Syntaxin 1A
Synaptobrevin - also known as VAMP or vesicle-associated membrane protein The structure of synaptobrevin, an intrinsic membrane protein of small synaptic vesicles from mammalian brain, was studied by purification and molecular cloning. Its
message in bovine brain encodes a 116 amino acid protein whose sequence reveals it to be the mammalian homolog of the ray Torpedo VAMP-1. Antibody probing
demonstrates that the protein is also present in Drosophila, and its Drosophila homolog was cloned. Alignment of the sequences of synaptobrevin/VAMP-1 from the
three species shows it to contain four domains, including a highly conserved central region of 63 amino acids that contains 75% invariant residues. The finding that a
membrane protein from vertebrate synaptic vesicles is conserved in Drosophila points toward a central role of this protein in neurotransmission and should allow a
genetic approach to neurotransmitter release (Sudhof, 1989).
VAMP (synaptobrevin) is a highly conserved membrane protein originally described as a component of brain synaptic vesicles. The Drosophila melanogaster
VAMP-encoding gene (syb) comprises five exons. Splicing exons 1,2,3,4,5 (syb-b) results in a protein with a C-terminal hydrophobic domain and a negligible
intraluminal domain. Splicing exons 1,2,3,5 (syb-a) predicts a protein with a 20-amino-acid luminal domain at the C terminus. The ratio of syb-a to syb-b transcripts
is highly regulated during development. The syb transcripts show no enrichment in the nervous system and are present in very early embryos, well before
neurogenesis. The greatest concentration of syb transcripts was found in cells of the gut and Malpighian tubules. Thus, syb may have a general role in membrane
trafficking and, perhaps, a role in the secretion of digestive enzymes (Chin, 1993).
Proteins associated with synaptic vesicles are likely to control the release of neurotransmitter. Because synaptic transmission is fundamentally similar between
vertebrates and invertebrates, vesicle proteins from vertebrates that are important for synaptic transmission should be present in Drosophila as well. This investigation
describes Drosophila homologs of vamp, synaptotagmin, and rab3 that are expressed in a pattern consistent with a function in Drosophila neurotransmission. One
previously reported candidate (syb), a Drosophila homolog of the vamp or synaptobrevin proteins, has been shown to be expressed at very low levels in neurons
and is most abundant in the gut. A neuronal Drosophila vamp (n-syb, neuronal-synaptobrevin) is described and is localized to chromosome band 62A. Northern analysis and in situ
hybridizations to mRNA indicate that the novel vamp, as well as the genes for synaptotagmin (syt) and rab3 (drab3), are all expressed in the Drosophila nervous system.
These genes are widely (perhaps ubiquitously) expressed in the nervous system and there is no evidence of additional neuronal isoforms of synaptotagmin, vamp, or
rab3. Immunoreactivity for synaptotagmin and vamp is located in synaptic regions of the nervous system. This distribution suggests that these molecules are
components of synaptic vesicles in Drosophila. The conserved structure and neuronal expression pattern of these genes indicate that they may function in processes
that are required for both vertebrate and invertebrate synaptic transmission. Because of their distribution in the nervous system and because n-syb, synaptotagmin,
and drab3 do not appear to be in a family of functionally redundant homologs, it is predicted that mutation of these genes will produce a profound neurological phenotype
and that they are therefore good candidates for a genetic dissection in Drosophila (DiAntonio, 1993b).
Tetanus toxin cleaves the synaptic vesicle protein synaptobrevin, and the ensuing loss of neurotransmitter exocytosis has implicated synaptobrevin in this process. To
further the study of synaptic function in a genetically tractable organism and to generate a tool to disable neuronal communication for behavioural studies, a gene encoding tetanus toxin light chain was expressed in Drosophila. Toxin expression in embryonic neurons removes detectable synaptobrevin and eliminates evoked,
but not spontaneous, synaptic vesicle release. No other developmental or morphological defects are detected. Correspondingly, only synaptobrevin (n-syb), but not
the ubiquitously expressed syb protein, is cleaved by tetanus toxin in vitro. Targeted expression of toxin can produce specific behavioral defects; in one case, the
olfactory escape response is reduced (Sweeney, 1995).
