Molecular insights into the mechanisms underlying the pleiotropic functions of Munc18-1 in dense-core vesicle exocytosis

Exocytosis refers to a cellular process in which an intracellular vesicle fuses its membrane with the plasma membrane to release its contents to the extracellular space. This process underlies diverse physiological processes such as communication between neurons, hormonal regulation, and immune responses. Munc18-1 is an indispensible protein that regulates exocytosis during neuronal communication and hormonal signaling. It is known to specifically interact with one or more SNARE proteins (syntaxin-1, SNAP-25, and synaptobrevin-2) through diverse binding modes. Multiple lines of evidence suggest the pleiotropic functions of Munc18-1 although they are sometimes contradictory: as a molecular chaperone of syntaxin-1, in priming of dense-core vesicles (DCVs) to fusion competent state, and in docking of DCVs to the plasma membrane. Munc18-1 consists of multiple domains: domain-1, -2, -3a, and -3b. However, the molecular mechanisms underlying the functions of Munc18-1 in exocytosis remain unclear. It was hypothesized that the multiple roles of Munc18-1 occur through its distinctive binding modes with SNARE proteins, which in turn are mediated by its specific domains. The structure and interactive properties underlying its essential roles were investigated through detailed phenotypic analyses of various domain-specific Munc18-1 mutants upon re-expression in Munc18-1/-2 double knockdown PC12 cells through lentivirus mediated infection. The results demonstrate that domain-1 and domain-3a of Munc18-1 are critical for a high affinity binary interaction with the closed form of syntaxin-1, which is crucial for syntaxin-1 trafficking and consequently in DCV docking and secretion. Furthermore, domain-3a of Munc18-1 additionally plays a crucial role in priming exocytosis by regulating SNARE complex interaction, which occurs independently from its role in syntaxin-1 stabilization and trafficking. Taken together, we postulate that Munc18-1 sequentially adopts distinctive binding modes at different stage of exocytosis that enable it to regulate each step through highly specialized mechanisms. The findings from this study provide novel insights into the obscure mechanisms underlying the functions of Munc18-1 in regulated exocytosis.Ph

The Hippo pathway kinases LATS1 and LATS2 attenuate cellular responses to heavy metals through phosphorylating MTF1.

Heavy metals are both integral parts of cells and environmental toxicants, and their deregulation is associated with severe cellular dysfunction and various diseases. Here we show that the Hippo pathway plays a critical role in regulating heavy metal homeostasis. Hippo signalling deficiency promotes the transcription of heavy metal response genes and protects cells from heavy metal-induced toxicity, a process independent of its classic downstream effectors YAP and TAZ. Mechanistically, the Hippo pathway kinase LATS phosphorylates and inhibits MTF1, an essential transcription factor in the heavy metal response, resulting in the loss of heavy metal response gene transcription and cellular protection. Moreover, LATS activity is inhibited following heavy metal treatment, where accumulated zinc directly binds and inhibits LATS. Together, our study reveals an interplay between the Hippo pathway and heavy metals, providing insights into this growth-related pathway in tissue homeostasis and stress response

Munc18-1 domain-1 controls vesicle docking and secretion by interacting with syntaxin-1 and chaperoning it to the plasma membrane

Striking correlations exist between the abilities of domain-1 cleft mutants of Munc18-1 to bind and chaperone syntaxin-1 and their ability to restore vesicle docking and secretion

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

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