4.4 Membrane Anchorage of SNAREs Potentiates the Power of αSNAP . 65
4.4.4 An αSNAP Mutant Lacking the Putative Membrane Interaction 72
The data collected so far made it tempting to speculate that a direct interaction between αSNAP and the membrane lipids is responsible for the increased effi-cacy on liposomes. If this were true, the lipid binding property might possible be mapped to a certain region of αSNAP. No crystal structure has been solved for αSNAP so far, but the structure of theαSNAP isoformγSNAP as well as the yeast αSNAP homolog Sec17 have been solved [30]. Based on the Sec17-structure and αSNAP/SNARE-complex interaction studies using various point-mutatedαSNAPs, a model of αSNAP bound to the SNARE complex [67] has been proposed by Marz et al.. Looking closely at the model one can observe that the very N-terminal 32 residues form an arm-like structure pointing away from the complex, the most far-out region of which includes a strand of mostly hydrophobic amino acids. If this region were the interaction site of αSNAP with the membrane, its deletion should abolish membrane binding and hence the higherαSNAP efficiency on membranes.
Marker Ni elution 1Ni beads Wash αSNAP
wt
Fraction 8 Fraction 1
1
Fraction 10
Fraction 9 Fraction 12Fraction 13Fraction 14Fraction 15Fraction 16
Figure 4.31: Purification of theαSNAP mutant ’αdelSNAP’. A sample after elution from the Ni column,αSNAP wildtype for size comparison as well as the elution fractions after anion-exchange chromatography are shown. Fraction 11 was used for all of the functional experiments.
A deletion mutant comprising aa 33-295 of αSNAP was hence cloned into the pET28a vector and subsequently expressed and purified. It will be referred to as
’αdelSNAP’ from now on. The purification of the His-tagged protein was carried out by Wolfgang Berning-Koch via Ni/NTA- followed by anion-exchange chromatogra-phy. It worked according to the standard protocol used for αSNAP 4.31.
First, I tested whetherαdelSNAP is able to disassemble SNARE complexes using the previously described FRET assay. Indeed, αdelSNAP is able to disassemble –
100 200 300 400 500 600 700 800
Figure 4.32: αdelSNAP efficiency is comparable on liposomes and in solution. Disassem-bly is shown∼70nM of FRET complexes (H3/SNAP25130OG/Sb28T R) either in solution or incorporated into liposomes via the TMD of Sb. At the start of the reaction the samples contained the amount ofαdel indicated. The reactions were triggered after 140s. 1,5µM of αdelSNAP were added twice more to the reactions represented in black and green, at 400s and 680s.
albeit at lower efficiency than αSNAP. At an αdelSNAP concentration of 1,5µM, disassembly was slow and only a third of the substrate was disassembled. In order to improve the rate of disassembly, moreαdelSNAP had to be added. 4,4µM succeeded to efficiently disassemble most of the complex (figure 4.32).
Next, to address the question of whether the membrane boost observed for wt-αSNAP in the previous sections is indeed abolished in the mutant, the kinetics of αdelSNAP mediated disassembly in solution and on liposomes were compared. As illustrated in figure 4.32, for all concentrations of αdelSNAP tested the kinetics in solution are identical to the kinetics on liposomes. This result indicates that the membrane mediated αSNAP-potentiation was indeed abolished by deletion of its N-terminal region.
To strengthen the finding that αdelSNAP does not differentiate between lipo-somal and soluble complexes with respect to disassembly efficiency, the anisotropy read-out was employed next andαdelSNAP directly compared to wildtypeαSNAP.
Since in the FRET-experiment it already became obvious that αdelSNAP is less effective than wildtype αSNAP in general, the extent of this difference was deter-mined at first. To do so, various amounts ofαdelSNAP andαSNAP were compared with respect to their ability to disassemble SNARE complexes in solution. As shown in figure 4.33, 3,6µM of αdelSNAP disassemble complexes with the same kinetics as 600nMαSNAP. Likewise, 1,8µM ofαdelSNAP are as efficient as 300nM αSNAP.
It can hence be concluded thatαdelSNAP is six times less efficient than αSNAP in solution (figure 4.33).
200 300 0,6
0,7 0,8 0,9 1,0 1,1
A/A0
α α α α
Figure 4.33: αdelSNAP is less efficient than αSNAP in solution. The fluorescence anisotropy changes upon disassembly of purified SNARE complexes (H3/SNAP25/Sb28T R) with either αSNAP or αdelSNAP as the NSF/SNARE-adaptor protein are shown. The signal at a given time is divided by the signal prior to the adaptor-addition (A/A0). The reaction was triggered after 120s as indicated.