Two modes of vesicular release of transmitter occur at a synapse: spontaneous release in the absence of a stimulus and evoked release that is triggered by Ca2+
influx. These modes often have been presumed to represent the same exocytotic apparatus functioning at different rates in different Ca2+ concentrations. To
investigate the mechanism of transmitter release, the role of synaptobrevin/VAMP, a protein involved in vesicular docking and/or fusion was studied. A series of mutations were examined, including null mutations, in neuronal-synaptobrevin (n-syb), the neuronally expressed synaptobrevin gene in Drosophila. Mutant
embryos completely lacking n-syb form morphologically normal neuromuscular junctions. Electrophysiological recordings from the neuromuscular junction of these
mutants reveal that the excitatory synaptic current evoked by stimulation of the motor neuron is abolished entirely. However, spontaneous release of quanta from
these terminals persists, although its rate is reduced by 75%. Thus, at least a portion of the spontaneous 'minis' that are seen at the synapse can be generated by a
protein complex that is distinct from that required for an evoked synaptic response (Deitcher, 1998).
Synaptobrevin is a key constituent of the synaptic vesicle membrane. The neuronal-synaptobrevin (n-syb) gene in Drosophila is essential for
nerve-evoked synaptic currents, but miniature excitatory synaptic currents (mESCs) remain even in the complete absence of this gene. To
further characterize the defects in these mutants, an examination was carried out of conditions that stimulate secretion. Despite the inability of an action
potential to trigger fusion, high K+ saline can increase the frequency of mESCs 4- to 17-fold in a Ca2+-dependent manner: the rate of
fusion approaches 25% of that seen in wild-type synapses under the same conditions. Similarly, the mESC frequency in n-syb null mutants
can be increased by a Ca2+ ionophore, A23187, and by black widow spider venom. Thus, the ability of the vesicles to fuse in response to
sustained increases in cytosolic Ca2+ persists in the absence of this protein. Tetanic stimulation also increases the frequency of mESCs,
particularly toward the end of a train and after the train of stimuli. In contrast, these mutants do not respond to an elevation of cAMP induced
by an activator of adenylyl cyclase, forskolin, or a membrane-permeable analog of cAMP, dibutyryl cAMP, which in wild-type synapses
causes a marked increase in the mESC frequency even in the absence of external Ca2+. These results are discussed in the context of models
that invoke a special role for n-syb in coupling fusion to the transient, local changes in Ca2+ and an as yet unidentified target of cAMP (Yoshihara, 1999).
To investigate a possible involvement of synaptic machinery in Drosophila visual system development, the effects of a
loss of function of neuronal synaptobrevin (n-syb), a protein required for synaptic vesicle release, were studied. Expression of tetanus toxin light chain
(which cleaves neuronal synaptobrevin) and genetic mosaics were used to analyze neuropil pattern formation and levels of
selected neural adhesion molecules in the optic lobe. Targeted tetanus toxin light chain (TeTxLC) expression in the developing optic lobe results
in disturbances of the columnar organization of visual neuropils and of photoreceptor terminal morphology. IrreC-rst
immunoreactivity in neuropils is increased after widespread expression of toxin. In photoreceptors, targeted toxin expression results in increased Fasciclin II and
chaoptin but not IrreC-rst immunoreactivity. Axonal pathfinding and programmed cell death are not affected. In genetic mosaics, patches of photoreceptors that lack
neuronal synaptobrevin exhibit the same phenotypes observed after photoreceptor-specific toxin expression. These results demonstrate the requirement of neuronal
synaptobrevin for regulation of cell adhesion molecules and development of the fine structure of the optic lobe. A possible causal link to fine-tuning processes that
may include synaptic plasticity in the development of the Drosophila CNS is discussed (Hiesinger, 1999).
The finding of an onset of n-syb expression in the first half of pupation poses the question of whether synapses actually start to function so early during optic lobe
development. Neuronal activity plays a major role during vertebrate visual system development. A critical period of 1 d after
eclosion has been demonstrated for experience-dependent developmental plasticity in the Drosophila. It has not yet been shown
whether synaptic plasticity in the Drosophila CNS extends to pupation or whether neurotransmitters are released before any form of neuronal activity. Assuming the
involvement of such processes, the following time scale would be expected: first, expression and localization of proteins of the vesicle release machinery; second,
release of neurotransmitter independent or dependent on spontaneous activity; and third, release of neurotransmitter dependent on evoked activity. Given the early
immunoreactivity of specific synaptic vesicle cycle proteins such as n-syb and synaptotagmin before P + 25% (25% through the pupal period), the synaptic vesicle cycle appears to be available for
more than half of pupal development before first evoked photoreceptor responses occur at P + 82%. Morphological analysis has revealed a brief interval of intense
synapse formation in the lamina of Musca starting ~P + 62% and peaking at P + 74%. Although this time window does
not necessarily correspond to the first occurrence of synapses in the optic lobe of Drosophila, and the heterogeneity of optic lobe neurons should also be taken into consideration, it
may indicate that n-syb is expressed long before synapses are morphologically recognizable (Hiesinger, 1999 and references).
