Optimization of lipid to protein ratio

To assess the effect of protein concentration in liposomes, I prepared Ubc6_A488, t-SNARE liposomes and Syb liposomes lacking Doa10 and incubated them to allow for SNARE-mediated fusion. I then measured the A488 fluorescence upon addition of anti-A488 antibody (anti-A488). If liposomes are stable, the antibody should only bind to wrongly oriented Ubc6_A488 and therefore quench about 50% of the total fluo-rescence. However, when a lipid:t-SNARE ratio of 400 is used (dashed black lines), the fluorescence decreases quickly to 45% but subsequently decreases further (Figure 4.1A and 4.1B). This further slow decrease could be due to antibody slowly accessing the liposome lumen and thus indicates liposome instability. Slight instability can be also observed when fusion is inhibited (Figure 4.1C and 4.1D). In contrast, at lower SNARE concentrations, the signal stays stable over 30 min. At a lipid:t-SNARE ratio of 1000 or 2000 (dashed blue and red lines), the signal stays stable at both tested lipid:Syb ratios (1000 and 2000) (Figure 4.1).

To test if these SNARE concentrations still ensure high co-reconstitution efficiency after fusion, I fused Ubc6_A488, t-SNARE liposomes with Doa10, Syb liposomes and again measured the A488 fluorescence upon anti-A488 addition. As Doa10-mediated re-lease is a readout for co-reconstitution of Ubc6 with Doa10, I tested for Doa10-mediated release at different SNARE-concentrations. Whereas a lipid:t-SNARE concentration of 2000 compromises the release efficiency (red solid lines), a lipid:t-SNARE concen-tration of 1000 leads to complete release efficiency at both tested Syb concenconcen-trations (blue solid lines), indicating co-reconstitution (Figure 4.1). SNARE concentrations

that led to complete release efficiency in the presence of Doa10 and no instability in the absence of Doa10 were chosen for all experiments (lipid:tSNARE 1000, lipid:Syb 2000). Importantly, the accessibility of anti-A488 to Ubc6_A488in the presence of Doa10 is not due to leakage but due the exposure of the carboxy-terminus of Ubc6 to the out-side, as the signal stays stable when a control protein is co-reconstituted with Doa10 (Ubc6_SybTM-A488, see Chapter 3, Figure 3.6C and 3.6D).

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lipid : Syb 2000 lipid : Syb 1000

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Figure 4.1: Titration of SNARE-concentration for SNARE-mediated fusion assay. Liposomes containing Ubc6A488and t-SNARE were incubated with Syb liposomes containing (solid) or lacking (dashed lines) Doa10 (lipid:Doa10 ra-tio of 2000) to allow for SNARE-mediated fusion. The A488 fluorescence upon incubation with anti-A488 antibody (+ab) was measured. Different lipid:protein ratios for reconstitution of SNAREs were tested. Graphs show results for recon-stitution of Syb using a lipid:protein ratio of 2000 (A, C) and 1000 (B, D).

Lipid:t-SNARE ratios of 400 (black), 1000 (blue) and 2000 (red) were tested.

Where indicated, fusion was inhibited with a soluble Syb fragment (C, D). Sam-ples without Doa10 are indicated with a dashed line. Where indicated, detergent was added (+det) to solubilize the liposomes.

These results show that the concentration of reconstituted proteins influences the stability of liposomes. Doa10 contains a large TM domain containing 14 TM segments.

The presence of Doa10 could therefore also affect the stability of liposomes due to its transmembrane domain. I tested next the effect of the presence of multiple

transmem-brane segments on liposome stability using the fluorescence-quenching assay again.

To be able to distinguish between liposome instability and release of Ubc6, a multi-pass protein was used as a control protein which does not interact with Ubc6. TF_oF₁ ATP synthase is a multi-subunit complex that consists of a soluble F₁ region (subunits α₃β₃γδ) and a membrane anchored F_o region (subunits ab₂c₁₀). The c-subunits form a ring consisting of 20 TM segments (Guo et al., 2019). I therefore chose the ATP synthase as a suitable multipass TM control protein to test the effect of the presence of multiple transmembrane segments on liposome stability.

I purified F_oF₁ ATP synthase from thermophilic Bacillus sp. PS3 (TF_oF₁) as described previously (Suzuki et al., 2002; Schenck et al., 2009), (Figure S1). ATP synthase was subsequently co-reconstituted with Syb into liposomes. ATP synthase and Syb co-floated in a Nycodenz step gradient indicating that they were both reconstituted into liposomes (see Figure 4.2).

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Figure 4.2: Co-reconstitution of TF_oF₁ ATP synthase with Syb into liposomes. Liposomes containing TFoF1 ATP synthase and Syb were floated in a Nycodenz step gradient and the flotation fractions analyzed by SDS-PAGE and Coomassie blue staining. Low (left) and high (right) exposure of the Coomassie staining are shown.

As observed for SNAREs, high concentrations of ATP synthase led to instability after fusion as measured again by fluorescence quenching upon anti-A488 addition (Fig-ure 4.3). However, when ATP synthase is reconstituted at a lipid:protein ratio of 8000, the A488 signal stays stable over time after an initial fast quenching of immediately accessible Ubc6_A488 indicating stable liposomes. When Doa10 is reconstituted with the same lipid:protein ratio (8000), Ubc6A488is fully accessible over time to anti-A488.

Assuming that the distribution of Doa10 and ATP synthase in liposomes is

compara-ble at the same lipid:protein ratio, this shows that liposome instability can be avoided without compromising co-reconstitution efficiency. Concluding, a lipid:protein ratio of 8000 is therefore optimal for reconstitution of multipass TM proteins like Doa10.

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Figure 4.3: Titration of ATP synthase and Doa10 for SNARE-mediated fusion assay. Different lipid:protein ratios (8000, 4000 and 2000) were used for reconstitution of Doa10 or ATP synthase into liposomes to test the influence of protein concentration on liposome stability. ATP synthase (A) or Doa10 (B) were co-reconstituted with Syb and those liposomes fused with liposomes con-taining Ubc6_A488 and t-SNARE. The A488 fluorescence upon incubation with anti-A488 antibody (+ab) was measured as a readout for liposome stability and co-reconstitution efficiency. Where indicated, detergent (+det) was added to sol-ubilize the liposomes.)