1 Sir William Dunn School of Pathology, Oxford University, South Parks Rd, Oxford, OX1 3RE, UK
2 Ludwig Institute for Cancer Research, University College, London, W1W 7BS, UK
* Authors contributed equally
Author for correspondence (e-mail: gillian.griffiths{at}path.ox.ac.uk)
Accepted March 20, 2001
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SUMMARY |
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Key words: Fas ligand, Secretory lysosomes, Sorting motif, SH3 domain, Proline-rich domain
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INTRODUCTION |
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Tight regulation of FasL surface expression is essential to prevent non-specific killing by T cells. Several different mechanisms are involved in controlling surface expression of FasL. Firstly, T cell activation leads to transcriptional up-regulation of FasL (Suda et al., 1995). The newly synthesised FasL is targeted to specialised secretory lysosomes, present in T cells, which undergo fusion with the plasma membrane when the T cell receptor (TcR) is activated (Bossi and Griffiths, 1999). Once at the cell surface, FasL surface expression is rapidly downregulated by a metalloprotease, which sheds the extracellular domain of the protein (Schneider et al., 1998; Tanaka et al., 1998).
FasL is thought to have a relatively short half-life as it does not accumulate in the secretory lysosomes (Bossi and Griffiths, 1999). FasL-mediated killing can be inhibited by cycloheximide (el-Khatib et al., 1995), demonstrating that de novo synthesis of protein is required during killing assays. Delivery of newly synthesised FasL to the surface of T cells via the secretory lysosomes has several physiological advantages. First, as degranulation is controlled by TcR cross-linking, delivery of the granule contents occurs only under conditions of activation. Second, secretion is polarised and consequently FasL is delivered exclusively to the immunological synapse between T cell and target, ensuring that only the target cell recognised is killed, thus providing an additional level of control for the appearance of FasL on the plasma membrane.
Our previous work demonstrated that the cytoplasmic tail of FasL contains the information required for sorting to secretory lysosomes, because swapping the FasL tail onto the transmembrane and extracellular regions of the type II cell surface protein CD69 targets this chimera to the secretory lysosomes (Bossi and Griffiths, 1999). Curiously, the cytoplasmic tail of FasL does not contain any of the well-characterised di-leucine (Letourneur and Klausner, 1992; Pond et al., 1995) or tyrosine-based (Trowbridge et al., 1993) lysosomal sorting motifs.
A prominent feature of the FasL tail is a polyproline-rich region flanked by di-arginine and di-lysine residues. Such proline-rich domains (PRDs) have been shown to be important in associating with various components of the AP-2-mediated endocytic machinery, such as the interaction between amphiphysin and dynamin via their SH3 domain and PRD, respectively (Simpson et al., 1999). The functional importance of this interaction has been shown by microinjection of the SH3 domain of amphiphysin, which inhibited synaptic vesicle endocytosis at the stage of invagination of clathrin-coated pits (Shupliakov et al., 1997). The PRD of FasL might therefore mediate trafficking of the protein to secretory lysosomes by endocytosis from the plasma membrane. Alternatively, the PRD may dictate a direct pathway to the lysosomes for FasL, as has been suggested for the metalloprotease disintegrins MDC9 and MDC15 (Howard et al., 1999).
In order to determine the region of the cytoplasmic tail of FasL required for sorting to secretory lysosomes, we have expressed a series of point and deletion mutants of FasL in cells with secretory lysosomes and cells with conventional lysosomes. We find that the PRD is required for sorting to secretory lysosomes. This sorting motif is not recognised in cells with conventional lysosomes and, in these cells, FasL travels by default to the plasma membrane. We suggest that FasL is sorted directly from the Golgi to the secretory lysosomes via the interaction of the PRD with an SH3-domain-containing cytosolic protein.
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MATERIALS AND METHODS |
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Transfection
1x107 cells (RBL, NRK, HeLa, Rat-1 and WR19L) were transfected by electroporation (500 µF and 250 V for RBL, NRK, Rat-1 and HeLa; 960 µF and 300 V for WR19L; BioRad, Richmond, California) with 35 µg DNA for WR19L and 20 µg for other cell lines. Transient transfectants were analysed 30-36 hours post-transfection. Stable transfectants were selected by supplementing the growth medium with 1 mg ml-1 G418 (Gibco) 24 hours post-transfection.