Apparently, not all processes between target selection and the establishment of functional connectivity are yet known. The demonstration of the dependence of
neuropil patterning on NO release (Gibbs, 1998) shows a process of terminal development in a similar time window as the neuropil patterning
defects observed in the current study. With regard to the current study, one possibility would be the involvement of n-syb in the release of neurotransmitters or other factors before or
during synapse development. Li (1997) observed that histamine is synthesized in photoreceptors extending from cultured imaginal disks. Histamine or
other substances released by growth cones after arrival in their target layers might exert functions necessary for the establishment of a regular terminal pattern (Hiesinger, 1999).
Lack of functional n-syb has no obvious influence on target selection and the development of largely overlapping terminals of R7 and R8 cells. In contrast, further
development of terminal fine structure between P + 25 and P + 50% is significantly disturbed, indicating its involvement in a fine-tuning process. This early onset of
n-syb function shows that either n-syb is involved in nonsynaptic processes taking place soon after target recognition, or synapses form earlier in the Drosophila
optic lobe than is generally believed. Because this time window occurs significantly before the observed upregulation of Fasciclin II in active toxin-expressing
photoreceptors, the morphological changes do not depend on this CAM (Hiesinger, 1999).
Cell adhesion molecules play multiple roles during optic lobe development. Most fruitfully investigated are functions during axon guidance and target recognition and synaptic plasticity. The finding of increased Fasciclin II immunoreactivity under conditions of blocked neurotransmitter release corresponds to previous studies that have shown the opposite
effect with an opposite approach: apCAM is downregulated after application of serotonin, and synaptic Fasciclin II is reduced in
mutants with abnormally high neuronal activity. Although it has been demonstrated that apCAM is downregulated via endocytosis, the mechanism of activity-dependent Fasciclin II downregulation at the Drosophila neuromuscular junction remains unknown. Possible
downregulation mechanisms to be considered include endocytosis, extracellular cleavage, and reduced transcription or translation in combination with a continuous
turnover of the protein. The upregulation of two different types of CAMs (Fasciclin II and chaoptin) in the same cell type under conditions of blocked neurotransmitter release poses the
question of the specificity of the mechanism. In the absence of functional n-syb, increased numbers of docked synaptic vesicles accumulate presynaptically. Assuming that this would result in a significant sequestration of membrane material and that the synaptic vesicle cycle is continuously
replenished from cell surfaces carrying adhesion molecules, deactivation of n-syb could result in decreased intake of CAMs and thus increased CAM
immunoreactivity. However, current understanding of the recycling mechanism in the synaptic vesicle cycle and different localization of CAM
isoforms does not support this hypothesis. Alternatively, specifically CAMs on active terminals and fibers could be
downregulated to serve as markers for the competence of the synapses for sprouting (Hiesinger, 1999).
In wild-type third instar larvae, Fasciclin II is found on R7 and R8 retinal axons. During parts of pupation, Fasciclin II is
detectable at low levels on photoreceptor cell bodies. It is possible that Fasciclin II is never completely downregulated from R7
and R8 terminals but is mostly below threshold for visualization with confocal microscopy. Upregulation of Fasciclin II levels in photoreceptors lacking functional
n-syb after P + 75% may thus be attributable to an accumulation of the protein, when its downregulation would normally occur via an n-syb-dependent mechanism
as part of a continuous protein turnover. The finding that IrreC-rst immunoreactivity remains unaltered in photoreceptors without functional n-syb but is increased in proximal neuropils after widespread
TeTxLC expression can be interpreted in two different ways: either IrreC-rst protein is not present on photoreceptor terminals at the addressed time of pupation, or
the n-syb-dependent CAM downregulation mechanism has a different molecular specificity in photoreceptors than in other optic lobe cells. During axonal pathfinding
IrreC-rst is expressed on photoreceptors. In pupal stages IrreC-rst is localized to
rhabdomeres but not to axons and cell bodies of photoreceptors during the second half of pupation. Because rhabdomeres are unique to this cell type and seem to
be a preferred localization for IrreC-rst in photoreceptors, a cell-specific distribution that excludes terminals appears more likely than a specific CAM regulation
mechanism for photoreceptors. Taken together, the results presented here clearly show a requirement of n-syb for optic lobe development. Either n-syb has a previously unknown activity-independent function, or
synaptic transmission is involved in optic lobe development, or both (Hiesinger, 1999 and references).
