sign in sign up
<<
Home , SNARE (protein), Vesicle fusion

The Journal of Neuroscience, August 19, 2015 • 35(33):11459–11461 • 11459

Journal Club

Editor’s Note: These short, critical reviews of recent papers in the Journal, written exclusively by graduate students or postdoctoral fellows, are intended to summarize the important findings of the paper and provide additional insight and commentary. For more information on the format and purpose of the Journal Club, please see http://www.jneurosci.org/misc/ifa_features.shtml.

Do SNARE Isoforms Determine Fusion Pore Characteristics?

X Linda van Keimpema1,2 and XTim Kroon3 1Sylics (Synaptologics B.V.), 1008 BA, Amsterdam, The Netherlands, and Departments of 2Functional Genomics and 3Integrative Neurophysiology, Center for Neurogenomics and Cognitive Research, VU University, 1081 HV, Amsterdam, The Netherlands Review of Chang et al.

Membrane fusion is a crucial element of and Fasshauer, 2012; Rizo and Su¨dhof, colleagues (2015) investigates the involve- many cellular processes, including the 2012). ment of the 2 transmem- release of from syn- One essential component of mem- brane domain in the fusion pore. In this aptic vesicles in and the release brane fusion is the formation of the fusion study, the authors used amperometry to of neuropeptides, neurotrophic factors, pore, an intermediate, temporary struc- measure the release of catecholamines and hormones from dense core vesicles ture that bridges the vesicular and plasma from DCVs. of a single DCV re- (DCVs) in brain and neuroendocrine membranes. The composition of this pore sults in a spike of current upon membrane cells. Vesicle secretion is mediated by the is currently under debate. One hypothesis fusion. However, just before this spike, formation of the soluble N-ethylmalei- is that the fusion pore is composed purely small amounts of cargo are secreted mide-sensitive factor (NSF) attachment of lipids of both membranes. According through the fusion pore. This shows up in protein receptor (SNARE) complex. to this hypothesis, the SNARE complex amperometric measurements as a pre- This SNARE complex comprises vesicle- accelerates pore formation by bringing spike foot (PSF) (Jackson and Chapman, associated (VAMP), the membranes in close proximity. An- 2006). For their experiments, Chang et al. , and synaptosomal-associated pro- other model proposes a proteinaceous fu- (2015) used chromaffin cells from synap- tein (SNAP). Different isoforms of these sion pore, in which the transmembrane tobrevin 2 and cellubrevin (VAMP2 and are involved in membrane fusion domains of the SNARE proteins are incor- VAMP3, respectively) double knock-out at different sites, with the canonical porated into the pore (Jackson and Chap- (DKO) mice, in which DCV fusion is al- SNARE complex of synaptic vesicles con- man, 2008; Fang and Lindau, 2014). The most completely abolished. Similar to the sisting of synaptobrevin 2, syntaxin 1, and latter hypothesis is supported by previous study by Han et al. (2004), 21 mutants of SNAP25 (Kasai et al., 2012). Both synap- work by Han et al. (2004), who showed synaptobrevin 2—each with a single tobrevin 2 and syntaxin 1 contain a that replacing any of three specific amino amino acid replaced with tryptophan in ␣ membrane-spanning -helix—the trans- acids of the syntaxin 1 transmembrane the transmembrane domain (positions membrane domain—that spans the vesi- domain with the large amino acid trypto- 96–116)—were individually expressed in cle membrane and the plasma membrane, phan altered flux through the fusion pore. the DKO cells. Expression of any of these respectively. In addition, these proteins These amino acids are located at the same constructs rescued DCV release frequency ␣ contain an -helical SNARE motif, while face of the ␣-helix, suggesting that this to wild-type levels (eight times higher ␣ SNAP25 contains two. These -helices ␣-helical face lines the fusion pore. These than DKO). In addition, four of these can form a tight bundle, which brings the data led the authors to propose a model in mutants (L99W, V101W, C103W, and vesicular and plasma membranes into which the fusion pore consists of a circu- I105W) showed reduced PSF amplitude. close contact. Subsequently, the mem- lar arrangement of five to eight syntaxin 1 Replacement of these amino acids with branes fuse and cargo is released (Jahn transmembrane domains (Han et al., the small amino acid glycine resulted in an 2004). This raises the question of whether increased PSF. Therefore, the authors Received May 31, 2015; revised July 16, 2015; accepted July 18, 2015. the transmembrane domain of synapto- concluded that the residues of these four Correspondence should be addressed to Linda van Keimpema, VU Uni- brevin 2 might also be involved in fusion amino acids influence flux through the fu- versity, De Boelelaan 1085, Room B-444, 1081 HV, Amsterdam, The Neth- erlands. E-mail: [email protected]. pore formation. sion pore in a size-dependent manner, DOI:10.1523/JNEUROSCI.2087-15.2015 A study recently published in The with larger residues obstructing the fusion Copyright © 2015 the authors 0270-6474/15/3511459-03$15.00/0 Journal of Neuroscience by Chang and pore. 11460 • J. Neurosci., August 19, 2015 • 35(33):11459–11461 van Keimpema and Kroon • Journal Club

