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Originally published In Press as doi:10.1074/jbc.M405333200 on July 15, 2004

J. Biol. Chem., Vol. 279, Issue 39, 41012-41017, September 24, 2004
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Exon Skipping of Cathepsin B

MITOCHONDRIAL TARGETING OF A LYSOSOMAL PEPTIDASE PROVOKES CELL DEATH*

Kathrin Müntener{ddagger}§, Roman Zwicky{ddagger}§, Gabor Csucs¶, Jack Rohrer||, and Antonio Baici{ddagger}**

From the {ddagger}Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland, the Light Microscopy Centre, Institute of Biochemistry, Swiss Federal Institute of Technology, CH-8093 Zurich, Switzerland, and the ||Department of Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

Received for publication, May 13, 2004 , and in revised form, July 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The alternatively spliced messenger RNA of the human cysteine peptidase cathepsin B missing exons 2 and 3 encodes a truncated form of the enzyme lacking the signal peptide and part of the inhibitory propeptide. This deletion results in a new N-terminal leader sequence characteristic of proteins predestined for transport into mitochondria. We determined enzyme targeting to intracellular organelles by transfecting HeLa cells with constructs containing segments of variable length of the N terminus of truncated cathepsin B fused to green fluorescent protein. Co-localization of the constructs with mitochondria and the endoplasmic reticulum was probed with specific markers. None of the chimeric products were found in the endoplasmic reticulum, showing that truncated cathepsin B is misrouted from its regular biosynthetic pathway and forced to enter the mitochondria instead of lysosomes as its final destination. The first 20 amino acids of the new N terminus were necessary and sufficient for mitochondrial targeting, but only cells expressing the complete truncated cathepsin B sequence died by nuclear fragmentation. This new and unexpected behavior draws attention to an additional extralysosomal role for a cysteine peptidase with several recognized important pathophysiological functions. Mitochondrial targeting of cathepsin B may have significant consequences on cell life in pathological or physiological situations characterized by excessive transcription of the cathepsin B message lacking exons 2 and 3, as observed for instance in osteoarthritic cartilage.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cathepsin B, one of the best characterized mammalian cysteine peptidases, is classically regarded as a typical lysosomal enzyme. Within lysosomes, it exerts beneficial physiological functions but it can be detrimental or even life-threatening when it reaches the extracellular space, as documented in pathological situations such as cancer (1, 2) and osteoarthritis (3, 4). The cathepsin B gene comprises 12 exons and its message consists of several variants produced by alternative exon splicing (5, 6). Among these, the splice variant lacking exons 2 and 3, CB(-2,3),1 has been originally detected as a rare species in human breast and colon carcinomas and in a human melanoma (6). While screening articular cartilage specimens, we ascertained the presence of CB(-2,3) in both healthy and osteoarthritic tissue and demonstrated the production of this message in cultures of normal chondrocytes and in chondrocytes modulated toward an osteoarthritic phenotype. Osteoarthritic cartilage from joints with active disease showed a preferential expression of the CB(-2,3) splice variant (7).

The CB(-2,3) message encodes a protein lacking the 17 amino acids of the signal peptide and the first 34 amino acids of the propeptide. In a study with four mammalian cell lines we demonstrated that the resulting truncated cathepsin B (tCB) is not processed as a regular preproenzyme via the endoplasmic reticulum (ER) and the Golgi apparatus and cannot be targeted to its final location, the lysosome (8). Moreover, cells expressing tCB died by nuclear fragmentation suggesting apoptosis (8, 9). In COS cells transfected with constructs encoding tCB, the CB(-2,3) splice variant was found to be a functional message (10). The authors suggested an association of tCB with nuclei and not yet identified intracellular membranes, possibly the ER (10).

Here we follow the fate of human tCB in HeLa cells transfected with constructs containing segments of variable length of the N-terminal region of tCB fused to green fluorescent protein (GFP). We demonstrate that the truncated form of the enzyme contains a new N-terminal leader sequence that delivers it to the mitochondria instead of the lysosomes. In addition, the short leader sequence, present within the first 20 amino acids, is able to target GFP, a reporter protein not related to cathepsin B, to the mitochondrion.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—HeLa cells, derived from a human cervix epithelioid carcinoma, were from the American Type Culture Collection (Manassas, VA). The cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% heat-inactivated fetal calf serum, 2 mM glutamine, 50 units of penicillin, and 50 ng of streptomycin/ml. Cells were grown at 37 °C in a humidified incubator with a 5% CO2, 95% air atmosphere. Maintenance of the cell lines was achieved by weekly passaging. A stable cell line, H2-HeLa, expressing an ER-localized monomeric red fluorescent protein was derived from HeLa cells transfected with the vector ProlacRFPKDEL using Polyfect transfection reagent according to the manufacturer's instruction (Qiagen, Basel, Switzerland). The resident ER protein consisted of a preprolactin leader sequence at the N terminus, the monomeric red fluorescent protein, and finally the KDEL sequence. Selection was carried out in 40 µg/ml G418 (Invitrogen).

