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Fas ligand is targeted to secretory lysosomes via a proline-rich domain in its cytoplasmic tail

Emma J. Blott^1,*, Giovanna Bossi^1,*, Richard Clark¹, Marketa Zvelebil² and Gillian M. Griffiths^1,

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

View larger version (43K): [in a new window]   Fig. 1. FasL is differentially sorted in haematopoietic cells with secretory lysosomes. FasLWT-GFP was transiently expressed in the non-haematopoietic cell lines, HeLa (A-C) and Rat-1 (D-F) and the haematopoietic cell lines, WR19L (G-I) and RBL (J-L). 36 hours post-transfection, the cells were fixed and co-stained for the lysosomal membrane proteins Lamp-1 (A,G) or Lgp-120 (D,J) and analysed by confocal microscopy. The images were merged (C,F,I,L) to demonstrate co-localisation of FasL and Lamp-1/Lgp120 in the lysosomal compartment. Bars, 10 µm except G-I, 5 µm.

View larger version (32K): [in a new window]   Fig. 2. (a) Map of FasL cytoplasmic tail showing mutations made during this study. The amino acid sequence of human FasL cytoplasmic tail. FasL is a type-II membrane protein and so the cytoplasmic tail represents the N terminus of the protein. The map outlines the deletions made to the tail during this study (37, 54, 67 and 74), the proline-rich domain, the two putative SH3-domain-binding motifs (arrows underneath denote the orientation), the KKR and RR residues (boxed), orientation of the GFP tag in the FasL-GFP construct, and the beginning of the transmembrane (tm) region of FasL. The first (M1) and last (G80) amino acid of the tail are numbered above the sequence. (b-d) Progressive N-terminal deletion of the cytoplasmic tail of FasL results in increased cell surface expression. FasL37-GFP, FasL54-GFP, FasL67-GFP and FasL74-GFP and FasLWT-GFP were transiently expressed in RBL cells and FasL cell surface expression was detected by confocal microscopy (b) and FACS analysis (c,d). For confocal analysis, the transfectants were fixed in ice-cold methanol 36 hours post-transfection and stained with Nok-1 antibody, which recognises the extracellular domain of FasL. The GFP and Nok-1 signals showed complete overlap; the images shown are from the Nok-1 signal. Unpermeabilised cells for FACS analysis were stained with Nok-1 as a measurement for membrane expression, and the total GFP signal was used as a measurement for total FasL. Double plots of GFP vs Nok-1 stain are shown (c). For each transfectant, the Nok-1 stain for cells expressing between 10 units and 150 units of GFP fluorescence (shown gated in c) was plotted (d). The percentage of cell surface FasL is shown in the top right-hand corner of the FACS plot.

View larger version (25K): [in a new window]   Fig. 3. A proline-rich region in the cytoplasmic tail of FasL is responsible for sorting to the secretory granules. FasLWT-GFP and FasLpro-GFP (lacking the proline-rich region, amino acids 45-74; Fig. 2a) were stably expressed in RBL cells and analysed by confocal microscopy (a) and FACS analysis (b) as described in Fig. 2.

View larger version (62K): [in a new window]   Fig. 4. The polyproline region of FasL can be modelled to bind the SH3 domain of Fyn. The Fyn SH3 domain is shown as a blue dot surface and the FasL peptide as gold van der Waals spheres. An area of negatively charged surface of the Fyn SH3 is illustrated in red (a). A magnified view of that area (b) shows that the Arg backbone (green) of the FasL peptide lies in proximity to the negatively charged area, enabling electrostatic interactions between the peptide Arg and the SH3 Asp. Also, the close fit of the peptide and SH3 are shown by the van der Waals and the SH3 dot surface.

View larger version (24K): [in a new window]   Fig. 5. The KKR or RR motif in the cytoplasmic tail of FasL contributes to the potential SH3-binding region. R45-R46 (RR-EE mutation) and K73-K74-R75 (KKR-EEE mutation) were mutated to EE or EEE, respectively, in the FasL-GFP construct, by site-directed mutagenesis, and were transiently expressed in RBL. The effects of these mutations on FasL surface expression was assayed by confocal (a) and FACS analysis (b) as previously described. The positive charge from the side chains of KKR and/or RR are thought to contribute to the binding of the proline-rich region of FasL to an SH3 domain.