Immunostaining and confocal microscopy
RBL, NRK, Rat-1 and HeLa cells were grown on coverslips for 36 hours post-transfection prior to immunostaining. YT and WR19L cells were resuspended in AIM V serum-free medium (Gibco) and plated on coverslips for 20 minutes at 37°C. Cells were fixed with ice-cold methanol for 5 minutes, then stained for 40 minutes with the relevant primary antibody diluted in 1% BSA/PBS. The antibodies used were: mouse anti-human FasL Nok-1 (10 µg ml-1; PharMingen, San Diego, CA), mouse anti-human lamp-1 (1:100 dilution of supernatant; clone H4A3, J. T. August, Developmental Studies Hybridoma bank, IA), rat anti-mouse lamp-1 (1:2 dilution of supernatant; clone IDB4, J. T. August), rabbit anti-human lamp (1:500 dilution of supernatant, clone AS120), mouse anti-rat Lgp-120 (sunshine; 1:100 dilution; Mark Marsh, University College, London, UK), mouse anti-human CD63 (1:2 dilution of supernatant; clone H5C6; J. T. August), mouse anti-human CD40L (10 µg ml-1; PharMingen) and mouse anti-rat MHC class II (1:2 dilution of supernatant, clone OX4; N. Barclay, Dunn School of Pathology, Oxford, UK). After washing, the cells were incubated with the relevant secondary antibody diluted in 1% BSA/PBS for 35 minutes. Final concentrations of secondary antibodies used were: FITC-conjugated goat anti-mouse IgG (14 µg ml-1), Texas-Red-conjugated goat anti-mouse IgG (14 µg ml-1), Texas-Red-conjugated goat anti-rat IgG (14 µg ml-1), FITC-conjugated goat anti-rabbit IgG (15 µg ml-1) and Texas-Red-conjugated goat anti-rabbit IgG (13 µg ml-1; Jackson Immunoresearch, West Grove, PA). The slides were mounted with 90% glycerol/PBS containing 2.5% DABCO (Fluka, Buchs, Switzerland). Cell staining was analysed using an MRC-1024 confocal microscope (BioRad, Hemel Hempstead, UK).
Green fluorescent protein constructs
pFasLWT-GFP was constructed as previously described (Bossi and Griffiths, 1999). This plasmid encodes full-length human FasL in the EGFP-C1 (Clontech) vector, in which the CMV promoter was replaced with the Bos promoter (pEGFP-C1-bos) from elongation factor 1 (Mizushima and Nagata, 1990; construct from K. Campbell, Fox Chase, PA). The vector also contains the neomycin resistance gene to enable selection of transfectants with G418. Deletions of the cytoplasmic tail of FasLWT-GFP were performed by PCR or direct subcloning. For PCR, the deletion mutants of FasL (
54,
67 and
74) were amplified from pFasLWT-GFP using Pfu polymerase (Stratagene, La Jolla, CA) and the same 3' primer encoding a BamHI site; 5'-CAGGTACCGGGAACCACAGCACAGG-3' and the following 5' primers, each of which encode a KpnI site: (
54) 5'-CAGGTACCCCACCTCCGCCGCCGCC-3', (
67) 5'-CAGGTA-CCCTGCCACCCCTGAAGAA-3', (
74) 5'-CAGGTACCGGGAA-CCACAGCACAGG-3'. The PCR products were subcloned into the pEGFP-C1-bos vector between the KpnI and BamHI sites. pFasL
37-GFP was constructed by digestion of pFasLWT-GFP with BglI and BamHI. The digestion product encoding FasL
37 was given blunt ends and subcloned in frame into pEGFP-C2 (Clontech) between the SmaI and BamHI sites. pFasL