Subsequently, the efficiencies ofαdelSNAP andαSNAP with respect to disassem-bly were compared on liposomes. First, I employed the concentrations ofαdelSNAP and αSNAP which led to comparable kinetics in solution. On liposomes, 600nM of αSNAP disassembled the SNARE complexes significantly faster than 3,6µM of αdelSNAP (figure 4.34). At lower αSNAP concentrations, which are more appro-priate for αSNAP disassembly on liposomes, the differences between αdelSNAP-and αSNAP-mediated kinetics at a ratio of 6:1 are even more pronounced: 280nM αdelSNAP do not permit any disassembly at all, whereas 45nM ofαSNAP mediate fast and complete disassembly (figure 4.35). Even after increasing the concentration ofαdelSNAP up to 2,3µM the rate of αdelSNAP mediated disassembly was slower than that of the wildtype reaction (data not shown). These results are in line with the outcome of the FRET assay, thatαdelSNAP does not preferentially disassemble on liposomes.
100 125 150 175 200 225 250 275 300 -0,2
0,0 0,2 0,4 0,6 0,8 1,0 1,2
parts of full disassembly
αα
Figure 4.34: The difference between αdelSNAP and αSNAP is far more pronounced on liposomes than in solution. Anisotropy changes during disassembly of∼70nM liposomal SNARE complexes (H3/SNAP25/Sb28T RTMD) at 3,6µM αdel or 600nM αSNAP in the presence of 5nM NSF. Disassembly was triggered at 130s by MgCl2. Anisotropy was normalized to parts of full disassembly to ease quantification.
125 150 175 200 225 250
0,70 0,75 0,80 0,85 0,90 0,95 1,00 1,05 1,10
1,15 αdel on liposomes (280nM)
αSNAP on liposomes (45nM)
A/A0
time / s
Figure 4.35: The low αdelSNAP efficacy on liposomes is even more obvious at low SNAP concentrations. Anisotropy traces of SNARE-complex disassembly (H3/SNAP25/Sb28T RTMD) at 280nM αdelSNAP or 45nM αSNAP in the presence of 5nM NSF. Disassembly was triggered at 125s by MgCl2.
Theoretically, from the findings we have so far, one can predict the factor by which wt-αSNAP should be more efficient than αdelSNAP during SNARE-complex
150 200 250 0,09
0,10 0,11 0,12 0,13 0,14 0,15 0,16
Anisotropy
αα
Figure 4.36: 45nM αSNAP disassemble liposomal SNARE complexes at a comparable speed as 3,6µM ofαdelSNAP. Anisotropy traces of liposomal SNARE-complex disassem-bly (H3/SNAP25/Sb28T RTMD) at 3,6µMαdel or 45nM αSNAP in the presence of 5nM NSF. Disassembly was triggered at 125s by MgCl2 as indicated.
disassembly on liposomes. We have seen in this section that αdelSNAP is approxi-mately six fold less efficient than αSNAP in solution, and in section 4.3.3 that the efficiency of wildtype αSNAP is increased by a factor of 10-20 in the presence of membranes. According to this, an efficacy factor in the range of 60-120 (roughly six times ten to twenty) would be expected on liposomes. Indeed, figure 4.36 illustrates that 45nMαSNAP disassemble liposomal SNARE complexes at similar speed as 3,6 µM ofαdelSNAP, indicating an 80-fold higher efficiency forαSNAP. These findings strongly suggest that the N-terminal arm-like structure observable in the crystal structure of αSNAP is essential for a protein/lipid-interaction, which mediates the potentiation of complex disassembly at low αSNAP concentrations.
4.4.5 Potentiation Through Membrane Anchorage – Gen-eral Feature of SNAPs or αSNAP-Specific Phenomenon?
The adaptor – αSNAP versus its brain specific isoform βSNAP
As described in the introduction, αSNAP is not the only adaptor protein of NSF.
Two more αSNAP isoforms are known, calledβ- andγSNAP, respectively. γSNAP is ubiquitously expressed like αSNAP, whereas βSNAP is a brain specific isoform.
Up to date there have been contradictory results about βSNAP function and its ability to disassemble SNARE complexes in particular and it yet remains unclear whether βSNAP functions as a positive or negative regulator or simply provides redundancy despite relatively low sequence homology [32, 33, 31, 34].