To verify these findings, Chang et al. aptobrevin 2, but it can partially take over 2013), cellubrevin regulates vesicle exocy- (2015) measured membrane capacitance its function when synaptobrevin 2 is ab- tosis in astrocytes (Li et al., 2015), and and pore conductance. Membrane capac- sent. Interestingly, while the SNARE do- synaptobrevin 1 mediates itance is a measure of the surface area of mains of synaptobrevin 2 and cellubrevin release in a subpopulation of hippocam- the plasma membrane. When a vesicle differ by only a single amino acid, the pal neurons (Zimmermann et al., 2014). fuses, its membrane is incorporated into transmembrane domain of cellubrevin is The isoform composition of the SNARE the plasma membrane, which increases only 44% homologous to the transmem- complex as a whole may thus determine the cell’s membrane capacitance. The brane domain of synaptobrevin 2. Of the the characteristics of fusion pores formed tryptophan substitutions that decreased four amino acids that Chang et al. (2015) by these proteins and therefore partly reg- PSF amplitude did not influence the found to influence fusion pore flux, three ulate temporal dynamics of release events changes in capacitance resulting from ves- differ between synaptobrevin 2 and cel- and the amount of cargo that is secreted icle fusion, which means that vesicle size lubrevin. At positions 101 and 103, cel- from a vesicle. At , characteristics was not affected. However, the substitu- lubrevin expresses a serine and leucine, of the fusion pore could therefore influ- tions did decrease conductance of the respectively. Compared to the residues ence kinetics of synaptic events, which can fusion pore, which indicates that the present in synaptobrevin 2 (valine at 101 in turn influence synaptic integration physical properties of the pore were al- and at 103), the polarity of these (Magee, 2000). Also, low levels of release tered. This further supports the idea that residues is altered (from nonpolar to polar through a fusion pore have been hypoth- these amino acids line the fusion pore and at residue 101 and from polar to nonpolar esized to act as an inhibitory signal, desen- influence its characteristics. The authors at residue 103). Residue polarity has been sitizing postsynaptic receptors (Jackson also asked whether the charge of residues linked to ion permeation through the and Chapman, 2008). One type of fusion lining the fusion pore affect the flux through pore of an ion channel (Juntadech et al., in which the fusion pore plays a funda- the pore. Indeed, they show that incorpora- 2014). Therefore, the altered residue po- mental role is kiss-and-run. Here, the fu- tion of negatively charged amino acid resi- larity might also directly influence the flux sion pore closes after release of cargo and dues increased the PSF amplitude, whereas of charged cargo through a fusion pore the vesicle retracts from the membrane, incorporation of positively charged amino formed by cellubrevin. In addition, cel- unlike full-collapse fusion, where the fu- acid residues decreased PSF amplitude and lubrevin contains a glycine at position 99. sion pore dilates and the vesicle mem- therefore the outflow of positively charged The authors report that making this spe- brane is incorporated into the plasma cathecholamines. cific substitution in synaptobrevin 2 membrane. Whether synaptic vesicles en- The data presented by Chang and col- (L99G) appeared to increase PSF ampli- gage in kiss-and-run release is still under leagues (2015) indicate that besides the tude, although the difference did not debate, but it has been well established for transmembrane domain of syntaxin 1, the reach statistical significance (Chang et al., DCV secretion (Alabi and Tsien, 2013), transmembrane domain of synaptobrevin 2015). Given