Hypomorphic mutations at the Drosophila synaptotagmin locus result in behavioral, electrophysiological, and morphological phenotypes similar to those seen in stn mutants. Synaptotagmin (Syt) is a synaptic vesicle
protein with a proposed role in both exocytotic and endocytotic functions in both mammals. The aim of these experiments was to determine whether either of
the viable stn alleles, when in combination with mutations at the synaptotagmin locus, enhances or suppresses the
syt phenotype. The stnC and
stnts alleles are homozygous
viable as adult flies and were isolated in the same genetic background,
and both possess the same insertional polymorphism as the original
Oregon-R strain. The flies used were heterozygous for two
stn null mutations, sytAD4 and
sytD27. The P-element-mediated transposition of a stn minigene to the third chromosome provides 10% of wild-type Syt protein levels and
allows the survival of flies with lethal null mutations on both chromosomes at the
syt locus to produce fertile adults. Double mutant combinations
were constructed, and the viability and behavior of the resulting flies were investigated. The data clearly indicate a genetic interaction between the
stnts mutations and syt. Because synaptotagmins are an integral component of synaptic
vesicle membranes, this data also suggests an interaction between the
stoned protein(s) and synaptic vesicles (Phillips, 2000).
Hydrophobicity analysis of both the StnA and StnB proteins
suggests that they should be soluble proteins. Wild-type fly head extracts, homogenized in both the presence and absence of calcium, were
subjected to differential centrifugation to produce P1 (1000 × g), P2 (25,000 × g), and P3 (125,000 × g) pellets and a final supernatant fraction, S3. Western blots prepared from these fractions were probed with anti-StnA, anti-StnB, and anti-Syt antibodies. The
anti-Syt antibodies were raised against the recombinant cytoplasmic
region of the Syt protein and recognize a number of isoforms of
Synaptotagmin (54-69 kDa). Neither the StnA nor StnB proteins could be visualized in the supernatant fraction. The StnB protein co-sediments with the synaptic
vesicle protein marker Syt, primarily in the P2 and P3 fractions. StnA,
in contrast, preferentially partitions into the P1 fraction,
although some StnA was found in both the P2 and P3 fractions. This indicates
that both stoned proteins preferentially partition into
either membrane fractions or fractions containing large protein
complexes. The association of StnA with the P1 fraction was investigated further. Solublization of StnA from P1 was not achieved with Triton X-100, deoxycholate, or high NaCl concentrations; however, the chaotropic agent KI effectively solubilizes all of the StnA protein from the P1 fraction. This indicates that StnA in the P1 fraction is not associated with heavy membranes but is more likely to be associated with a large protein complex (Phillips, 2000).
As expected, the Syt isoforms were associated with both the P2 and P3
fractions, plasma membrane, and vesicle-enriched fractions, respectively. Also observed was a coincidental shift of Syt and StnB
from the P3 to the P2 fraction when homogenization was performed in the
presence of Ca2+. The supernatant fraction
from the P1 centrifugation (S1) was applied to a glycerol gradient and
centrifuged to separate membrane components. A peak of Syt,
corresponding to the synaptic vesicle fraction, was observed. StnB protein
co-sediments with the Syt peak, whereas StnA, although entering the
gradient, peaks in fractions 3-6. These
two results, the coincident redistribution of StnB and Syt in the
presence of Ca2+ and their
co-sedimentation in glycerol gradients, are consistent with an
association of the StnB protein with synaptic vesicles. The
plasma membrane marker syntaxin was also present in the gradients, probably indicating fragmentation of plasma membrane during
homogenization, although its distribution does not mirror that of
synaptotagmin/StnB or StnA (Phillips, 2000).
To determine whether the StnB present in the P3 fraction is associated
with synaptic vesicles, anti-StnB antibodies were attached to Protein
A-coated magnetic beads, and incubated with a P3 fraction prepared from
Drosophila head homogenates in the absence of calcium. These
beads were then analyzed for the presence of StnB and the synaptic
vesicle protein markers Syt, Csp, and Syb as well as the plasma
membrane marker Syx. The results indicate that a major proportion of
the StnB protein in the P3 fraction is immunoprecipitated. The StnB antibodies
coprecipitate all three synaptic vesicle markers (Syt, Csp, and Syb),
but not Syx. Although multiple species of Syt can
be identified in P3 fractions, only the 69 kDa Syt isoform is
present in these precipitates (Phillips, 2000).
