Two distinct secretory vesicle–priming steps in adrenal chromaffin cells

Liu, Yuanyuan; Schirra, Claudia; Edelmann, L; Matti, Ulf; Rhee, Jeong−Seop; Hof, Detlef; Bruns, Dieter; Brose, Nils; Rieger, Heiko; Stevens, David R.; Rettig, Jens

doi:10.1083/jcb.201001164

Cited by 58 publications

(71 citation statements)

References 39 publications

(82 reference statements)

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“…For CAPS2d, we failed to generate a full-length protein, most likely due to the high GC content of the unique C terminus of this construct ( Figure S1). Since all CAPS2 splice variants are expressed in adrenal glands, we first tested their ability to rescue the exocytosis deficit in chromaffin cells derived from CAPS1/CAPS2 DKO mice (Liu et al, 2008;Liu et al, 2010). For this purpose, we performed Ca 2+ uncaging experiments and recorded LDCV fusion by measuring the resulting increase in membrane capacitance.…”

Section: Resultsmentioning

confidence: 99%

Secretory Vesicle Priming by CAPS Is Independent of Its SNARE-Binding MUN Domain

et al. 2014

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Priming of secretory vesicles is a prerequisite for their Ca(2+)-dependent fusion with the plasma membrane. The key vesicle priming proteins, Munc13s and CAPSs, are thought to mediate vesicle priming by regulating the conformation of the t-SNARE syntaxin, thereby facilitating SNARE complex assembly. Munc13s execute their priming function through their MUN domain. Given that the MUN domain of Ca(2+)-dependent activator protein for secretion (CAPS) also binds syntaxin, it was assumed that CAPSs prime vesicles through the same mechanism as Munc13s. We studied naturally occurring splice variants of CAPS2 in CAPS1/CAPS2-deficient cells and found that CAPS2 primes vesicles independently of its MUN domain. Instead, the pleckstrin homology domain of CAPS2 seemingly is essential for its priming function. Our findings indicate a priming mode for secretory vesicles. This process apparently requires membrane phospholipids, does not involve the binding or direct conformational regulation of syntaxin by MUN domains of CAPSs, and is therefore not redundant with Munc13 action.

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Section: Resultsmentioning

confidence: 99%

Secretory Vesicle Priming by CAPS Is Independent of Its SNARE-Binding MUN Domain

et al. 2014

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“…For successful vesicle priming, we therefore need the 3a domain of Munc18-1, N-terminal SNARE assembly (Walter et al, 2010), the very N-terminal end of syntaxin-1 (Shen et al, 2010;Zhou et al, 2013), a Munc13 protein (Ma et al, 2015), which in the case of chromaffin cells is ubMunc13-2 (Man et al, 2015). Finally, synaptotagmin-1 (Nagy et al, 2006) and calcium activator protein for secretion (CAPS) are required for the transition between the SRP and RRP state (Liu et al, 2010), although the relative importance of CAPS binding to PI(4,5)P 2 and SNAREs is currently under discussion (Khodthong et al, 2011; Nguyen Truong et al, 2014). Overall, the opening of syntaxin-1 within Munc18-1 by the extension of helix 12 appears to be the central event, which is regulated by other factors, including Munc13 proteins, in a fashion, which has yet to be unraveled.…”

Section: Discussionmentioning

confidence: 99%

Extension of Helix 12 in Munc18-1 Induces Vesicle Priming

Munch

Kedar

Weering

et al. 2016

Journal of Neuroscience

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Munc18-1 is essential for vesicle fusion and participates in the docking of large dense-core vesicles to the plasma membrane. Recent structural data suggest that conformational changes in the 12th helix of the Munc18-1 domain 3a within the Munc18-1:syntaxin complex result in an additional interaction with synaptobrevin-2/VAMP2 (vesicle-associated membrane protein 2), leading to SNARE complex formation. To test this hypothesis in living cells, we examined secretion from Munc18-1-null mouse adrenal chromaffin cells expressing Munc18-1 mutants designed to either perturb the extension of helix 12 (⌬324 -339), block its interaction with synaptobrevin-2 (L348R), or extend the helix to promote coil-coil interactions with other proteins (P335A). The mutants rescued vesicle docking and syntaxin-1 targeting to the plasma membrane, with the exception of P335A that only supported partial syntaxin-1 targeting. Disruptive mutations (L348R or ⌬324 -339) lowered the secretory amplitude by decreasing vesicle priming, whereas P335A markedly increased priming and secretory amplitude. The mutants displayed unchanged kinetics and Ca 2ϩ dependence of fusion, indicating that the mutations specifically affect the vesicle priming step. Mutation of a nearby tyrosine (Y337A), which interacts with closed syntaxin-1, mildly increased secretory amplitude. This correlated with results from an in vitro fusion assay probing the functions of Munc18-1, indicating an easier transition to the extended state in the mutant. Our findings support the notion that a conformational transition within the Munc18-1 domain 3a helix 12 leads to opening of a closed Munc18-1:syntaxin complex, followed by productive SNARE complex assembly and vesicle priming.

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“…Sapphire discs were frozen in a high-pressure freezer and subjected to freeze substitution and embedding as previously described (Liu et al, 2010). Ultrathin (70 nm) sections were cut parallel to the cell monolayer, collected on copper grids, stained with uranyl acetate and lead citrate, and analyzed on the Tecnai 12 Biotwin electron microscope (Philips, Eindhoven, The Netherlands).…”

Section: Electron Microscopymentioning

confidence: 99%

Differential effects of Sec61α-, Sec62- and Sec63-depletion on transport of polypeptides into the endoplasmic reticulum of mammalian cells

Lang

Benedix

Fedeles

et al. 2012

Journal of Cell Science

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SummaryCo-translational transport of polypeptides into the endoplasmic reticulum (ER) involves the Sec61 channel and additional components such as the ER lumenal Hsp70 BiP and its membrane-resident co-chaperone Sec63p in yeast. We investigated whether silencing the SEC61A1 gene in human cells affects co-and post-translational transport of presecretory proteins into the ER and post-translational membrane integration of tail-anchored proteins. Although silencing the SEC61A1 gene in HeLa cells inhibited co-and post-translational transport of signal-peptide-containing precursor proteins into the ER of semi-permeabilized cells, silencing the SEC61A1 gene did not affect transport of various types of tail-anchored protein. Furthermore, we demonstrated, with a similar knockdown approach, a precursor-specific involvement of mammalian Sec63 in the initial phase of co-translational protein transport into the ER. By contrast, silencing the SEC62 gene inhibited only post-translational transport of a signal-peptide-containing precursor protein.

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Two distinct secretory vesicle–priming steps in adrenal chromaffin cells

Abstract: The calcium-dependent activator proteins for secretion, CAPS1 and CAPS2, facilitate syntaxin opening during synaptic vesicle priming.

Cited by 58 publications

References 39 publications

Secretory Vesicle Priming by CAPS Is Independent of Its SNARE-Binding MUN Domain

Secretory Vesicle Priming by CAPS Is Independent of Its SNARE-Binding MUN Domain

Extension of Helix 12 in Munc18-1 Induces Vesicle Priming

Differential effects of Sec61α-, Sec62- and Sec63-depletion on transport of polypeptides into the endoplasmic reticulum of mammalian cells

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