Plasmids—The human cathepsin B mRNA code, to which all sequences in this study refer, is the sequence of 1996 nucleotides under accession number L16510 [GenBank] at EMBL/GenBankTM/DDBJ. Plasmids were constructed by PCR using the primers listed in Table I (Microsynth, Balgach, Switzerland) and vector 5'-(-2,3)CBL16GFP (8) containing the tCB-GFP fusion sequence as a template. The PCR products were ligated into the vector 5'-(-2)CBL16GFP, which was first digested with the appropriate restriction enzymes. Cloning procedures were performed as described (8). Plasmids were prepared using Plasmid Midi kits (Qiagen) according to the manufacturer's instructions. For transfection experiments, plasmids were purified using the EndoFree plasmid maxi kit of Qiagen. In all experiments, the start codon of GFP was omitted by PCR as described previously (8) to guarantee the use of the start codons of cathepsin B rather than that of GFP in the fusion protein.


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  		TABLE I
Primers used for plasmid construction

 
The sequence for vector tCB-GFP was obtained by PCR with primers CBcd4forXhoI and CBcdrevBamHI using 5'-(-2,3)CBL16GFP as a template, followed by digestion with XhoI and BamHI. This sequence was inserted in the 5'-(-2)CBL16GFP, which was previously cut with XhoI and BamHI.

The sequences for vectors 10aatCB-GFP, 15aatCB-GFP, 20aatCB-GFP, 38aatCB-GFP, and 65aatCB-GFP were obtained by PCR with the forward primer CBcd4forXhoI and the corresponding reverse primers SignaltCB10r, SignaltCB15r, SignaltCB20r, SignaltCB38r, and SignaltCB65r, respectively, using 5'-(-2,3)CBL16GFP as a template, followed by digestion with XhoI and BamHI. These sequences were individually inserted in the 5'-(-2)CBL16GFP, which was cut previously with XhoI and BamHI.

The sequence for vector PreprolacRFPKDEL was obtained with the forward primer ProLactGFPXhofor and the reverse primer mRFPKDELNotrev using vector ppL-mRFP1-UCE as a template.2 All products were digested with XhoI and NotI and ligated into pEGFP-N3 (Clontech) previously digested with XhoI and NotI. The sequences of all vectors were confirmed by sequencing.

Differential Centrifugation and Western Blot Analysis—Cells were seeded at a density of 5 x 105 cells in a 75-cm2 culture flask. After 24 h of incubation, cells were transfected using polyethyleneimine (molecular mass 25 kDa). A 1 mg/ml polyethyleneimine stock solution (Polysciences, Eppelheim, Germany) was prepared. Plasmids (17 µg/25-cm2 flask) were diluted in normal medium up to a total volume of 527 µl. 70 µl of polyethyleneimine were mixed with medium up to the same volume. Finally, the DNA- and polyethyleneimine-containing media were merged and incubated for 15 min at room temperature. Then the media were diluted with 9.2 ml of medium and slowly added to the cells after having removed the entire medium from the cells. 15 h after transfection, the cells were harvested, washed twice with phosphate-buffered saline, and lysed in phosphate-buffered saline by passing 10 times through a 25-gauge needle. The lysates were centrifuged at 700 x g for 10 min at 4 °C, and the supernatant was separated from the pellets containing unbroken cells and nuclei. A protease inhibitor mixture containing benzamidine, pepstatin A, leupeptin, antipain, and chymostatin was added to the supernatants, and the suspension was centrifuged at 100,000 x g for 30 min at 4 °C. Supernatants were concentrated by precipitating proteins with trichloroacetic acid. Pellets were resuspended in an equal volume of SDS-PAGE reducing sample buffer and boiled for 5 min at 100 °C. SDS gel electrophoresis was carried out on a 12% polyacrylamide gel under reducing conditions. The same volume of each sample was loaded to be able to directly compare the intensity of the bands. Western blotting was carried out using the monoclonal mouse antibody G1/139 against the lysosomal-associated membrane protein (LAMP-1) (11) and the mouse anti-GFP IgG (Roche Applied Science). Chemiluminescence detection was performed with anti-mouse antibodies using a light-emitting non-radioactive method (BM chemiluminescence POD, Roche Applied Science) and image capturing on Agfa Curix Ortho HTA films.

