Rescue of Munc18-1 and -2 Double Knockdown Reveals the Essential Functions of Interaction between Munc18 and Closed Syntaxin in PC12 Cells

Abstract

Munc18-1 binds to syntaxin-1A via two distinct sites referred to as the "closed" conformation and N terminus binding. The latter has been shown to stimulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis, whereas the former is believed to be inhibitory or dispensable. To precisely define the contributions of each binding mode, we have engineered Munc18-1/-2 double knockdown neurosecretory cells and show that not only syntaxin-1A and -1B but also syntaxin-2 and -3 are significantly reduced as a result of Munc18-1 and -2 knockdown. Syntaxin-1 was mislocalized and the regulated secretion was abolished. We next examined the abilities of Munc18-1 mutants to rescue the defective phenotypes. Mutation (K46E/E59K) of Munc18-1 that selectively prevents binding to closed syntaxin-1 was unable to restore syntaxin-1 expression, localization, or secretion. In contrast, mutations (F115E/ E132A) of Munc18-1 that selectively impair binding to the syntaxin-1 N terminus could still rescue the defective phenotypes. Our results indicate that Munc18-1 and -2 act in concert to support the expression of a broad range of syntaxins and to deliver syntaxin-1 to the plasma membrane. Our studies also indicate that the binding to the closed conformation of syntaxin is essential for Munc18-1 stimulatory action, whereas the binding to syntaxin N terminus plays a more limited role in neurosecretory cells.

Crucial role of the hydrophobic pocket region of Munc18 protein in mast cell degranulation

“…We previously found that Munc18-1/2 DKD causes not only a strong reduction in syntaxin-1 level but also perturbation of its plasmalemmal localization in PC12 cells (15,16). We therefore examined whether the subcellular localization of syntaxin-11 can be significantly altered by Munc18-1/2 DKD in RBL-2H3 cells ( Fig.…”

“…The lack of rescue by the hydrophobic pocket mutants in Munc18-2 is surprising, as we previously found that the expressions of Munc18-1 with the identical mutations showed 70-80% of the rescue ability compared with the wild type, using Munc18-1 KD and Munc18-1/2 DKD PC12 cells (15,31). Furthermore, the same (F115E) and a similar (L130K) mutant have shown a complete rescue ability in autaptic neurotransmission of Munc18-1-deficient neurons (32).…”

“…The Munc18 family of proteins has been revealed to play multiple functions, which include both chaperoning cognate syntaxins (14)(15)(16)(17)(18)(19) and priming/stimulating membrane fusion through interaction with the SNARE complex (20-23) (reviewed in ref. 24).…”

“…The chaperoning function of Munc18 contributes to stabilizing the expression level of cognate syntaxins and, in some cases, trafficking them to the appropriate intracellular compartments. For instance, in Munc18-1-deficient neurons, the syntaxin-1 level is reduced by 50-75% (3), whereas in Munc18-1 single-knockdown (KD) and Munc18-1/2 double-knockdown (DKD) pheochromocytoma (PC) 12 cells, not only the reduction of syntaxin-1 expression level but also its localization at the plasma membrane are severely perturbed (14,15). In some patients with FHL5 whose functional Munc18-2 protein is absent, a strong reduction in syntaxin-11 level was found in their immune cells (11,12).…”

“…In contrast, the Munc18-1 hydrophobic pocket has been suggested to play limited or dispensable roles in exocytosis. For example, in the rescue experiments of Munc18-1 single-KD and Munc18-1/2 DKD PC12 cells as well as Munc18-1 knockout neurons, various mutants (F115E, E132A, F115E/ E132A, and L130K) in the hydrophobic pocket region of Munc18-1 have exhibited either very mild or no impairment in their ability to rescue exocytosis in comparison with the wild type (15,31,32), leaving the function of the hydrophobic pocket region of Munc18 unclear.…”