On the basis of its deduced amino acid sequence, StnB does not contain
any putative transmembrane segments and is unlikely to be an integral
membrane protein. What then is the molecular nature of the StnB/vesicle
association? To address this question, fractions of the StnB
immunoprecipitations were washed extensively with 1% Triton X-100. The
presence of the detergent entirely removes Csp from the precipitates
and considerably reduces the amount of Syb present. However, Triton
X-100 has no effect on the amount of Syt bound to the beads. This result suggests that StnB is not associating with
the lipid components of the vesicle membrane and that the interaction
may be via Syt (Phillips, 2000).
There was relatively little StnA seen in the P3 fraction on the Western
blots. However, StnA protein was observed on the glycerol gradients, and although
there was no coincidence of the peak fractions, there was overlap
between StnA and the synaptic vesicle peak. The immunoprecipitations
were therefore further probed for the presence of StnA. The StnA
protein was found to be associated with the immunoprecipitates and to
be insensitive to the Triton X-100 washes. When
immunoprecipitations were performed using anti-StnA antibodies attached
to beads, again Syt and Csp were coprecipitated. Therefore, both StnB and StnA can be found associated with synaptic
vesicles in the P3 fraction (Phillips, 2000).
The continued association of Syt with immunoprecipitated StnB
even after Triton X-100 treatment suggests a direct interaction between StnB and Syt. It appeared likely that this interaction would
be via the µ2-like domain of StnB. To investigate this, a 621 residue
protein containing the µ2-like region, residues 883-1089, and
adjacent sequence up to residue 1261
was expressed in the pGEX expression vector to produce a GST fusion.
The resin-bound GST/µ2-like fusion protein was incubated overnight
with a crude extract of bacterial cells expressing the cytoplasmic
portion of Drosophila Syt as a 6x-His (pET) fusion protein
of 39 kDa. When proteins eluted from the resin were Western-blotted and
the blot was exposed to anti-Syt antibody, the expected 39 kDa Syt fusion protein was identified. The GST protein itself was unable to bind Syt in this assay. A series of fusion constructs with different regions of the µ2-like domain of StnB were similarly assayed. In repeated experiments, fusion proteins terminating at amino acid residue 930 were unable to bind Syt. However, Syt binds to all fusion proteins containing amino acid residues 847-1108 of the StnB protein. Because residues 883-1138 of StnB constitute the µ2-like domain, this domain is sufficient for Syt binding in vitro. It is possible, however, that residues outside the µ2-like domain influence the strength of this interaction in vivo (Phillips, 2000).
StnA can be found associated with
synaptic vesicles immunoabsorbed by the StnB antibodies. To determine
whether the StnA protein might be associated directly with
synaptotagmin, Syt binding to StnA fusion proteins was analyzed. Three constructs were used. The first was a fusion protein that included the first 290 residues of StnA (GST/5'StnA), the second included residues 26-350
(GST/StnA Xho-p33), which removes most of the N-terminal region that
would be missing if the methionine in the stnts mutant acted as a novel translation initiation site, and the third was identical to GST/5'StnA
but contained the sequence encoding the K to M substitution found in
the stnts flies. The results indicate
that residues 26-290 of the amino terminal region of StnA can bind
Syt. This region includes the sequence altered in the
stnts mutation. Binding of Syt to the GST/5'StnA construct containing the stnts mutation shows that this
mutant protein also binda Syt. The affinity of StnA for Syt appears less than that seen with the StnB constructs; however, the binding of StnA to Syt in vitro is observed consistently (Phillips, 2000).
There are two C2 domains, C2A and C2B, in the Syt monomer, and
the two domains have been found to interact with different intracellular components. Two Syt protein constructs were produced, each containing one of the C2 domains, and the ability of these protein constructs to bind to both the StnA/GST and StnB/GST fusions was investigated. The protein containing the
cytoplasmic sequences including the C2A domain (residues 137-327) but
excluding the C2B domain failed to bind to either StnA or StnB.
However, the protein containing only the C2B domain (residues 317-473)
was found to bind to both StnA and StnB (Phillips, 2000).
The immunoprecipitation studies indicate that only the 69 kDa Syt isoform is coprecipitated with StnB. However, there are other synaptotagmin species in Drosophila, including a SytV homolog of 55 kDa that are associated with synaptic vesicles. The C2B domain (residues 325-374) of SytV was expressed as a pMAL fusion protein and shown to cross-react with the polyclonal anti-Syt antibodies. Therefore, it was asked whether StnB could interact with SytV in vitro. The SytV fusions were then assayed for their ability to bind to StnB. The SytV C2B domain indeed interacts with the µ2-homology region of StnB (Phillips, 2000).
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