these differences in amino for which kiss-and-run is used almost 2 also lines the fusion pore. Interestingly, acid residues at pore-lining sites between exclusively (Chiang et al., 2014). The the four residues in the synaptobrevin 2 synaptobrevin 2 and cellubrevin, we sug- composition of the SNARE complexes in- transmembrane domain that alter the PSF gest that fusion pores formed by these volved in different types of fusion may are located two-by-two on opposite faces proteins will have different characteris- therefore be important in determining of the ␣-helix, and therefore cannot line tics, and that pores formed by cellubrevin the release of cargo through the fusion the fusion pore simultaneously. This is will have a higher PSF amplitude than pore from both dense core and synaptic unlike the findings for syntaxin 1, where pores formed by synaptobrevin 2. Consis- vesicles. the residues that alter fusion pore flux are tent with this, Borisovska et al. (2005) Currently, little is known about the on one face of the ␣-helix. However, syn- showed that PSF amplitude was increased structure of the fusion pore, and its exact aptobrevin 2 is known to dimerize (Fdez in a synaptobrevin 2 KO. This function remains to be determined. How- et al., 2010). Therefore, the authors pro- was rescued by synaptobrevin 2 overex- ever, the data shown by Chang et al. pose a model in which three to four asym- pression, but not by overexpression of cel- (2015) support the view of the fusion pore metric dimers of synaptobrevin 2 line the lubrevin (Borisovska et al., 2005). as an arrangement of proteins and may fusion pore (Chang et al., 2015; their Fig. Because the composition of trans- provide further insight into the functional 5B). These would line up with six to eight membrane domains differs across VAMP role of the fusion pore and of different syntaxin 1 monomers as proposed by Han isoforms, fusion pore characteristics may SNARE protein isoforms in regulating fu- et al. (2004) in a gap junction-like manner depend on the particular isoform present sion events. to form a protenaceous fusion pore on different types of vesicles. Further- (Chang et al., 2015; their Fig. 5C). more, there are various isoforms of syn- References It is worth noting that although knock- taxin, which also show variability in their Alabi AA, Tsien RW (2013) Perspectives on kiss- ing out both synaptobrevin 2 (VAMP2) transmembrane domains. Importantly, and-run: role in , , and . Annu Rev Physiol 75: and cellubrevin (VAMP3) abolishes DCV the three residues that alter fusion pore 393–422. CrossRef Medline release, knocking out synaptobrevin 2 flux upon tryptophan substitution (I269, Borisovska M, Zhao Y, Tsytsyura Y, Glyvuk N, alone reduces, but does not abolish, DCV G276, I283) (Han et al., 2004) are differ- Takamori S, Matti U, Rettig J, Su¨dhof T, release, and knocking out cellubrevin ent in multiple syntaxin isoforms. The Bruns D (2005) v-SNAREs control exocyto- alone does not significantly influence composition of the SNARE complexes in- sis of vesicles from priming to fusion. EMBO J DCV release. Moreover, cellubrevin is up- volved in membrane fusion differs be- 24:2114–2126. CrossRef Medline regulated in synaptobrevin 2 KO cells tween vesicles and cell types. For example, Chang CW, Hui E, Bai J, Bruns D, Chapman ER, Jackson MB (2015) A structural role for the (Borisovska et al., 2005). This suggests syntaxin 6 or syntaxin 16 together with synaptobrevin 2 transmembrane domain in that cellubrevin does not play a significant VAMP4 regulate early to trans- dense-core pores. J Neurosci 35: role in DCV release in the presence of syn- Golgi network trafficking (Shitara et al., 5772–5780. CrossRef Medline van Keimpema and Kroon • Journal Club J. Neurosci., August 19, 2015 • 35(33):11459–11461 • 11461