The Drosophila stoned locus was identified on the basis of stress-sensitive behavioral mutants. The locus is dicistronic and encodes two distinct proteins: stoned A and stoned B, that are expressed
specifically in presynaptic terminals at central and peripheral synapses. Several stoned mutant alleles cause embryonic
lethality, suggesting that these proteins are essential for synaptic function. Physiological analyses at the stoned synapse
reveal severe neurotransmission defects, including reduced and asynchronous neurotransmitter release and rapid fatigue after repetitive stimulation. At the EM
level, stoned synapses show a depletion of synaptic vesicles (SV) and a concomitant increase in membrane-recycling intermediates. Mutant terminals also display a
specific mislocalization of the SV protein Synaptotagmin.
Instead of the punctate bouton localization of Syt
observed in wild type, the stoned mutants display reduced Syt expression in the boutons and the protein aberrantly dispersed throughout the presynaptic
terminal. This mislocalization is not resolved with development in the viable stnC allele because Syt expression remains mislocalized at the third instar
NMJ. Double-labeling assays with other presynaptic markers indicate this mislocalization is specific to Syt,
because CSP, the neuronal membrane marker HRP, another SV protein (Syb), and the membrane protein Syx display normal patterns of
expression in all mutants. These results suggest that stoned mutants specifically mislocalize the SV protein Syt in the synaptic terminal and retain other
synaptic proteins properly. These results suggest that the stoned proteins are essential for the recycling of SV membrane and are required for the proper sorting of synaptotagmin during endocytosis (Fergestad, 1999).
Mature SV have a specific complement of proteins required for a variety of functions. Each protein must be selectively recruited to the maturing
SV during endocytosis. The mislocalization of Syt in stoned synapses is not accompanied by a loss of other SV proteins (e.g., synaptobrevin and CSP),
suggesting that the stoned proteins may be involved in the specific recycling of Syt from endocytosed membrane. It is hypothesized that the Stoned proteins
normally function at a choice point segregating recycled Syt protein into maturing synaptic SVs and away from the multivesicular body degradative pathway. Such a role has been suggested previously for the AP3 complex, shown to be
required for localization of an SV transporter protein and to be required for the generation of SVs. Similarly, the Drosophila gene LAP, which encodes AP180 (associated with clathrin-dependent endocytosis with the AP2
complex), has recently been shown to be involved in regulating SV size and the proper recruitment of the vesicle coat protein clathrin. In the C. elegans AP180 mutant (UNC-11), the protein also seems to have a specific role in recruiting synaptobrevin to the recycled vesicle. These AP180 data, combined with the observation that SV size is not altered in mutant animals lacking
synaptobrevin, suggest that AP180 has two distinct functions: structural budding of membrane and the specific recycling of
synaptobrevin. The similarities between these studies led to the hypothesis that there may be separate mechanisms required for the recycling of each distinct SV protein and
that these mechanisms may be intimately integrated into the membrane-budding machinery. Such a coupled mechanism would guarantee that newly generated
SVs have the correct functional complement of SV proteins. Clearly, within this general mechanism, the Stoned proteins couple the specific recruitment of Syt
to proper SV biogenesis. The Stoned proteins may participate in the AP2-mediated plasma membrane mechanism or, alternatively, act in a separate and/or
later site such as AP3-mediated endosomal sorting to direct the recruitment and/or localization of Syt into mature SVs. Ongoing experiments are aimed at
testing the site and/or mechanism of Stoned function by determining the exact location of Stoned function in the SV-recycling pathway (Fergestad, 1999 and references therein).
