To investigate the sorting signal requirements of membrane proteins for targeting to lysosome-related organelles, it is crucial to establish whether there is an
additional population of conventional lysosomes in the same cell. This is pertinent since if secretory lysosomes are the sole lysosomal population of the cell, then conventional lysosomal sorting signals would be sufficient for the sorting of
membrane proteins, whether “secretory” or “lysosomal” in nature. This hypothesis is supported by the localisation of resident secretory lysosomal proteins to
conventional lysosomes when heterologously expressed (Green et al. 1994, Marks et al. 1995, Blagoveshchenskaya et al. 1998a, Simmen et al. 1999, Calvo et al.
1999). This is in contrast with the data from the Griffiths laboratory, who provide evidence that Fas ligand targeting to secretory lysosomes (also termed lytic granules) of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells utilises sorting signals that are insufficient for sorting to conventional lysosomes of other cell types (Bossi and Griffiths 1999, Blott et al. 2001). This suggests that although lytic granules of CTL and NK cells are the sole lysosomal population, different sorting signals that are specific to secretory lysosomes are used. The requirement for separate sorting mechanisms to conventional lysosomes and secretory
lysosomes becomes more apparent in cell types that possess both types of organelle. An example of this scenario is in melanocytes, where melanosomes coexist with lysosomes, but certain lysosomal membrane proteins are either shared or segregated between the two populations (Raposo et al. 2001).
In this chapter, Rbl-2H3 cells have been used to investigate the targeting requirements for localisation to secretory lysosomes that constitute the only lysosomal population of the cell. Rbl-2H3 cells contain secretory lysosomes that possess both ‘lysosomal’ and ‘granule’ markers, which are synchronously released upon activation (Bonifacino et al. 1986, Suarez-Quian 1987, Bonifacino et al. 1989, Xu et al. 1998a, Dragonetti et al. 2000). To investigate whether lysosomal targeting signals are sufficient for secretory lysosome localisation in Rbl-2H3 cells, I have
used the membrane protein P-selectin. P-selectin resides in the membranes of the lysosome-related organelles, dense granules of platelets (Israels et al. 1992) and WPB of endothelial cells (Bonfanti et al. 1989, McEver et al. 1989, Johnston et al.
1989). Upon physiological stimulation of both platelets and endothelial cells, P- selectin is rapidly mobilised to the cell surface (Hattori et al. 1989) and then either internalised or shed by proteolysis (Michelson et al. 1996, Berger et al. 1998, Hartwell et al. 1998). Following internalisation, P-selectin is either recycled back to WPB or targeted to lysosomes (Subramaniam et al. 1993, Arribas and Cutler
2000). The 35 amino acid cytoplasmic tail of P-selectin is necessary for targeting to WPB and heterologous expression studies of P-selectin have allowed for definition of granule, lysosomal and synaptic-like microvesicle (SLMV) targeting signals (see Figure 1.4 from Chapter One). The defined sorting signals of P-selectin have been utilised to investigate two questions: firstly, is the cytoplasmic tail of P-selectin sufficient for delivery to secretory lysosomes of Rbl-2H3 cells, and secondly, if so, then does delivery rely on lysosomal, granule or SLMV targeting signals?
3.2 Enrichment for the secretory Ivsosome
Since morphological colocalisation of Rbl-2H3 endogenous markers such as the granule membrane protein 5G10 (Bonifacino et al. 1989), serotonin, Igp120 and P- hexosaminidase has been reported within a single population of organelles (Xu et al. 1998a, Dragonetti et al. 2000) I expected to find a single peak containing these markers by fractionation. However, evidence also suggests that Rbl-2H3 cells contain a conventional lysosome population (Baram et al. 1999). This conventional lysosome pool was identified by the co-distribution of p-hexosaminidase with pro- cathepsin D (53kDa), a precursor usually associated with the Golgi in Rbl-2H3 cells (Baldassarre et al. 2000, Dragonetti et al. 2000). This disparity was investigated by using a similar subcellular fractionation method to that used by Baram et al. To mark the twin characteristics of secretory lysosomes, I chose to assay the endogenous activity of the lysosomal enzyme P-hexosaminidase, and monitor serotonin-containing secretory compartments with [^H]-serotonin (5-
I first established that [^H]-serotonin was taken up specifically into Rbl-2H3 cells using the inhibitor reserpine, which blocks the vesicular uptake of catecholamines and serotonin. Figure 3.1 shows the levels of [^H]-serotonin and p-hexosaminidase activity in cells treated without (left) and with (right) reserpine. The uptake of [^H]- serotonin is completely inhibited in the presence of reserpine. I used 10 times the concentration of [^H]-serotonin than was used in subsequent experiments, to establish an upper limit to the concentration of [^H]-serotonin. The overnight treatment of cells with reserpine does not cause the death of cells, as levels of p- hexosaminidase activity are comparable in cells treated with and without this inhibitor.
Sucrose equilibrium gradients were used to determine the distribution of a number of different organelle markers of Rbl-2H3 cells. The identification of these
organelles by subcellular fractionation would enable the determination of the localisation of HRP-P-selectin and mutations in the cytoplasmic tail that may affect targeting to secretory lysosomes. In addition to marking secretory lysosomes (p- hexosaminidase activity and [^H]-serotonin), Rbl cells were also incubated with [^^^l]-transferrin under various conditions to identify endosomal compartments and the plasma membrane. Holotransferrin binds to its receptor at the plasma
membrane, and is rapidly internalised and delivered to endosomes (Marsh et al. 1986, Hopkins et al. 1990, Ghosh et al. 1994). An equilibrium between the influx and efflux of f ^^l]-transferrin into endosomes is thought to be reached after about 45 minutes incubation at 37°C (Knight 2002). The incubation of cells with [^^^1]- transferrin labels both the plasma membrane and internal endosomal stores of transferrin. To distinguish [^^®l]-transferrin at the plasma membrane from that of internal stores, either f ^®l]-transferrin bound at the cell surface can be removed by cell surface stripping, which results in the loss of plasma membrane labelling, or [^^^l]-transferrin can be labelled at the plasma membrane alone, by incubation of cells at 4°C.