-Hex. Taken together, these results suggest that the major location for CLN3p is the Golgi apparatus, and lysosomes contain only a very small proportion of CLN3p. This result is in agreement with the fluorescence microscopy data. The enzymatic activity peak corresponding to endosomes overlapped with the Golgi enzymatic activity peak. Consequently, the possibility that in addition to lysosomes, some of the vesicles containing CLN3p in the stable transfectants are endosomes cannot be discounted. It is therefore likely that the small number of vesicles staining with CLN3p/GFP and the Lysotracker dye in our stable transfectants are mainly lysosomes and endosomes.

Although our ‘one copy’ stable transfectants represent a more physiological model than the transient tranfectants, there is still a possibility that the CLN3-GFP gene construct is expressed at higher levels than the endogenous CLN3. This is because the CLN3-GFP construct is under the control of a promoter that may be stronger than the endogenous CLN3 promoter. Consequently, the CLN3p found in endosomal/lysosomal vesicles may represent excess CLN3p/GFP protein that the cell targets to these organelles for degradation.

Based on its amino acid sequence CLN3p contains six transmembrane regions with its N- and C-termini oriented in the lumenal side of the membrane (14) and contains only four potential N-glycosylation sites. This motif does not have the general structural features of lysosome membrane-associated proteins which usually consist of a heavily N-glycosylated lumenal domain, a single transmembrane domain and a short cytoplasmic tail (10). In analysis using the PSORT World Wide Web server (for analyzing and predicting protein sorting signals coded in amino acid sequences; http://psort.nibb.ac.jp/ ) the Golgi bodies were assigned the second highest score for localization of CLN3p (plasma membrane 0.6, Golgi body 0.4, endoplasmic reticulum 0.3, mitochondrial inner membrane 0.03). PSORT did not predict lysosomal localization.

In our biochemical fractionation experiments CLN3p appeared to be associated with the microsomal membrane pellet in a detergent-sensitive but alkaline pH-resistant manner. This finding demonstrates that CLN3p behaves biochemically as an integral membrane protein, as predicted from its primary amino acid sequence (14).

Recent observations have demonstrated the involvement of the yeast CLN3p homologue BTN1 in a growth phenotype involving the compound d-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP) (16). Yeast growth sensitivity to ANP has been associated with the gene ANP1 (17). The corresponding protein ANP1p is part of a Golgi protein complex that is involved in the transport of the mannosyltransferase Mnt1p and the protease DPAP-A (18). Although there is currently no direct evidence of a possible association between ANP1p and BTN1 our findings indicate that CLN3p, like ANP1p, is also a Golgi protein. It is possible that, like ANP1p, CLN3p may be involved in intracellular protein transportation.

In Batten patient cells mitochondrial F1F0-ATP synthase subunit c accumulates in cytosomes exhibiting lysosomal markers. This observation has led to the suggestion of a possible association between the disease cellular phenotype and the process of mitochondrial degradation by macroautophagy (13). Macroautophagy involves the engulfment of mitochondria by endoplasmic reticulum/Golgi-derived membranes that subsequently fuse with primary lysosomes to form autophagosomes (19-21). It is possible that the observation of some CLN3p localization in vesicles carrying lysosomal/endosomal markers is a reflection of CLN3p localization to autophagosomes. Macroautophagy is stimulated in cultured cells when they reach confluence (22). Interestingly, we observed localization of CLN3p to vesicular structures of lysosomal phenotype in transient transfectant cultures where the cells were very confluent.

In conclusion, we demonstrate that CLN3p is an integral membrane protein of the Golgi apparatus. We also observed very small localization in lysosomal/endosomal vesicles which may either be a reflection of excess CLN3p degradation or CLN3p transportation in autophagosome vesicles via the process of macroautophagy. High mitochondrial content and dependence on mitochondrial turnover by the process of macroautophagy in certain cell types may account for the cell-specific degeneration observed in Batten patients. The possible involvement of CLN3p in the process of mitochondrial degradation by macroautophagy remains to be studied.

MATERIALS AND METHODS

DNA constructs

The CLN3 open reading frame sequence was amplified from a previously described fetal library cDNA clone (4) and cloned into the mammalian expression vectors pEGFP-N1 (Clontech, Palo Alto, CA) and pEGFP-C1 (Clontech) using a modification of the ‘hetero-stagger’ cloning strategy (23). The primer combinations used were: (i) 5[prime]-aat tcc ctc ggg gga cct gaa ctt gat g-3[prime] with 5[prime]-gga gag ctg gca gag gaa gt-3[prime]; and (ii) 5[prime]-ccc tcg ggg gac ctg aac ttg atg-3[prime] with 5[prime]-gat cgg aga gct ggc aga gga agt-3[prime]. Two of these primers include EcoRI and BamHI cohesive ends (nucleotides in bold). The CLN3-FLAG construct was generated by amplification of the CLN3 open reading frame using a reverse primer (5[prime]-TCA ctt gtc atc gtc gtc ctt gta gtc gga gag ctg gca gag gaa gt-3[prime]) that included the nucleotide sequence coding for the FLAG peptide (nucleotides in bold) and a stop codon (nucleotides in upper case). The PCR product was directly ligated into the TA mammalian expression vector pTargeT (Promega, Madison, WI). All DNA constructs were checked by sequencing.