MitoTracker Staining—HeLa cells were seeded on round coverslips (15-mm diameter, Assistant, Germany) in a medium lacking phenol red and left for 48 h to adhere. Transfection was carried out as described above by proportional scaling reagents to the area of a 12-well plate using a phenol red-deprived medium. 15 h after transfection, the cells were washed twice with phosphate-buffered saline and incubated for 30 min at 37 °C with 50 µl of fresh medium containing 100 nM MitoTracker Red (CM-H2XRos) solution (Molecular Probes, Leiden, the Netherlands). After washing with phenol red-deprived medium, incubating with fresh medium for another 45 min at 37 °C, washing twice with phosphate-buffered saline, and fixing for 15 min in 4% paraformaldehyde, cells were washed twice with phosphate-buffered saline/glycine and mounted in ProLong® Antifade (Molecular Probes).

Microscopy—Fluorescence microscopy of the GFP-transfected and red-labeled cells was performed using a 100x/1.4 NA Plan Apochromat objective mounted on a Zeiss Axiovert 100M microscope and using the Zeiss LSM510 confocal scanning module (excitation at 488 and 543 nm).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The cathepsin B sequence with the signal peptide, the propeptide, and the mature enzyme, is shown in Fig. 1A to illustrate the components of the structure of the enzyme relevant to this study. The constructs containing the segments of variable length of the N-terminal region of tCB-fused GFP used in this study are summarized in Fig. 1B together with their acronyms.



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  		FIG. 1.
Structure of human cathepsin B. A, schematic representation of the cathepsin B primary structure showing the relationship between regular procathepsin B and tCB. B, structure of the fusion constructs to GFP used in this study and their acronyms. The cathepsin B sequence is based on the structure under accession number L16510 [GenBank] at EMBL/GenBankTM/DDBJ. GFP was fused C-terminal to tCB (tCB-GFP) or to parts of it as indicated. Thus, for instance, 10aa-tCB-GFP consisted of residues 52-61 fused to GFP and 15aa-tCB-GFP consisted of residues 52-66 fused to GFP.

 
tCB Constructs Are Not Targeted to the ER—We attributed the final cellular destination of the fusion proteins in HeLa cells by co-localization with specific markers of the ER and mitochondria using confocal fluorescence laser microscopy. We first investigated any possible association of tCB with the ER for tracking the classical pathway of lysosomal enzyme biosynthesis and processing. Depending on the length of the GFP constructs, a transfection of HeLa cells revealed two different locations of the fusion protein (Fig. 2, green, GFP). When only 10 or 15 amino acids of the tCB N terminus were present, the fluorescence product showed a typical cytosolic distribution. Fusion proteins containing 20, 38, or 65 amino acids of tCB, as well as the entire tCB, manifested a clear distribution as prolate and fibrous intracellular structures besides the cytosolic distribution. The percentage of cells showing these structures increased from ~20% with the 20 aa construct to 50% with the 38 aa and to 60% with the 65 aa-tCB-GFP construct. Approximately 50% of the cells expressing the whole tCB protein contained a clear patch distribution of GFP-fluorescence. Compared with the 38aa-tCB-GFP and 65aa-tCB-GFP constructs, the fluorescence intensity of the product of the tCB-GFP construct was weaker, suggesting either a lower rate of expression or a shorter lifetime for this species. The red channel in Fig. 2 (ER) indicates the distribution of the ER monitored by stably transfecting HeLa cells with monomeric red fluorescent protein (12) made resident in the ER by attaching the ER recycling motif KDEL. No co-localization of the green GFP signal and the red ER signal, which should produce yellow staining in the merge panels in Fig. 2, right column, could be detected. Cells showing only red color in the merge channel are those that do not express the cathepsin B-GFP proteins. Three-dimensional imaging clearly revealed that the green and red staining was not coincident.