pro-GFP was constructed by mutagenesis using pFasLWT-GFP as a template; the restriction site BsshII was introduced either side of the R43-K71 region by two rounds of site-directed mutagenesis (QuickChange, Stratagene). The first round introduced a BsshII site at R42-R43, but also introduced a conservative amino acid change R42-A (5' primer, 5'-AGGCCTGGTCAAGCGCGCCCACCACCACCA-3'; 3' primer, 5'-TGGTGGTGGTGGGGCGGCTTGACCAGGCCT-3'). The second round introduced a BsshII site at K71-K72 and also a conservative amino acid change K72-A (5' primer, 5'-CCACCCCTG-AAGGCGCGCGGGAACCACAGC-3'; 3' primer, 5'-GCTGTGG-TTCCCGCGCGCCTTCAGGGGTGG-3'). The resulting mutant was digested with BsshII to remove the R43-K71 region and religated. The plasmids pFasL(R43R44-EE)-GFP and pFasL(K72K73R74-EEE)-GFP were also constructed by site-directed mutagenesis, using pFasLWT-GFP as a template. The primers used for RR-EE were: 5' primer, 5'-AGGCCTGGTCAAGAGGAGCCACCACCACCA-3'; 3' primer, 5'-TGGTGGTGGTGGCTCCTCTTGACCAGGCCT-3'. The primers used for KKR-EEE: 5' primer, 5'-CTGCCACCCCTGG-AGGAGGAAGGGAACCACAGC-3'; 3' primer, 5'-GCTGTGGTT-CCCTTCCTCCTCCAGGGGTGGCAG-3'. pCD63-EGFP-bos was constructed by cloning CD63 into the BamH1 site of the pEGFP-C1-bos promoter vector. This construct encodes a chimera with GFP at the N-terminus of CD63 with a 22 amino acid spacer encoded by the multiple cloning site of pEGFP-C1 before the first methionine of CD63. The sequences of all constructs were confirmed using AP Prism 377 DNA sequencer (Perkin-Elmer, Norwalk, CT).
FACS analysis
RBL FasL-GFP transfectants were plated in 6-well dishes 12 hours prior to fluorescence-activated cell sorting (FACS) staining. 2 hours prior to FACS staining, the cell medium was supplemented with 10 µM BB3013 metalloprotease inhibitor (British Biotech, Oxford, UK), after which the dishes were placed on ice for 30 minutes. The medium was replaced with 1 µg ml-1 Nok-1 antibody diluted in medium plus 10 µM BB3013 and incubated for 30 minutes on ice. The cells were washed with FACS buffer (1% FCS/PBS with 1 µM sodium azide), then fixed with 1% paraformaldehyde (Electron Microscopy Sciences, Washington, PA) in FACS buffer for 10 minutes at room temperature. The cells were washed further, and then incubated for 30 minutes with phycoerythrin-conjugated goat anti-mouse secondary antibody (final concentration 50 µg ml-1 in FACS buffer; Jackson Immunoresearch). The cells were washed three times in FACS buffer and scraped from the dishes into FACS tubes and resuspended in a final volume of 300 µl FACS buffer. The samples were analysed using a FACScalibur flow cytometer (Becton Dickinson). PE fluorescence was measured at 575 nm and GFP fluorescence at 525 nm. Fluorescence data was collected for 20,000 events on a four-decade log scale and analysed using CELLQuest software (Becton Dickinson).
Flow cytometric endocytosis assay
NRK transfectants were released from flasks by incubating with 10 mM EDTA/PBS for 10 minutes at 37°C. The cells were washed once with complete media and then incubated with the relevant primary antibody (Nok-1; 10 µg ml-1 or anti-CD63; 1:2 dilution) diluted in FACS buffer (see above) for 30 minutes on ice. Cells were washed three times with ice-cold complete media and one sample of each transfectant was removed at time 0. The remaining cells were divided into three aliquots and incubated in complete medium at 37°C. Cells were harvested after 15 minutes, 30 minutes and 120 minutes, and stained with PE-conjugated goat anti-mouse secondary antibody (final concentration 50 µg ml-1 in FACS buffer) for 30 minutes on ice. The cells were washed three times in FACS buffer and then fixed by resuspending in 1% paraformaldehyde in FACS buffer. The samples were analysed by flow cytometry as above. To calculate internalisation, the PE fluorescence of each sample was plotted and gated to determine the mean fluorescence intensity (MFI) of each sample outside that obtained with background staining (secondary antibody alone). The resulting numbers were divided by the MFI at time 0 to obtain the percentage remaining at the cell surface.