Chiang HC, Shin W, Zhao WD, Hamid E, Sheng J, cytosis. Annu Rev Biophys Biomol Struct 35: trocyte VAMP3 vesicles undergo Ca(2ϩ)- Baydyuk M, Wen PJ, Jin A, Momboisse F, Wu 135–160. CrossRef Medline independent cycling and modulate glutamate LG (2014) Post-fusion structural changes Jackson MB, Chapman ER (2008) The fusion transporter trafficking. J Physiol 593:2807– and their roles in exocytosis and endocytosis pores of Ca(2ϩ)-triggered exocytosis. Nat 2832. CrossRef Medline of dense-core vesicles. Nat Commun 5:3356. Struct Mol Biol 15:684–689. CrossRef Medline Magee JC (2000) Dendritic integration of excit- CrossRef Medline Jahn R, Fasshauer D (2012) Molecular machines atory synaptic input. Nat Rev Neurosci 1:181– Fang Q, Lindau M (2014) How could SNARE governing exocytosis of synaptic vesicles. Na- 190. CrossRef Medline proteins open a fusion pore? Physiology 29: ture 490:201–207. CrossRef Medline Rizo J, Su¨dhof TC (2012) The membrane fusion 278–285. CrossRef Medline Juntadech T, Kanintronkul Y, Kanchanawarin C, enigma: SNAREs, Sec1/Munc18 proteins, and Katzenmeier G, Angsuthanasombat C (2014) Fdez E, Martínez-Salvador M, Beard M, Wood- their accomplices—guilty as charged? Annu rev Importance of polarity of the ␣4–␣5 loop res- man P, Hilfiker S (2010) Transmembrane- cell dev biol 28:279–308. CrossRef Medline idue—Asn166 in the pore-forming domain of domain determinants for SNARE-mediated Shitara A, Shibui T, Okayama M, Arakawa T, the Bacillus thuringiensis Cry4Ba toxin: im- membrane fusion. J Cell Sci 123:2473–2480. plications for ion permeation and pore open- Mizoguchi I, Shakakura Y, Takuma T (2013) CrossRef Medline ing. Biochim Biophys Acta 1838:319–327. VAMP4 isrequired to maintain the ribbon Han X, Wang CT, Bai J, Chapman ER, Jackson CrossRef Medline structure of the . Mol Cell MB (2004) Transmembrane segments of Kasai H, Takahashi N, Tokumaru H (2012) Dis- Biochem 380:11–21. CrossRef Medline syntaxin line the fusion pore of Ca(2ϩ)- tinct initial SNARE configurations underlying Zimmermann J, Trimbuch T, Rosenmund C triggered exocytosis. Science 304:289–292. the diversity of exocytosis. Physiol Rev 92: (2014) Synaptobrevin 1 mediates vesicle CrossRef Medline 1915–1964. CrossRef Medline priming and evoked release in a subpopula- Jackson MB, Chapman ER (2006) Fusion pores Li D, He´rault K, Zylbersztejn K, Lauterbach MA, tion of hippocampal neurons. J Neurophysiol and fusion machines in Ca2ϩ-triggered exo- Guillon M, Oheim M, Ropert N (2015) As- 112:1559–1565. CrossRef Medline