View larger version (37K): [in a new window]   Fig. 6. NRK transfectants expressing CD63-GFP, FasLWT-GFP, FasLpro-GFP, and FasL74-GFP were incubated for 30 minutes on ice with the relevant primary antibody (anti-CD63 for cells expressing CD63-GFP and Nok-1 for cells expressing FasL-GFP (WT, pro and 74)) or an irrelevant isotype-matched control antibody (data not shown). The antibody was replaced with fresh media and the cells were returned to 37°C for 120 minutes, after which they were stained with the relevant Texas-Red-conjugated secondary antibody and analysed by confocal microscopy. The images have been merged (C,F,I,L); yellow signal demonstrates endocytosed antibody. No endocytosis was seen with the control antibody. Bars, 10 mm.

View larger version (44K): [in a new window]   Fig. 7. As described in Fig. 6, NRK transfectants expressing CD63-GFP, FasLWT-GFP, FasLpro-GFP, and FasL74-GFP were incubated for 30 minutes on ice with the relevant primary antibody (anti-CD63 for cells expressing CD63-GFP and Nok-1 for cells expressing FasL-GFP (WT, pro and 74)) or an irrelevant isotype-matched control antibody (data not shown). The antibody was replaced with fresh media and the cells were returned to 37°C for 0 minutes, 15 minutes, 30 minutes or 120 minutes, after which they were stained with the relevant PE-conjugated secondary antibody and analysed by FACS. The values were calculated as described in the Materials and Methods.

View larger version (45K): [in a new window]   Fig. 8. Deletion of the proline-rich region of FasL does not prevent internalisation to the lysosomal compartment in NRK. NRK transfectants expressing CD63-GFP (A-C) and FasLWT-GFP (D-F), FasLpro-GFP (G-I) and FasL74-GFP (J-L) were stained for the lysosomal compartment with an anti-Lgp-120 antibody (A,D,G,J). The images have been merged (C,F,I,L) to show co-localisation of intracellular CD63-GFP and FasL-GFP (WT, pro and 74) with the lysosomal compartment. Bars, 10 µm.

View larger version (57K): [in a new window]   Fig. 9. FasL is targeted directly to the secretory lysosomes in NK cells. FasL endocytosis was studied in the human NK cell line YT. To demonstrate expression of CD63 and FasL in YT, the cells were stained with anti-CD63 (B) or Nok-1 (E) and co-stained with anti-lamp-1 (A,D). C and F show the merge of the two signals, demonstrating co-localisation of CD63 and FasL with the granules. For endocytosis, YT cells were incubated with an anti-CD63 antibody (G-I), Nok-1 (J-L) or an isotype-matched control antibody (data not shown) in complete medium supplemented with the metalloprotease inhibitor BB3013 for 2 hours at 37°C. The cells were fixed in ice-cold methanol and stained with an anti-lamp antibody (G,J). The anti-lamp and endocytosed antibody signals were detected with the relevant Texas-Red- and FITC-conjugated secondary antibodies, respectively. (I,L) The merged signals demonstrate that most endocytosed antibody was located in the Lamp-positive compartment. All samples were analysed by confocal microcopy. Bars, 5 µm (A-F), 10 µm (G-I).

View larger version (23K): [in a new window]   Fig. 10. Model of FasL sorting pathways in cells with secretory lysosomes versus conventional lysosomes. FasL exits the biosynthetic pathway at the trans-Golgi network (TGN). In secretory cells, an SH3-domain-containing protein sorts FasL, via its proline-rich domain, to the secretory lysosome. The absence of this interaction in non-secretory cells results in sorting of FasL to the plasma membrane via the default exocytosis pathway. Delivery of FasL to the plasma membrane in haematopoietic secretory cells only occurs during activation (e.g. stimulation via the TcR).

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