cAMP is thought to be involved in learning process and known to enhance transmitter release in various systems. In two Drosophila memory mutants, dunce and rutabaga, the cAMP cascade is defective, and no post-tetanic facilitation is observed at the neuromuscular junction. Thus, changes in synaptic efficacy were suggested for the molecular mechanism of memory. In Aplysia, cAMP mediates changes in synaptic transmission during dishabituation, sensitization, and classical conditioning. cAMP blocks various types of K+ channels, which in
turn leads to membrane depolarization and/or a prolongation of presynaptic action potentials, and finally results in an activation of voltage-gated Ca2+ channels. The long-term potentiation (LTP) at the bullfrog sympathetic ganglion and at the rat hippocampal CA3 is also mediated by cAMP and requires Ca2+ influx at presynaptic terminals. In other cases, cAMP directly enhances Ca2+ influx through modulation of Ca2+ channels. The Ca2+-independent effect of cAMP has also been demonstrated in the crayfish neuromuscular junction and in cultured mammalian CNS neurons. Thus, the effects of cAMP on synaptic transmission are diverse. Multiple mechanisms might be
operating in parallel in one synapse (Yoshihara, 2000).
Using Drosophila genetics it is possible to separate the multiple mechanisms involved in the effects of cAMP on synaptic transmission. Synaptic transmission has been examined in Drosophila embryos lacking a synaptic vesicle protein, neuronal-synaptobrevin (n-syb), which is a v-soluble
NSF attachment protein receptor (SNARE) protein, and required for nerve-evoked transmitter release. Even though evoked release is absent, miniature synaptic currents (mSCs) are readily observed in n-syb null mutants. Their
frequency increases in response to an increase of Ca2+ concentrations in high K+ saline. A Ca2+ ionophore, A23187, also increases the mSC frequency in the
presence of external Ca2+. These findings indicate that the n-syb null mutants are still capable of responding to elevations of internal Ca2+. Furthermore, in wild-type
embryos cAMP increases the frequency of mSCs in the absence of external Ca2+, but does not in the n-syb null mutants. Thus,
requirements for two modes of vesicle fusion, spontaneous transmitter release and nerve-evoked release, seem to be different (Yoshihara, 2000).
The preceding results, showing that in the absence of external Ca2+ cAMP enhances spontaneous transmitter release, suggest two basic features regarding the effects
of cAMP on spontaneous transmitter release. (1) This pathway involves n-syb, a protein that is essential for evoked release (n-syb-dependent pathway). (2)
cAMP enhancement of release is not dependent on external Ca2+. It has been asked, using the n-syb null mutant, whether cAMP also enhances spontaneous
transmitter release through an increase of intracellular Ca2+ when Ca2+ is available. Further is has been asked whether PKA encoded by DC0 is involved in this enhancing
effect of cAMP on transmitter release (Yoshihara, 2000).
cAMP has been shown to enhance spontaneous transmitter release in the absence of extracellular Ca2+ and n-syb is required in this enhancement (n-syb-dependent). The cAMP-induced enhancement of transmitter release was examined in the presence of external Ca2+. The intracellular concentration of cAMP was raised by application of either forskolin, an activator of adenylyl cyclase, or by 4-chlorophenylthio-(CPT)-cAMP, a
membrane-permeable analog of cAMP, in the presence of external Ca2+, while recording miniature synaptic currents (mSCs) at the neuromuscular junction in n-syb
null mutant embryos. The frequency of mSCs increases in response to elevation of cAMP, and this effect of cAMP is completely blocked by Co2+
(n-syb-independent pathway). In contrast, in wild-type embryos the cAMP-induced mSC frequency increase is partially blocked by Co2+. In DC0, a mutant
defective in protein kinase A (PKA), nerve-evoked synaptic currents are indistinguishable from the control, but mSCs are less frequent. In this mutant the
enhancement by cAMP of both nerve-evoked and spontaneous transmitter release is completely absent, even in the presence of external Ca2+. Taken together,
these results suggest that cAMP enhances spontaneous transmitter release by increasing Ca2+ influx (n-syb-independent) as well as by modulating the release
mechanism without Ca2+ influx (n-syb-dependent) in wild-type embryos, and these two effects are mediated by PKA encoded by the DC0 gene (Yoshihara, 2000).
