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.

journal_name

Mol Biol Cell

authors

Han L,Jiang T,Han GA,Malintan NT,Xie L,Wang L,Tse FW,Gaisano HY,Collins BM,Meunier FA,Sugita S

doi

10.1091/mbc.e09-08-0712

subject

Has Abstract

pub_date

2009-12-01 00:00:00

pages

4962-75

issue

23

eissn

1059-1524

issn

1939-4586

pii

E09-08-0712

journal_volume

20

pub_type

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