With the following experiments I sought to answer, whether βSNAP is capable of disassembling SNARE-complexes and if so, whether its properties differ from those ofαSNAP. For instance, it is as yet not known, whetherαSNAP has the same affinity for all three binding sites and whether all three binding sites need to be occupied to allow for disassembly. If the SNAP binding sites are not exchangeable there might be differences ofβ- andαSNAP affinities to any of the binding sites. If so, it is for instance feasible thatα- andβSNAP are more or less efficient in different concentration ranges, respectively and that mixing of both SNAP isoforms would lead to different kinetics than the sum of both isoforms alone. Furthermore, an interesting question would be, whether the membrane boost observed for αSNAP on liposomes is conserved between SNAP isoforms.
The properties of α- and βSNAP are similar in solution To first estab-lish, whetherβSNAP disassembles neuronal SNARE complexes in general, I purified recombinantβSNAP and used it for disassembly experiments exploiting the fluores-cence anisotropy.
As can be seen in figure 4.37, βSNAP was able to disassemble the SNARE com-plex. Like previously observed for αSNAP, the binding of βSNAP to the SNARE-complex results in an increase of fluorescence anisotropy. All experiments were also performed with αSNAP as a direct comparison. It should be noted that βSNAP turned out to be fragile during purification, making it impossible to estimate the true power ofβSNAP as compared toαSNAP. The following experiments are hence not intended to provide absolute numbers but only aim to investigate whether there are mechanistical differences between the SNAP isoforms.
As is shown in figure 4.37, 3,4µM βSNAP disassembled the complexes with the same kinetics as 600nMαSNAP. TheβSNAP preparation used was hence about four times less effective thanαSNAP at this concentration. To see, whether this difference is reproducible at much lower concentrations, a second experiment using only 600nM βSNAP and 150nM αSNAP was carried out. Again, the kinetics were comparable.
Further experiments were pursued using different β- and αSNAP concentrations, all of which lead to comparable kinetics of α- and βSNAP-containing reactions, when for times moreβSNAP preparation than αSNAP was used (data not shown).
Furthermore, mixing of limiting amountsαSNAP andβSNAP resulted in a reaction rate which would be expected for the sum of the two isoforms (data not shown). None of these solution-experiments hence pointed towards a different function ofβSNAP andαSNAP, rather suggesting that both isoforms are mechanistically exchangeable in solution. Whether the efficiencies of the isoforms differ, can not be judged from these experiments, as pointed out above.
0 200 400 600 800 1000 1200
400 600 800 1000 1200 1400 1600 1800
0,07
Figure 4.37: βSNAP is approximately four times less effective than αSNAP during SNARE-complex disassembly in solution. Anisotropy changes during disassembly of
∼70nM FRET-complex (Sb28T R/H3/SN AP25) at high adaptor-protein concentrations (as indicated in A) and low adaptor-protein concentrations (as indicated in B) with 6nM NSF are shown. The reactions were triggered by MgCl2 at 780s (A) or 650s (B).
The membrane boost observed for αSNAP is conserved for βSNAP To now address the question of whether the potentiation ofαSNAP efficiency by incor-poration of the complexes into liposomes is conserved for βSNAP, disassembly of the two SNAP isoform was compared on liposomes as well. In order to exclude that putative differences between liposomes and solution are caused by different qualities
of the preparations used, the same preparations of αSNAP and βSNAP as in the last experiment were used and reactions carried out on the same day. As illustrated in figure 4.38, 45nM ofαSNAP disassembled almost as efficiently as 160nMβSNAP.
The rates of disassembly in presence of α- or βSNAPs are thus comparable when about four times as much βSNAP as αSNAP was used like previously observed in solution. The fact that the efficiency-difference between the two SNAPs is the same on liposomes as in solution, indicates that the efficiency ofβSNAP on liposomes in-creases to the same extent as has earlier been determined for αSNAP. The positive effect of complex incorporation into liposomes hence is conserved amongst at least two of the SNAP isoforms, namely αSNAP and βSNAP.
0 100 200 300 400 500 600
200000 400000 600000 800000 1000000 1200000 1400000
Fluorescence 520nm cps / r
time / s
βSNAP 160nM αSNAP 45nM more βSNAP
more αSNAP
MgCl2
Figure 4.38: βSNAP versusαSNAP on liposomes. 160nMβSNAP or 45nMαSNAP were pre-incubated with ∼70nM FRET-complex (H3/SNAP25130OG/Sb28Alexa594TMD), NSF and ATP and the disassembly initiated at t= 180s by MgCl2.