In a DC0 mutant, the amplitude and Ca2+ dependency of nerve-evoked synaptic currents are not significantly different from those in wild-type, whereas the mSC frequency is lower. Conversely, in a n-syb null
mutant, n-syb DeltaF33B, no
nerve-evoked synaptic currents are detected, whereas mSCs are readily
observable. Thus, it appears that these two modes of vesicle fusion, nerve-evoked and
spontaneous, seem to have distinct requirements (Yoshihara, 2000).
Under various conditions there is a good correlation between the
frequency of mSCs and the number of quanta released by nerve stimulation. In rat cerebellar synapses, there is a clear correlation between
the frequency of mSCs and the amplitude of evoked synaptic currents in
preparations treated with various concentrations of forskolin. In
accordance with this, in wild-type embryos, forskolin increases
the frequency of mSCs and the quantal content in a similar time course.
Furthermore, in the DC0 mutant, the effect of forskolin is observed neither in spontaneous transmitter release nor in nerve-evoked release.
These results suggest that these two modes of transmitter release are
similarly affected by cAMP-PKA (Yoshihara, 2000).
cAMP is shown at the Drosophila neuromuscular junction in
third instar larvae to increase the size of exo-endo cycling pool (readily releasable pool) of synaptic vesicles. The size of this pool is closely correlated with the
quantal content of synaptic potentials evoked by nerve stimulation at a
low frequency. Forskolin increases the mSC
frequency in wild-type embryos and in newly hatched wild-type larvae. Thus, it is likely that this pool supplies vesicles for
both modes (nerve-evoked and spontaneous) of transmitter release. However, the two modes dichotomize after
this step. For nerve-evoked release n-syb protein is required, whereas
for spontaneous fusion this protein is not of absolute necessity,
although its presence facilitates spontaneous transmitter release. The cAMP-PKA cascade
seems to influence the vesicle fusion process at multiple levels: (1)
vesicle mobilization and translocation, which increase the size of
exo-endo cycling pool; (2) modification of
Ca2+ influx through voltage-gated
Ca2+ channels (n-syb-independent pathway), and (3) modulation of transmitter vesicle fusion
(n-syb-dependent pathway). It is likely that
the first mechanism affects both modes of vesicle fusion similarly.
However, the second and third mechanisms may act differentially on the
two modes of vesicle fusion, which may explain the phenotype of the
DC0 mutant, namely at the resting state
the cAMP-PKA cascade might not be affecting the nerve-evoked
transmitter release, whereas spontaneous release might be supported by
the baseline activity level of the cascade (Yoshihara, 2000).
The V0 complex forms the proteolipid pore of an ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been proposed largely based on yeast vacuolar fusion experiments. Mutations were isolated in the largest V0 component vha100-1 in flies in an unbiased genetic screen for synaptic malfunction. The protein is required only in neurons, colocalizes with markers for synaptic vesicles as well as active zones, and interacts with t-SNAREs. Both GST-V100 and GST-Syx retrieve His-tagged SNAP-25. Furthermore, GST-V100 also interacts with His-Syx. In contrast, only GST-Syx, but not GST-V100, pulls down His-n-Syb, indicating that V100 directly interacts with Syx and SNAP-25, the SNAREs at the target membrane, but not with the vesicle-SNARE n-Syb. Loss of vha100-1 leads to vesicle accumulation in synaptic terminals, suggesting a deficit in release. The amplitude of spontaneous release events and release with hypertonic stimulation indicate normal levels of neurotransmitter loading, yet mutant embryos display severe defects in evoked synaptic transmission and FM1-43 uptake. The data suggest that Vha100-1 functions downstream of SNAREs in synaptic vesicle fusion (Hiesinger, 2005).
Vesicular or vacuolar ATPases are the most prominent intracellular proton pumps, consisting of at least 12 subunits in two sectors (V0 and V1). Acidification is important for many cellular functions, including receptor-ligand dissociation, degradative pathways, and the generation of intercompartment proton motive forces that are in turn utilized as driving forces for numerous secondary transport processes. This study reports the consequences of the selective disruption of a V0 subunit a1 homolog in neurons. The V0 subunit a is encoded by four homologous genes in flies, worms, mouse, and human. The data from yeast and C. elegans indicate a crucial role of V0 subunit a proteins for specific functions in distinct intracellular compartments and different cell types (Hiesinger, 2005).