Confocal microscopy endocytosis assay
Prior to the assay, cells (NRK transfectants or YT) were adhered onto glass coverslips as previously described. The coverslips were incubated with the relevant antibody (Nok-1; 10 µg ml-1, anti-CD63; 1:2 dilution, or an isotype-matched control antibody (OX4 for YT cells and anti-CD40-L for NRK transfectants) diluted in medium. NRK transfectants were incubated with the antibody for 30 minutes at 4°C, after which they were washed and incubated with fresh medium for 2 hours at 37°C. The cells were then fixed with ice-cold methanol and incubated with the relevant Texas-Red-conjugated secondary antibody. YT cells were incubated with the primary antibody for 2 hours at 37°C, then fixed as above and stained with an anti-human Lamp antibody (clone 120). The endocytosed antibody and the anti-lamp signal were detected with the relevant FITC-conjugated and Texas-Red-conjugated secondary antibodies, respectively. All samples were analysed by confocal microscopy.
Modelling of the FasL peptide complex
The crystal structure of the Fyn SH3 domain complexed with a synthetic peptide (PPAYPPPPPVP) solved to 2.3 Å (Musacchio et al., 1994) was taken as a template from which to construct the FasL-Fyn model. The synthetic peptide in the structure was mutated to represent the FasL peptide. The model was then subjected first to CHARMM Steepest Descent minimisation followed by soaking the model (adding water molecules) and carrying out a DISCOVER Steepest Descent minimisation from within InsightII (1300 steps) until the average absolute derivate converged below at 0.8 kcal mol-1 Å-1.
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RESULTS |
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Endogenous FasL is targeted directly to the secretory lysosomes in secretory cells
The NRK transfectants demonstrate the ability of FasL to undergo endocytosis and so we asked whether endogenous FasL is sorted directly to the lysosomal compartment in cells with secretory lysosomes. The human NK cell line YT constitutively expresses FasL, which, like CD63, can be detected in the secretory lysosomes (Fig. 9C,F). In order to ask whether FasL in the secretory lysosomes has transited the cell surface and entered the lysosomal compartment via the endocytic pathway, YT cells were cultured with antibody (in the presence or absence of metalloprotease inhibitor for 2 hours). This assay differs from that performed on NRK cells in that the antibody was cultured with the cells for an extended time (2 hours) to allow for constitutive internalisation. CD63 but not FasL demonstrated endocytosis of surface bound antibody (Fig. 9H,K), which co-localises with the lysosomal compartment in the case of CD63 (Fig. 9I). No internalisation with an isotype-matched control antibody was seen (results not shown). These results indicate that FasL follows a direct pathway to the secretory lysosomes rather than indirectly transiting the plasma membrane via endocytosis.
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DISCUSSION |
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In this paper, we have identified the sorting motif required for FasL to reach the secretory lysosomes and asked how this affects sorting in the different cell types. We find that sorting is mediated by a PRD, which facilitates sorting of FasL directly to the secretory lysosomes from the trans-Golgi network without transiting the cell surface. Because FasL is such a potent mediator of cell death, this is an important mechanism in preventing constitutive release of FasL. We propose a model for a specialised sorting mechanism based on the interaction of the PRD of FasL with an SH3-domain-containing protein expressed selectively in cells with secretory lysosomes (Fig. 10). According to our model, FasL is sorted from the TGN by virtue of the PRD that recognises an SH3-domain-containing protein, which facilitates its sorting to the secretory lysosomes. In cells lacking this compartment, we propose that the SH3-domain-containing protein is absent and, in the absence of this component of the sorting machinery, FasL travels by default to the plasma membrane. A number of SH3-domain-containing proteins specific to cells of the haematopoietic lineage have been identified (Thomas and Brugge, 1997). Because most cells with secretory lysosomes are derived from this lineage, they are likely to express such proteins.
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Another critical aspect of our model is that FasL is sorted directly to the secretory lysosomes without transiting the plasma membrane. Our previous work supported this model, as FasL can only be bound by antibody at the plasma membrane after exocytosis in T cells in the presence of metalloprotease inhibitors, demonstrating the presence of highly effective metalloproteases in these cells. The fact that an antibody against the extracellular domain detected FasL within the secretory lysosomes (Bossi and Griffiths, 1999) indicates that FasL must reach this compartment directly. If FasL had been expressed on the cell surface prior to the lysosomal compartment then its extracellular domain would have been cleaved by a metalloprotease. In this paper, we have demonstrated that endogenous FasL cannot bind antibody and be internalised from the plasma membrane even in the presence of metalloprotease inhibitors, further supporting the model that FasL is sorted directly from the TGN to the secretory lysosomes.