RT-PCR and FISH

One-step RT-PCR was carried out on total RNA obtained from the CLN3p/GFP-HeLa.8 cell line. The primer combinations used were: (i) 5[prime]-tca ctc tcg gca tgg acg-3[prime] (GFPC-f) and 5[prime]-cgc tct ctg ctt ctt ctt cc-3[prime] (CLN3.880-861); (ii) 5[prime]-gaa gaa gaa gca gag agc gc-3[prime] (CLN3.862-881) and 5[prime]-aca aat gtg gta tgg ctg-3[prime] (GFPC-r); and (iii) 5[prime]-tca ctc tcg gca tgg acg-3[prime] (GFPC-f) and 5[prime]-aca aat gtg gta tgg ctg-3[prime] (GFPC-r). One-step RT-PCR was carried out using the reagents included in the Superscript One-Step RT-PCR System kit (Gibco BRL, Grand Island, NY).

Metaphases from CLN3p/GFP-HeLa.8 cells were hybridized in situ as described (24) using the vector pEGFP-C1 DNA as a probe. Images of metaphase preparations were captured by a CCD camera using the CytoVision Ultra image collection and enhancement system (Applied Imaging International, Newcastle upon Tyne, UK).

Cell culture and transfections

HeLa and COS-7 cells were obtained from the American Type Culture Collection (Rockville, MD). All cell cultures were carried out in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and antibiotics. Transfections were carried out using the lipids Lipofectamine (Gibco BRL), Lipofectamine Plus (Gibco BRL) or Superfect (Qiagen, Valencia, CA) following the guidelines supplied by the manufacturers. Briefly, cells were seeded at 4 × 105 (HeLa), 1.5 × 105 (fibroblasts) or 2 × 105 (COS-7) in 35 mm wells on coverslips 18-24 h before transfection. Cells were exposed to lipid/DNA complexes for 3-5 h in serum/antibiotic-free medium. Subsequently the cells were incubated in DMEM with 10% fetal calf serum. Transfectants were examined at 28, 48 and 72 h post-transfection.

Fluorescence/immunofluorescence

Cells expressing GFP chimeras were mounted in DMEM medium and studied live or fixed with formaldehyde and mounted in glycerol/DABCO medium. Counterstaining of lysosomes, endoplasmic reticulum and mitochondria was performed on live cells. Lysosomes were stained with 75 nM LysoTracker Red DND-99 (Molecular Probes, Leiden, The Netherlands), endoplasmic reticulum with 100 nM ER-Tracker Blue-White DPX (Molecular Probes) and mitochondria with 25 nM MitoTracker Red CMXRos (Molecular Probes). Counterstaining of nuclei and Golgi was performed on fixed cells using DAPI (Sigma, St Louis, MO) and WGA/Texas Red (Molecular Probes), respectively.

Immunofluorescence analysis was carried out on fixed cells using routine protocols. Briefly, formaldehyde-fixed cells were incubated with the anti-FLAG antibody M2 (Kodak Scientific Imaging Systems, New Haven, CT) followed by incubation with sheep anti-mouse Ig FITC (Silenus, Melbourne, Australia) or goat anti-mouse Ig CY3 (Jackson Immunoresearch). Images of cells were captured by a cooled CCD camera using the CytoVision Ultra image collection and enhancement system (Applied Imaging International).

Subcellular fractionation and western blot analysis

Separation of crude organelle fractions was performed on cells using the differential centrifugation method of Ohtsuka et al. (25). Pellets were resuspended in SDS-sample buffer or TES buffer (10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 0.25 M sucrose). The amount of total protein was estimated using the Bradford assay (Bio-Rad, Hercules, CA). Fifty micrograms of protein were loaded per lane. Membrane extractions with 100 mM sodium carbonate were performed as previously described (11).

Refined fractionation of microsomal fractions was carried out on a Percoll continuous density gradient using a modification of the method described by Miekle et al. (26). Harvested cells were rinsed with phosphate-buffered saline resuspended in 3 vol of sucrose solution (0.25 M sucrose, 10 mM HEPES, pH 7.4, 1 mM EDTA) and lysed by hypobaric shock in a sealed syringe. Nuclei and cytosolic proteins were removed by differential centrifugation. The remaining pellet fraction containing endoplasmic reticulum, Golgi, lysosomes and mitochondria was overlayed onto 17 ml of Percoll solution (20% Percoll, 0.25 M sucrose, 10 mM HEPES, pH 7.4, 1 mM EDTA) and centrifuged for 1 h at 40 000 g. One milliliter fractions were removed and used for western blot analysis and activity assays for [beta]-Hex (26), galactosyl transferase (26), [alpha]-ketoglutarate dehydrogenase (27) and acid phosphatase (26,28).

Western blot analysis was carried out using standard SDS-PAGE followed by transfer to nitrocellulose membranes. The CLN3p/GFP (carboxy) protein was detected using a mouse anti-GFP antibody (Clontech), labeled with HRP-conjugated sheep anti-mouse (Silenus). Signal was developed by chemiluminescence reaction (ECL kit; Amersham, Buckinghamshire, UK).

ABBREVIATIONS

ANP, d-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol; [beta]-Hex, N-acetyl-[beta]-d-glucosaminidase; DMEM, Dulbecco’s modified Eagle’s medium; FISH, fluorescence in situ hybridization; GFP, green fluorescence protein; NCL, neuronal ceroid lipofuscinose; RT-PCR, reverse transcription-polymerase chain reaction; WGA, wheatgerm agglutinin.

ACKNOWLEDGEMENTS

We thank Elizabeth Baker and Helen Eyre for cheerful instruction with the Cytovision image capture and processing equipment and Cathy Derwas and Lynne Hobson for assistance with routine cell work. We are indebted to Professor Heddy Zola and Sylvia Nobbs for generous access to and assistance with the FACS equipment. This work was supported by research grants from the Adelaide Women’s and Children’s Hospital Foundation and the National Health and Medical Research Council of Australia.

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*To whom correspondence should be addressed. Tel: +61 8 8204 7333; Fax: +61 8 8204 7342; Email: gkremmid@pulse.adelaide.edu.au

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