Mutations were isolated in v100 based on the specific defect of photoreceptor neurons to evoke a postsynaptic response. Photoreceptors are an excellent 'test tube' because they are not required for viability of the fly, and numerous assays can be used to assess morphology and function. Since loss of v100 does not affect photoreceptor specification, development, viability, and the ability to sense light, it is surmised that most intracellular vesicle trafficking and acidification processes are unaffected. Indeed, mutations that affect two key protein components of the V1 subcomplex (subunits A and B) are cell lethal when removed in photoreceptors, and acidification as measured with LysoSensor in v100 mutant photoreceptor cell bodies is unaffected. Hence, if acidification is the cause of the observed phenotypes, it is likely to only affect synaptic vesicles (Hiesinger, 2005).
Several methods can be used to directly or indirectly assess the acidification of synaptic vesicles. The pH-sensitive dye LysoSensor or genetically encoded pHluorin, a pH-sensitive GFP fusion protein that localizes within vesicles, can be used in Drosophila neurons to directly assess synaptic vesicle acidification. Unfortunately, the intensity differences at embryonic NMJs are too small to be observed with the confocal system used. However, several lines of evidence allow an assessment of the contribution of a possible acidification defect to the observed phenotypes. Several results are not consistent with a defect in synaptic vesicle acidification alone. These include (1) the accumulation of vesicles in mutant terminals, (2) the normal mEPSC amplitude and frequency combined with a severely reduced evoked response in a hypomorphic allelic combination, and (3) the impairment of FM1-43 uptake. In addition, several lines of evidence are not readily explained with the function of V100 as part of a proton pump: (1) selective partial rescue of vesicle trafficking but not acidification in yeast, (2) selective interaction with t-SNAREs, and (3) localization at active zones. Taken together, these results indicate that V100 exerts additional or alternative functions to synaptic vesicle acidification at Drosophila synapses (Hiesinger, 2005).
There is wide support for the hypothesis that SNAREs form the basic molecular apparatus that forces lipid bilayers to fuse. However, this does not imply that SNAREs alone are required to induce synaptic vesicle exocytosis. SNAREs alone are sufficient to induce fusion of liposomes, but the kinetics of these events does not mimic the kinetics that has been observed in synaptic vesicle fusion in vivo. The yeast vacuolar fusion assay is the only system in which evidence for an additional component downstream of SNARE function has been identified. Interestingly, the isolation of a proteolipid pore complex from synaptosomes from electroplaques of Torpedo, named the 'mediatophore' has been isolated. This pore complex contains a subunit of the V0 complex and transfection of this component in some cells allows quantal release of neurotransmitter. Subunit a1 localizes to synaptic terminals and interacts with the v-SNARE n-Syb. While the localization is in agreement with the current findings at Drosophila synapses, selective interactions of V100 with the t-SNAREs Syx and SNAP-25 were found in agreement with the observations made in yeast (Hiesinger, 2005).
These analyses have revealed many similarities between v100 and n-syb mutants: both die as late embryos without coordinated movement, accumulate vesicles at synapses, exhibit reduced spontaneous vesicle release and FM1-43 uptake, contain vesicles with normal transmitter content that is poorly released upon stimulation, and interact with t-SNAREs. The only assay in which n-syb mutants behaved differently from v100 is the hypertonic stimulation. Vesicle release induced with hypertonic solution in Drosophila SNARE mutants is largely abolished. In contrast, v100 mutants exhibit some responses with a reduced number of events but normal amplitude. This implies (1) the presence of neurotransmitter-loaded vesicles in mutant terminals and (2) that SNARE function required for hypertonic release is at least partially possible. V100 is not crucial for this step, placing its role downstream of SNARE-dependent priming, congruous with the findings in yeast vacuolar fusion. A function downstream of priming is also supported by the observation that Syx overexpression phenotypes are suppressed by the loss of v100 (Hiesinger, 2005).
The results show only a late exocytic role for the V0 subunit a1, while the implication of other V0 components remains to be tested. Hence, the data is formally consistent with a role of the V0 subunit a1 outside of the V0 complex in association with SNAREs. However, no role outside the V0 complex has so far been shown for a subunit a in any system. In summary, the data indicate a function for V100 and possibly the V0 proteolipid pore as a mediator of vesicle release efficiency downstream of SNARE-dependent priming (Hiesinger, 2005).
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Syntaxin 1A:
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