Do the two binding sites on Sly1p work together for regulating SNARE assembly?

Inferred from the studies of Burkhardt et al., the two binding sites of the Munc18a/Syntaxin 1a pair collaborates during SNARE assembly (Burkhardt et al., 2008).

When both binding sites are available, Munc18a seem to inhibit SNARE complex formation, whereas, the block is relieved when N-peptide is removed. Similarly, when a double mutation (L165A/E166A) is introduced on the linker region of Syntaxin 1a to destabilize its closed conformation, the mutant protein could still bind Munc18a, but is not rendered inaccessible for SNARE assembly anymore (Burkhardt et al., 2008).

Therefore, depending on the binding status of Munc18a to the two distinct sites, the conformation of the Munc18a/Syntaxin 1a is altered such that the accessibility of the Syntaxin 1a SNARE motif is modulated. These findings suggest that Munc18a makes use of its both binding sites when regulating the neuronal SNARE assembly.

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If the two binding sites of the Sly1p/Sed5p pair collaborate in analogy to the Munc18a/Syntaxin 1a pair, regulation of SNARE assembly by Sly1p could be disturbed or altered upon mutating one of the two binding sites of Sed5p. In order to test this, the F10A mutant of Sed5p was used in the SNARE assembly assays in the presence or in the absence of Sly1p. It needs to be kept in mind that, the F10A mutation in yeast was demonstrated to have no effect on the transport kinetics of protein cargo between the ER and Golgi (Peng and Gallwitz, 2004). Hence, it would be important to know whether this mutation can severely affect the regulation of Sly1p on SNARE assembly or not. The F10A mutant was used in two types of SNARE assembly assays using either anisotropy or FRET approach (see Figure 3.23). Somewhat different results were obtained upon using the two different approaches. Using the anisotropy approach, only a slight decrease was observed in the promoting effect of Sly1p on SNARE assembly. However, a severe defect was observed using the FRET approach, very likely due to the overall lower SNARE protein concentrations used in the experiments, and thus due to a reduced likelihood for the formation of the QabR intermediate. One interpretation of these results could be that in conditions favoring the formation of the QabR intermediate, the role of the Sed5p N-peptide is perhaps less critical for regulation of SNARE assembly. Thus, perhaps it is not very surprising that the F10A mutant causes no trafficking defects in yeast, since intracellular conditions might favor formation of the QabR-intermediate through different factors, e.g., COPII coat or accessory proteins or lipids or spatial segregation of the SNAREs on membranes. Alternatively, there could be regulatory factors in yeast that are functionally redundant with the N-peptide.

Since it was observed using the FRET approach that, the F10A mutant perturbs the SNARE complex-promoting activity of Sly1p, one could suggest that the two binds sites of Sly1p collaborate during SNARE assembly. About 5 μM of the F10A mutant and about 7.5 μM of Sly1p were used in the FRET assays, indicating that Sly1p and the F10A mutant could interact in the experimental conditions based on their binding affinity (Kd ≈ 234 nM). Since the observed effect cannot be attributed to abolution of the Sly1p/Sed5p interaction, disruption of N-peptide binding must be involved. It is possible that when the N-peptide binding is weakened, Sly1p cannot that easily render the SNARE motif of Sed5p accessible. FRET approach might allow to observe this effect better compared to anisotropy approach, since assembly of the QabR-intermediate and thus, the SNARE complex, might be less efficient due to the low amount of the fluorescent R-SNARE (~ 50

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nM) used in the FRET experiments. Supporting the notion that N-peptide plays a critical role, the effect of Sly1p was completely abolished in the presence of ∆N-Sed5p, lacking the N-peptide, no matter whether the anisotropy or FRET approaches were used (see Figure 3.24). However, in this case, it cannot be ruled out that Sly1p and Sed5p interaction was not abolished. There was no detectable binding between Sly1p and ∆N-Sed5p using ITC, thus, it might be possible that Sly1p could not bind ∆N-Sed5p in experimental conditions and thereby could not orchestrate SNARE assembly.

In addition to the N-peptide, Sly1p interacts with the C-terminal part of Sed5p, possibly in a closed conformation. I wanted to introduce mutations in Sed5p to destabilize its closed conformation in order to observe whether these mutations would alter the SNARE complex-promoting activity of Sly1p. I designed mutations which are in analogy to the ones that were previously introduced in Sso1p (Munson et al., 2000). However, none of the mutations destabilized the closed conformation drastically as monitored by SNARE assembly assays. Then, I replaced the entire linker region with a flexible linker and generated the mutant protein Sed5p-linked in order to observe whether Sly1p requires the linker region to promote SNARE assembly. Interestingly, Sly1p effect on SNARE assembly remained unperturbed in the presence of Sed5p-linked, suggesting that Sly1p does not modulate the linker region during SNARE complex formation. Thus, it remains unclear how Sly1p could rearrange the structural configuration of Sed5p using the two binding sites. It is also possible that the coupling between the two binding pockets of Sly1p is not primarily required for rearranging the configuration of Sed5p, but is rather needed for modulating configuration and/or stability of the folding intermediate.

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Chapter 5