**5. Conclusions: Lipids as sorting receptors**

54 Crosstalk and Integration of Membrane Trafficking Pathways

phospholipid across the membrane, and PLSCR1 is present on neutrophil secretory vesicle membranes (Frasch et al. 2004). Un-phosphorylated PtdIns can be translocated by a flippase, however, none of the translocase families have been tested for the ability to flip PtdInsPs.

Our data suggest that PtdIns(3,4)P2 may be present on the luminal face of the parotid granule membrane as a binding site for PSP, however, it is unclear how it would get there. Therefore, intact rat parotid secretory granules were incubated with fluorescent NBDtagged PtdInsP to measure flipping, according to (Natarajan and Graham 2006; Natarajan et al. 2009). In this method, added NBD-lipids integrate rapidly into the outer leaflet of the granule membrane on ice. After incubation at 37 oC to allow flipping, label remaining in the outer leaflet is quantitatively destroyed by addition of BSA and dithionite, however, NBDlipids which had translocated to the luminal leaflet are protected by the membrane. We found that incubation for 1 hour at 37 oC allowed 15% of integrated NBD-PtdIns(3,4)P2 to translocate to the protected inner leaflet (Fig. 10). Less than 2% of the PSP leaked from the granules after incubation at 37 oC for 1 h, indicating that the granules remained sealed

Unphosphorylated PtdIns is translocated by a flippase at a rate similar to phosphatidylcholine (Vishwakarma et al. 2005). This provides us with a benchmark for comparison with the extent of flipping of PtdInsPs. In our experiments, approximately 10% of PtdIns was flipped to the inner leaflet of parotid granules, whereas 15-18% of PtdIns(4)P, PtdIns(3,4)P2, or PtdIns(3,5)P2 was translocated (Fig. 10). Hence, the flippase activity is relatively non-selective, and the phosphate groups do not inhibit translocation. Parotid granule membranes support flipping of phosphorylated forms to a greater extent than PtdIns. Taken together, our results demonstrate that PtdIns(3,4)P2 is present in the granule membrane, and can flip to the inner face of the membrane. Further, the presence of PtdInsPs on the luminal leaflet of membranes raises the possibility that other intra-organelle proteins

Fig. 10. A. Parotid secretory granules translocate PtdIns(3,4)P2. Purified intact parotid secretory granules were incubated with fluorescent NBD-labeled PtdIns(3,4)P2 to measure translocation, as described (Natarajan and Graham 2006; Venkatesh et al. 2011). Data show the amount of lipid flipped to the inner leaflet. Data are Mean±SE of 3 experiments in triplicate. \*p<0.01 compared with 0h. B. Several PtdInsPs translocate parotid granule membranes. Data are the amount of flipped lipid at 0 or 1 hour. Data are Mean ± SE of 3-6

experiments in triplicate. \*p<0.05, \*\*\*p<0.01 compared to PtdIns.

during the assay.

may be localized by PtdInsP anchors.

As discussed above, a central aspect of selective trafficking is ensuring that the vesicle targeting information encoded by the granule coat matrix directs the choice of which cargo proteins fill the lumen of the granule. Current models for selective sorting all rely on interactions with a transmembrane protein to convey that information, directly or indirectly. Our results with parotid secretory granules suggest the possibility of a variant of this model. Rather than a transmembrane sorting receptor, translocation (flipping) of a rare lipid, PtdIns(3,4)P2, may convey the character of the coat matrix (Fig. 11).

Both PI3-kinase and PI4-kinase are present on the TGN or vesicles in several cell types, and create specific PtdInsPs in the outer leaflet of the membrane. In addition, lipid translocases, or flippase activity, has been reported on mammalian secretory granules. Our results demonstrate that PtdIns(3,4)P2 is present in parotid secretory granule membranes. We observe that PtdIns(3,4)P2 can translocate to the luminal bilayer of the granule membrane. Further, we find that PSP binds strongly to PtdIns(3,4)P2 in the membrane. Since the translocase likely is recruited and localized by the coat proteins, we suggest that the translocase may create a local region of higher concentration of PtdIns(3,4)P2 in the luminal leaflet within the forming vesicle bud, compared to other areas of the TGN. This may serve to localize PSP within the budding vesicle, thereby sorting it for secretion. The membranebound PSP may in turn act as a sorting chaperone for other cargo proteins. This hypothetical model has the advantage that it suggests specific interactions which can be tested for a role in sorting for secretion.

Fig. 11. A hypothetical model for how PtdIns(3,4)P2 could mediate sorting of PSP.