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  		FIG. 2.
Trafficking of cathepsin B chimeras encoded by cathepsin B constructs lacking exons 2 and 3 in H2-HeLa cells. This cell line stably expresses ER-localized monomeric red fluorescent protein. ER localization of the red fluorescent protein-KDEL construct was confirmed by co-labeling with calnexin (24) and the anti-binding protein Bip (25) (not shown). The labels of the rows correspond to the constructs shown in Fig. 1 with simplified abbreviations and indicate whole tCB or GFP fusion constructs coding 10, 15, 20, 38, and 65 amino acids of tCB, starting with position 52 of the preprocathepsin B sequence. GFP, green channel showing the GFP fluorescence. ER, red channel showing ER resident red fluorescent protein. Merge, merged green and red channels. Cells with only red color in the merged channels are those that were not transfected with the GFP-containing constructs.

 
Mitochondrial Targeting of tCB Constructs—The red channel (MitoTracker) in Fig. 3 shows fluorescence staining of mitochondria in HeLa cells. The MitoTracker dye is a specific marker that becomes fluorescent after oxidation in active mitochondria. The GFP channel illustrates the same staining pattern as seen in Fig. 2, i.e. cytoplasmic fluorescence with the constructs coding the first 10 or 15 amino acids of the tCB sequence, with an increasing number of fluorescent patches when the number of those amino acids is 20 or more. Comparison of the GFP, MitoTracker, and merge channels of the constructs containing 10 and 15 aa reveals that the cytoplasmic GFP-fluorescence and MitoTracker-stained mitochondria do not co-localize. However, with 20 or more amino acids of the tCB sequence, the number of yellow intracellular structures in cells successfully transfected with the cathepsin B-GFP constructs increases and becomes predominant, indicating a co-localization of the chimerical cathepsin B products with mitochondria (Fig. 3, merge). This property is not only demonstrated by the yellow color in the merge channel but also by direct comparison of the GFP and MitoTracker channels. Mitochondrial staining with MitoTracker is only possible with living cells. Because transfection with the tCB-GFP construct resulted in cell death of the cells expressing the chimerical protein after a given period, staining as shown in Fig. 3 was performed at a time point preceding the onset of cell death symptoms.



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  		FIG. 3.
Co-localization of human tCB with mitochondria. Cathepsin B chimeras encoded by constructs lacking exons 2 and 3 were transfected into HeLa cells. The labels of the rows correspond to the constructs shown in Fig. 1 with simplified abbreviations and indicate entire tCB or GFP fusion constructs coding 10, 15, 20, 38, and 65 amino acids of tCB starting with position 52 of the preprocathepsin B sequence. GFP, green channel showing the GFP fluorescence. MitoTracker, red channel showing fluorescence produced by treating the cells with MitoTracker red (CM-H2Xros), a specific stain for active mitochondria. Merge, merged green and red channels.

 
tCB Constructs Are Targeted to the Membrane Fraction of the Cell—A further proof for the presence of tCB in the membrane fraction is provided in Fig. 4 where Western blots of the cytosolic and the membrane fractions, separated by differential centrifugation, are shown. The membrane fraction contained proteins associated with intracellular organelles such as mitochondria and lysosomes. The cytosolic fraction (Fig. 4, 1) contained mainly the expression product of the 15aa-tCB-GFP construct and traces of the 65aa-tCB-GFP construct, whereas the product of the tCB-GFP construct could not be determined because of either low expression or rapid degradation. The membrane fraction (Fig. 4, 2) also contained the products of the 15aa-tCB-GFP and 65aa-tCB-GFP constructs but in this case with a much stronger signal for the latter. The band around 58 kDa revealed the presence of the tCB-GFP protein product in the membrane fraction, whose faint signal confirmed its low rate of expression (or rapid degradation) as commented in the experiments discussed above (Figs. 2 and 3). The blots shown in Fig. 4, 3 were stained for the LAMP-1 to compare the relative concentrations of the membrane fraction in samples A-D. Staining for LAMP-1 in Fig. 4, 1 was negative for all samples. The main band in Fig. 4, 2 corresponds to the protein encoded by the 65aa-tCB-GFP construct, and the two bands at lower molecular mass represent fragments of this protein generated by limited proteolysis. We submitted the bands shown in Fig. 4, 2 to N-terminal sequencing after immunoprecipitation, SDS-PAGE, and transfer to a sequencing membrane. However, even using a 30-fold higher protein load as that shown in Fig. 4, 2, we were unable to determine the sequence because, the proteins turned up to be N terminally protected. This experiment aimed at exploring any processing of the N-terminal leader sequence for mitochondrial import, which is usually cleaved off during transport of proteins into mitochondria (13).



