4.5 How is the Reaction Being Regulated?
4.5.2 Does Phosphorylation of NSF Have an Impact on Function? . 84
The competition betweenαSNAP and Complexin discussed in the last section affects the interface between the SNAPs and the SNARE complex. An additional level of regulation is feasible at the interaction sites between the SNAPs and NSF. For example, phosphorylation of NSF at a tyrosine at position 83 has been reported to reduce NSF’s affinity for αSNAP [36]. This hypothesis is best to be tested under conditions which allow for the use of limiting amounts of αSNAP, making the fluorescence assays on liposomes ideally suited.
To this end, two NSF mutants were expressed and purified: First, a wildtype construct of NSF was expressed in anE. colistrain in which proteins can be in vitro-phosphorylated directly inside the bacteria during expression. The strain contains a second plasmid coding for a kinase whose expression is under control of a trypto-phane operon and can hence be induced independently. Expression of the kinase is triggered by changing the expression medium to a medium containing indole-acrylic-acid (IAA) as an inducing agent after three hours of NSF production. The kinase then phosphorylates NSF which can afterwards be purified according to the proto-cols used for unphosphorylated NSF. Phosphorylation was subsequently confirmed
by Western blotting (data not shown). Using this approach the degree of phos-phorylation can however not be determined. Therefore, we also employed a second approach to have a control which should behave like NSF phosphorylated to satura-tion: A phosphomimetic point-mutant (NSFY83E) of NSF was expressed, which was purified according to the protocols for wildtype NSF as well. During purification of NSFY83E it became evident that the monomeric peak was relatively higher, whereas the hexameric was relatively smaller compared to wildtype NSF preparations (data not shown). This may have a physiological background, but could as well simply be due to an increased fragility of the mutant under the conditions used during purification.
As shown in section 4.1.2, the phosphomimetic mutant indeed performed less efficient than wtNSF in the gel-based disassembly assay. However, as mentioned in section 4.1.2 as well, this approach does not allow to differentiate between incomplete disassembly and partial re-assembly. The interpretation of such a finding is thus more difficult than in the ’real-time’ fluorescence approach.
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Figure 4.44: NSFY83E efficiently disassembles liposomal complexes (H3/SNAP25130OG/Sb28T R) incorporated into liposomes via the Sb-TMD (A) or the H3-TMD (B), respectively. FRET complexes were disassembled using 60nMαSNAP and NSF or NSFY83E as indicated. The reaction was triggered by MgCl2 after 180s.
Consequently, both of these mutants were tested in the FRET assay, where they behaved similarly. Therefore, disassembly on liposomes is exemplified only for NSFY83E (figure 4.44). Liposomal SNARE complexes were incorporated either via the Sb- or the H3-transmembrane domain and afterwards disassembled by either wildtype NSF or NSFY83E. For both complexes the mutant performed slightly less efficient than the wildtype. In case of the Sb-TMD complex, disassembly was only slightly slowed down whereas for the H3-TMD complex, only half of the complex was disassembled by the mutant at all. Increasing the mutant’s concentration by 50%
anyhow led to disassembly comparable to, or in case of the H3-TMD complex even better than, the wildtype indicating that the difference was not very pronounced.
On liposomes, the differences between wildtype and phosphorylated NSF can be diminished by increasing the mutant’s concentration by 50%, a difference which
might as well result from reduced stability of the mutant. At this stage it should again be pointed out that NSF is known to be a fragile enzyme and it is difficult to compare the preparations of the wildtype and the point mutation quantitatively, since it can never be excluded that differences between the enzymes stem from buffer-ing conditions which, even though optimized for NSF as described in section 4.1.3, do not mimic the enzymes natural environment.
To next investigate, whether the reported phosphorylation-mediated defect of NSF function is dependent on the Habc domain of Syntaxin, the experiments were also performed with liposomal complexes containing full-length Syntaxin, again once incorporated via the Sb- and once via the Syntaxin-transmembrane domain. Again, using 1,5-fold the amount of mutant sufficed to disassemble at comparable speeds as the wildtype, indicating that the Sx Habc domain does not influence the reaction (see figure 4.45).
Figure 4.45: NSFY83E efficiently disassembles liposomal complexes containing full-length Syntaxin. Everything was carried out as in figure 4.44 except that complexes contained full-length Syntaxin (including the Habc domain) (Sx/SNAP25130OG/Sb28T R) (A) or the Sx-TMD (B), respectively.
To clarify, whether the disassembly differences observed in the gel-based assay only show up in solution, the FRET assay was carried out in solution next. As shown in figure 4.46, phosphorylated NSF once more disassembled slower and less efficiently than wildtype when used at identical concentrations. As opposed to the liposomal findings however, this deficiency could not be overcome by adding more phosphorylated NSF. Here, it is therefore less likely that the imperfect disassembly by pNSF is due to a lower concentration or stability. Due to the reported defect in αSNAP binding, I subsequently tested whether disassembly would improve in presence of more αSNAP. Indeed, the defect is almost completely abrogated by increasing the αSNAP concentration from 1,1µM to 2,2µM. Whether this is due to a reducedαSNAP affinity of NSFY83E in solution remains to be elucidated. Further experiments hence need to be performed in order to get further insights into whether the phosphorylation indeed leads to a mechanistical difference of the mutant or whether the mutant is simply less stable in my hands than the wildtype.
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F/F0 520nm
αα α
Figure 4.46: pNSF performance at 1,1µMαSNAP is less efficient in solution. Everything was carried out as in figure 4.44 except that soluble complexes and NSF andαSNAP as indicated were used. The reaction was triggered by MgCl2 after 130s. The Black arrows indicate further additions of pNSF after equilibrium has been reached.