Recently PRD/SH3-domain interactions have been shown to play a critical role in endocytosis. Disruption of these interactions perturbs function, as demonstrated by microinjection of the SH3 domain from amphiphysin, which inhibits endocytosis of synaptic vesicles (Shupliakov et al., 1997). In this study, we find no gross disruption of endocytosis upon removal of the PRD of FasL. In NRK cells, FasL endocytosis is unaffected by removal of the PRD (Figs 6, 7), demonstrating that the PRD is not an essential endocytic determinant.
The ability of all of the deletion mutants to undergo endocytosis explains one of the surprising results of expression of these mutants in RBL (Fig. 2b). Although we find that disruption of the PRD leads to increased cell surface expression, demonstrating that this region is required for correct intracellular sorting, some intracellular localisation is also observed. Our studies on endocytosis explain these findings. When FasL reaches the cell surface by mutation of the PRD, it is then able to undergo endocytosis even in cells with secretory lysosomes. We have shown that RBL cells endocytose CD63 efficiently, whereas WR19L cells do so poorly (data not shown). This suggests that the degree of intracellular staining of FasL may correlate with the efficiency of endocytosis in these cell types. In RBL cells, intracellular FasLpro-GFP and FasL
74-GFP co-localises with the Lgp-120-positive compartment (data not shown), consistent with it reaching this compartment via endocytosis from the plasma membrane.
From our results, we cannot determine whether the SH3 protein chaperones FasL to the secretory lysosomes directly or by other interactions with known sorting complexes, such as AP-3 (which has been shown to mediate direct sorting of proteins from the trans-Golgi network to the yeast vacuole) (Odorizzi et al., 1998) and mammalian lysosomes (Le Borgne et al., 1998). Two lines of evidence suggest that FasL itself does not interact directly with any of the adaptor complexes used during protein trafficking. First, the cytoplasmic tail of FasL does not interact with any of the µ subunits of AP-1, AP-2 or AP-3 in a yeast two-hybrid assay (D. Stephens and G. Banting, unpublished). Second, mutation of minimal tyrosine or di-leucine motifs in the tail of FasL (7Y-9Y-13YAAA and 29V-30L
AA) does not result in mis-sorting of FasL (G. Bossi, unpublished). We currently favour the interpretation that interaction of the PRD of FasL with an SH3-domain-containing protein allows formation of a protein complex that may allow interaction with AP-3.
Which SH3-domain-containing protein(s) are required for sorting? We have shown by computer modelling that the proline-rich region of FasL is capable of binding a Fyn-like SH3 domain. Hane et al. demonstrated an in vitro interaction between the SH3 domains of Fyn and Lck with peptides of the proline-rich region of murine FasL (Hane et al., 1995). The in vivo importance of the FasL-Fyn/Lck interaction is still unknown and there is currently no evidence to support the idea that Fyn or Lck themselves are important in the sorting of FasL. As the specificity and binding strength of an SH3 domain-ligand interaction is governed by neighbouring residues to the proline-rich core sequence (Feng et al., 1994; Ren et al., 1993), it is possible that an in vitro binding peptide partner for FasL may not reflect an in vivo partner.
We have defined a novel lysosomal-sorting motif based on a PRD found in FasL. It is likely that other membrane proteins will use the same mechanism to reach the secretory lysosomes in lymphoid cells. Although FasL is the only member of the TNF family with SH3-binding domains in the cytosolic tail, several members of the ADAMS (a disintegrin and metalloprotease) family of metalloproteases contain polyproline sequences in their tails (Rosendahl et al., 1997). The metalloprotease responsible for extracellular cleavage of FasL is thought to be a member of this family (Schneider et al., 1998; Tanaka et al., 1998), and it is tempting to speculate that the metalloprotease might use a similar sorting motif. Simultaneous sorting of FasL and its metalloprotease could enable subsequent delivery of both molecules to the cell surface provide yet another mechanism to control FasL activity precisely.
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ACKNOWLEDGMENTS |
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