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  		FIG. 4.
Western blots of extracts of HeLa cells separated by differential centrifugation. A, HeLa cells transfected with tCB-GFP; B, untransfected cells as control; C, transfection with the 15aa-tCB-GFP construct; D, transfection with the 65aa-tCB-GFP construct. Following homogenization and differential centrifugation, the cytosolic (1) and the membrane fractions (2 and 3) were subjected to SDS-PAGE and Western blot analysis. The pellets of the membrane fraction were resuspended in an equal volume of SDS-PAGE buffer and equal volumes were applied to the individual lanes of gels 2 and 3. Gels 1 and 2 were stained with anti-GFP antibodies, and gel 3 was stained with antibodies against LAMP-1, the lysosomal associated membrane protein characteristic of late endosomes and lysosomes. The staining of gel 3 shows the relative content of membranous material in lanes A-D based on the content of endosomes and lysosomes. The membrane fraction also contains mitochondria. Lines and numbers (kDa) on the left indicate the positions of molecular mass standards.

 
Structural Evidence for Mitochondrial Import of tCB—Besides obtaining direct experimental evidence for a localization of tCB within mitochondria and not within lysosomes, we verified the theoretical potential of constructs containing 10, 15, 20, 38, and 65 amino acids of tCB fused to GFP to be targeted to mitochondria using the program Target P (14). This procedure considers the presence of mitochondrial targeting peptides, which are thought to form an amphipathic {alpha}-helix of hydrophobic and basic amino acids positioned in a particular sequence within the first 65 residues of human tCB, and calculates the import scores of the constructs, i.e. their ability to code polypeptides targetable to mitochondria. The score for mitochondrial import of cathepsin B chimeras increased with the length of the N-terminal amino acid sequence derived from cathepsin B up to 38aa-tCB-GFP, and tCB was determined to be a mitochondrial protein in contrast to regular cathepsin B. Thus, after deleting the first 34 amino acids of the cathepsin B propeptide, the resulting truncated enzyme acquires an N-terminal sequence containing the characteristic signal of proteins predestined for mitochondrial import. This signal motif is typified by folding of part of the N-terminal segment into an amphipathic {alpha}-helix, with the hydrophobic surface on one side and the positively charged surface on the opposite side (13).

The published three-dimensional structure of human procathepsin B (15) clearly shows an {alpha}-helical segment beginning at Met-52, i.e. the starting sequence of tCB (Fig. 5). This helix is amphipathic with three residues (Met-52, Leu-55, and Leu-58) forming a hydrophobic surface on one side of the helix and two residues (Lys-56 and Arg-57) forming a positively charged surface at the opposite side. Using a computational approach with the program PSIPRED (16) we tested the ability of the N-terminal segment of tCB to form this type of helix even in the absence of residues 18-51, i.e. in tCB. This calculation afforded the highest probability score for Leu-55 and Leu-58 to be present in an {alpha}-helical secondary structure in constructs bearing 20 or more amino acids of the N-terminal tCB sequence.



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  		FIG. 5.
Three-dimensional structure of human cathepsin B. Based on published data (15), the {alpha}-helix starting at residue 52 (see also Fig. 1) is represented here as a cylinder viewed along its main axis. Three residues (Met-52, Leu-55, and Leu-58) form a hydrophobic surface on one side of the helix (black side chains pointing to the right of the cylinder), and two residues (Lys-56 and Arg-57) form a positively charged surface at the opposite side (white side chains with black contour).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The majority of mitochondrial proteins are encoded in the nucleus and are transported into mitochondria with the help of characteristic leader sequences. Precursor proteins possessing amphipathic helices in their N-terminal presequence are recognized by the Tom20 receptor of the translocase of the outer mitochondrial membrane (TOM complex) and are then transported into the mitochondrial matrix through the translocase of the inner mitochondrial membrane (TIM23). Only three hydrophobic residues of the leader sequence are necessary for binding in a hydrophobic groove of the Tom20 domain to promote transport (reviewed in Ref. 13).

GFP expressed in human cells is not targeted to organelles and remains in the cytosol. However, adding a leader sequence composed of at least 20 amino acids of the N terminus of the truncated human cathepsin B to GFP conveys GFP to the mitochondria. Thus, human tCB generated by the cathepsin B mRNA splice variant missing exons 2 and 3 possesses all prerequisites for being targeted to mitochondria, a property that is not shared with regular preprocathepsin B. This property is because of the presence of a latent mitochondrial import leader sequence, which is hidden in regular procathepsin B but becomes available when the new N terminus of tCB is created. Besides direct evidence from co-localization measurements, our claim is supported by the three-dimensional structural data of the cathepsin B molecule (15) and from theoretical prediction using the most reliable software available to date (14, 16). The structural motif highlighted in Fig. 5, in particular the three hydrophobic residues defining a hydrophobic surface on one side of the amphipathic {alpha}-helix starting at residue 52, strongly suggests the role of this segment of the tCB sequence as a mitochondrial leader peptide.

