Grasas S.A

2.2.3.1 Preparation of pig brain cytosol for proteo-liposome recruitment

Pig brains were washed with recruitment buffer and then homogenised in an electric blender with an equal volume of recruitment buffer (w/v) containing the protease inhibitors aprotinin, E-64, PMSF and pepstatin at final concentrations of 3.2 µg/ml, 4 µg/ml 1 mM and 1 µg/ml respectively. The suspension was centrifuged at 2500 xg for 30 minutes at 4˚C. The supernatant was harvested and centrifuged at 100000 xg for 1 hour at 4˚C to remove endogenous membranes. The resulting supernatant was aliquoted and the samples were flash frozen in liquid nitrogen and stored at -80˚C.

2.2.3.2 Production of liposomes with coupled cytoplasmic receptor tails

Recombinant 6xHis MBP tagged cytoplasmic receptor tails were expressed in E.coli (Bl21DE3) and were purified as explained in 2.2.2.1. 500 µg of each receptor tail was digested with 5 µg of purified recombinant tobacco etch virus protease (TEV) (2.2.2.2) and 1 mM tris(2-carboxyethyl)phosphine (TCEP) overnight at 25˚C to remove the 6xHis MBP tag.

Liposomes were prepared from a mixture of the following lipids; PC, PE, PS, C, PE MCC and one of the phosphatidylinositol containing lipids (either PI, PI(3)P, PI(3,5)P2

or PI(4,5)P2) unless stated otherwise (2.1.2). The final amounts of each lipid were 200

nmol, 150 nmol, 50 nmol, 50 nmol, 50 nmol and 5 nmol respectively. The lipid mixture was dried under nitrogen gas and re-suspended in 0.65 ml of recruitment buffer. The re-suspended lipid film was subjected to 5 freeze/thaw cycles in liquid nitrogen and warm water. 100 µl of the liposome suspension was incubated with one TEV digested protein sample for 60 minutes in the dark at room temperature. The coupling of the cytoplasmic receptor tails to the liposomes is due to the formation of a sulphydryl bond between an extra cysteine residue at the N terminus of the receptor tail and the activated maleimide head group on the anchor lipid (PE-MCC) (Pocha et al., 2011).

The liposomes containing the coupled receptor tail will be referred to as proteo- liposomes. The proteo-liposomes were pelleted by centrifugation at 100000 xg for 20 minutes at 4˚C, re-suspended in 1 ml of 10 mM L-cysteine (in RB) and incubated for 15 minutes in the dark at room temperature. This step was important for quenching unreactive maleimide head groups, and for the creation of cysteine control samples. The sample was subjected to centrifugation (as above) the supernatant discarded and the pellet re-suspended in 100 µl of recruitment buffer.

2.2.3.3 Proteo-liposome recruitment experiments

Pig brain cytosol was centrifuged for 1 hour at 100000 xg and at 4˚C to remove any remaining endogenous membranes. The GTP analogue GTPγS (or GMPNP) was added to the cleared pig brain cytosol (unless stated otherwise) at a final concentration of 0.15 mM. 900 µl of cleared pig brain cytosol was added to 100 µl of proteo- liposomes and incubated for 30 minutes at 37˚C. Here the pig brain cytosol acts as a protein reservoir. Recruitment was also carried out using purified protein instead of the pig brain cytosol. Here GTPγS (or GMPpnp) was not added to the recruitment mixture. The proteo-liposomes were separated from the cytosol or purified recombinant protein using a sucrose cushion (2 ml 60% sucrose in RB and 8 ml 5% sucrose in RB) in a Beckman Coulter Optima L-100k ultracentrifuge (SW40 rotor) at 38000 rpm for 90 minutes at 4˚C. The interface between the two sucrose layers containing the proteo- liposomes was removed and re-suspended in 11 ml of RB and centrifuged in a Beckman Coulter Optima L-100k ultracentrifuge (SW40 rotor) at 38000 rpm for 1 hour at 4˚C. The supernatant was removed and the pellet re-suspended in 100 µl of 2x Laemmli buffer (unless stated otherwise). The samples were analysed by SDS-PAGE and western blotting for interacting proteins.

2.2.3.4 Flow cytometry analysis of liposomes

Liposomes were produced as explained in 2.2.3.2 except they also contained 2 mol% of the fluorescent lipophilic dye DiI, and the liposomes were re-suspended in 1 ml instead of 100 µl. Six sets of liposomes were prepared containing 66 µg, 164 µg, 329 µg, 657 µg, 1350 µg and 2694 µg of coupled GFP. These liposomes were analysed using flow cytometry in a Beckman Coulter Cell Lab Quanta SC flow cytometer. GFP was excited at 498 nm and DiI at 549 nm and 10000 events were counted.

2.2.3.5 Pull downs with purified His-Vac14

20 µg of purified recombinant, MBP-AICD, MBP-Tr1, MBP-Tr2, MBP-Tr3, MBP-Tr4 and MBP (negative control) were incubated with 10 µg of purified HIS-Vac14 for 1 hour rocking on ice. The volumes were adjusted to 200 µl with wash buffer. The samples were centrifuged in a bench top centrifuge at maximum speed for 5 minutes to remove any aggregates. The supernatant was transferred to a tube containing 20 µl of amylose resin beads (New England BioLabs) which had been washed 3 x in wash buffer. The samples were incubated rocking on ice for 1 hour and the amylose beads washed 5 times with wash buffer. The proteins were eluted by incubating the beads with 50 µl of PD elution buffer for 10 minutes on ice. The samples were centrifuged at 2000 rpm in a bench top centrifuge for 1 minute at 4˚C. The supernatant was transferred to a new tube containing 12.5 µl of 4 x Laemmli buffer. The sample was heated at 95˚C for 5 minutes before being subjected to SDS-PAGE and western blotting. 25 µl of each pull down sample was loaded on an 8% acrylamide gel.

2.2.3.6 Clathrin recruitment assay

Clathrin recruitment assays were based on those used in Kelly et al. (2014). Liposomes were created comprising 10% cholesterol, 5% PE-MCC, 5% PI(4,5)P2

in 182 µg of TEV digested purified receptor tail (AICD and Crbs2) or cysteine (negative control). The liposomes were centrifuged at 50000 rpm for 10 minutes at 4ºC in a (Beckman Coulter Optima TLX Ultracentrifuge) and the pellet re-suspended in 1 ml of 10 mM cysteine in 1 x HKM buffer to saturate any free anchor lipid. The liposomes were centrifuged again and re-suspended in 1 ml of 1 x HKM buffer to obtain a final lipid concentration of 0.4 mg/ml. Purified AP-2 (2.2.2.6) was added at a final concentration of 0.8 µM to 50 µl of the liposomes and incubated for 30 minutes on ice. The liposomes were centrifuged at 50000 rpm for 10 minutes at 4ºC in a (Beckman Coulter Optima TLX Ultracentrifuge) the supernatant removed and the pellet re- suspended in 50 µl of 1 x HKM buffer. Purified clathrin (2.2.2.5) previously dialysed against de-polymerisation buffer to disassembly the clathrin cages was added to the liposomes at a final concentration of 0.2 µM and incubated for 30 minutes on ice and then 15 minutes at 37ºC. The sample was centrifuged at 50000 rpm for 10 minutes at 4ºC in a (Beckman Coulter Optima TLX Ultracentrifuge) and the pellet and supernatant adjusted with 2 x Laemmli buffer. The samples were subjected to analysis by western blotting and SDS-PAGE stained with Coomassie.