A pivotal role for Pro335 in balancing the dual functions of Munc18-1 domain-3a in regulated exocytosis [Neurobiology]

October 17th, 2014 by Han, G. A., Park, S., Bin, N.-R., Jung, C. H., Kim, B., Chandrasegaram, P., Matsuda, M., Riadi, I., Han, L., Sugita, S.

Munc18-1 plays essential dual roles in exocytosis: (i) stabilizing and trafficking the central SNARE protein, syntaxin-1 (i.e., chaperoning function) by its domain-1 and (ii) priming/stimulating exocytosis by its domain-3a. Here, we examine whether or not domain-3a also plays a significant role in the chaperoning of syntaxin-1 and, if so, how these dual functions of domain-3a are regulated. We demonstrate that introduction of quintuple mutations (K332E/K333E/P335A/Q336A/Y337L) in domain-3a of Munc18-1 abolishes its ability to bind syntaxin-1, fails to rescue the level and trafficking of syntaxin-1 as well as to restore exocytosis in Munc18-1/2 double knockdown cells. By contrast, a quadruple mutant (K332E/K333E/Q336A/Y337L) sparing Pro335 residue retains all of these capabilities. A single point mutant of P335A reduces the ability to bind syntaxin-1 and rescue syntaxin-1 levels. Nonetheless it surprisingly outperforms the wild-type in the rescue of exocytosis. However, when additional mutations in the neighboring residues are combined with P335A mutation (K332E/K333E/P335A, P335A/Q336A/Y337L), the ability of the Munc18-1 variants to chaperone syntaxin-1 and to rescue exocytosis is strongly impaired. Our results indicate that residues from Lys332 to Tyr337 of domain-3a are intimately tied to the chaperoning function of Munc18-1. We also propose that Pro335 plays a pivotal role in regulating the balance between the dual functions of domain-3a. The hinged conformation of the α-helix containing Pro335 promotes the syntaxin-1 chaperoning function whereas the P335A mutation promotes its priming function by facilitating the α-helix to adopt an extended conformation.