We illustrated in two previous contributions that the majority of cells transfected with the tCB construct died by nuclear fragmentation indicating an apoptotic cell death (8, 9). We confirm that this phenomenon always occurred after cell transfection with the construct containing the entire tCB sequence but was not observed when cells were transfected with constructs coding for 10-65 amino acids starting from the N terminus of tCB. Thus, twenty amino acids of the tCB sequence were necessary and sufficient for mitochondrial targeting but the entire tCB molecule was required for promoting cell death.

Lysosomal cathepsin B has been recognized as the dominant executioner peptidase in tumor necrosis factor-induced apoptosis of fibrosarcoma cells (17) and hepatocytes (18). The last study (18) highlighted a beneficial role of cathepsin B, as opposed to its well known function in malignancy by facilitating invasion (1). The participation of cathepsin B in the mechanism of apoptosis was identified in the upstream death signals as a link between the death receptor and the mitochondrial loop of caspase activation (19). Although cathepsin B has been suggested to mediate apoptosis through the proteolytic processing of the proapoptotic Bcl-2 family member Bid in vitro (20), this view was challenged in a subsequent paper based on studies in vivo (21). The key for understanding the mechanisms of lysosomal peptidase-dependent apoptosis relies thus far in the assumption of enzyme translocation from lysosomes to cytosol and nucleus, as seen for example in liver cell apoptosis (19, 22).

The results discussed in our present contribution represent a new concept and a possible alternative mechanism of cell death. In essence, an abnormal form of a cysteine peptidase designed for lysosomes can reach the mitochondria through modification of its biosynthetic route, which imitates the regular processing pathway of nuclear-encoded mitochondrial proteins. This situation is different from enzyme leakage or translocation from lysosomes, which can potentially reach the external surface of mitochondria and elicit undesired effects but does not possess the structural characteristics to be imported inside these organelles (17, 18). Alternative trafficking of another cysteine peptidase, cathepsin L, was recently shown to be responsible for enzyme targeting to the nucleus (23). This was accomplished through initiation of translation at a downstream AUG site leading to a cathepsin L form devoid of the signal peptide. Taken together, the newly discovered unusual properties of cathepsin B (this work) and of cathepsin L (23) point to a much wider role of "lysosomal" cysteine peptidases than previously expected and suggests new pathophysiological functions at extralysosomal sites.

Establishing a link between tCB biosynthesis triggered by the cathepsin B mRNA splice variant missing exons 2 and 3 and cell death in vivo or in cell cultures is still elusive. We were not yet able to set up clear-cut experiments aimed at distinguishing morphologically and biochemically the mechanisms of cell death induced by known means or by the new one described in the present work. However, the regular occurrence of cell death following transfection with tCB-encoding constructs (8, 9), and the preferred occurrence of the CB(-2,3) message in pathological tissues undergoing degeneration accompanied by cell death (4, 7), suggests a pathophysiological function for tCB at least in osteoarthritis. This notion well deserves further attention in other pathologies.


   FOOTNOTES
 
* This work was supported by grants from the Swiss National Science Foundation (32-61312-00, 32-102108/1 (to A. B.), and 31-67274 (to J. R.)) and from the Lydia-Hochstrasser, Hartmann-Müller, and Olga Mayenfisch Foundations (to A. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

** To whom correspondence should be addressed. Tel.: 41-1-635-55-42; Fax: 41-1-635-68-05; E-mail: abaici{at}bioc.unizh.ch.

1 The abbreviations used are: CB(-2,3), cathepsin B lacking exons 2 and 3; tCB, truncated cathepsin B; ER, endoplasmic reticulum; GFP, green fluorescent protein; aa, amino acid; LAMP, lysosomal-associated membrane protein. Back

2 J. Rohrer, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank H.-P. Hauri for kindly providing the monoclonal antibody G1/139 against LAMP-1, R. Y. Tsien for providing the monomeric red fluorescent protein, A. Mezzacasa for antibodies against ER markers, and Ch. Briand for help with Fig. 5.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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