Genetic Modifiers of the Drosophila NSF Mutant, comatose, Include a Temperature-Sensitive Paralytic Allele of the Calcium Channel 1-Subunit Gene, cacophony

Corresponding author: Richard W. Ordway, Department of Biology, 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802., rwo4{at}psu.edu (E-mail)

Communicating editor: T. C. KAUFMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The N-ethylmaleimide-sensitive fusion protein (NSF) has been implicated in vesicle trafficking in perhaps all eukaryotic cells. The Drosophila comatose (comt) gene encodes an NSF homolog, dNSF1. Our previous work with temperature-sensitive (TS) paralytic alleles of comt has revealed a function for dNSF1 at synapses, where it appears to prime synaptic vesicles for neurotransmitter release. To further examine the molecular basis of dNSF1 function and to broaden our analysis of synaptic transmission to other gene products, we have performed a genetic screen for mutations that interact with comt. Here we report the isolation and analysis of four mutations that modify TS paralysis in comt, including two intragenic modifiers (one enhancer and one suppressor) and two extragenic modifiers (both enhancers). The intragenic mutations will contribute to structure-function analysis of dNSF1 and the extragenic mutations identify gene products with related functions in synaptic transmission. Both extragenic enhancers result in TS behavioral phenotypes when separated from comt, and both map to loci not previously identified in screens for TS mutants. One of these mutations is a TS paralytic allele of the calcium channel 1-subunit gene, cacophony (cac). Analysis of synaptic function in these mutants alone and in combination will further define the in vivo functions and interactions of specific gene products in synaptic transmission.

UNDERSTANDING electrical signaling in neurons requires in vivo analysis of the underlying molecular mechanisms. A classical approach to this problem has been the use of temperature-sensitive (TS) paralytic mutants of Drosophila (SUZUKI et al. 1971 ; GRIGLIATTI et al. 1973 ; SIDDIQI and BENZER 1976 ). These mutants typically develop and function normally at permissive temperature and can be shifted to restrictive temperatures to examine the physiological function of a specific gene product. Drosophila is ideal for this purpose given that it is ectothermic and amenable to sophisticated genetic, molecular, electrophysiological, and behavioral analysis.

Our recent work has extended some early studies on TS paralytic mutants (SIDDIQI and BENZER 1976 ) by examining synaptic function in comatose (comt) mutants. These studies show that the dNSF1 protein encoded by comt (ORDWAY et al. 1994 ; PALLANCK et al. 1995 ) functions in neurotransmitter release by priming synaptic vesicles for fast, calcium-triggered exocytosis (KAWASAKI et al. 1998 ; KAWASAKI and ORDWAY 1999A ). Studies of adrenal chromaffin cell exocytosis following N-ethlymaleimide treatment (XU et al. 1999 ) suggest an analogous function for N-ethylmaleimide-sensitive fusion protein (NSF) in this system. Somewhat similar conclusions were drawn from experiments utilizing presynaptic injection of NSF peptides at the squid giant synapse, with some important differences (SCHWEIZER et al. 1998 ). All of the above studies have extended earlier biochemical analysis in semi-intact pheochromocytoma PC12 cells, which indicated a role for NSF in priming vesicles for regulated exocytosis (BANERJEE et al. 1996 ).

This recent progress in analyzing the function of NSF at synapses adds to a substantial body of work on the biochemical interactions between NSF and other synaptic proteins. NSF is a cytosolic ATPase thought to regulate the interactions of several proteins required in neurotransmitter release. These include a class of membrane proteins called soluble NSF attachment protein receptors (SNAREs). The characteristics and proposed interactions of these proteins are reviewed elsewhere (HANSON et al. 1997A ; HAY and SCHELLER 1997 ) and are discussed briefly below.

SNARE proteins are present on the synaptic vesicle membrane as well as the presynaptic plasma membrane to which these vesicles are targeted. After a vesicle docks at the target membrane, a 7S SNARE protein complex is thought to form by assembly of vesicle SNAREs (v-SNAREs, e.g., synaptobrevin) and target SNAREs (t-SNAREs, e.g., syntaxin and SNAP-25). This complex may function directly in membrane fusion (WEBER et al. 1998 ; CHEN et al. 1999 ) or, alternatively, it may serve a transient function preceding fusion (COORSSEN et al. 1998 ; UNGERMANN et al. 1998 ). Although the precise role of SNARE proteins in neurotransmitter release remains unresolved, it is generally agreed that they serve an important role. After assembly of the 7S SNARE complex, NSF and the soluble NSF attachment proteins (SNAPs) can join this complex to form a 20S particle (SOLLNER et al. 1993 ; ROTHMAN and WIELAND 1996 ). NSF can then disassemble the complex in an ATP-dependent fashion (SOLLNER et al. 1993 ). Consistent with this biochemical action of NSF in vitro, in vivo experiments indicate that plasma membrane SNARE complex accumulates at restrictive temperature in comt (TOLAR and PALLANCK 1998 ). Together, the above observations indicate that NSF is a central biochemical component of the neurotransmitter release apparatus.

Some progress has been made in determining the structural basis of NSF function. NSF is a homomultimer composed of six subunits arranged in a barrel structure (HANSON et al. 1997B ). Each subunit contains an N-terminal domain followed by two repeats, D1 and D2, and each of these repeats includes motifs for ATP binding and hydrolysis. Structure-function analysis has been carried out using an in vitro Golgi transport system and suggests differences in the functional roles of the D1 and D2 repeats (see DISCUSSION). Crystal structures have been determined for the N domain (YU et al. 1999 ) as well as the D2 domain (LENZEN et al. 1998 ; YU et al. 1998 ). These structures define the residues involved in ATP binding in D2 (LENZEN et al. 1998 ; YU et al. 1998 ) and suggest a structural basis for SNARE complex disassembly (YU et al. 1999 ).

Because NSF can disassemble SNARE complexes, it may influence the biochemical interactions of SNAREs with other proteins. For example, SNAREs interact directly with the voltage-gated calcium channels that provide the calcium trigger for neurotransmitter release. These are typically non-L type, high voltage-activated calcium channels (WHEELER et al. 1995 ; CATTERALL 1998 ), composed of a primary structural subunit, 1, as well as 2-, ß-, and -subunits. 1-Subunits implicated in neurotransmitter release contain a synaptic protein interaction (SYNPRINT) domain that binds several synaptic proteins, including both v- and t-SNAREs (reviewed in SHENG et al. 1998 ). Recently, an analogous synaptic protein binding domain has been characterized in an L_c-type calcium channel 1-subunit as well (WISER et al. 1999 ). The binding interactions between 1-subunits and synaptic proteins are proposed to link the neurotransmitter release apparatus to the calcium channel and thus couple calcium influx to fast synaptic vesicle fusion (MOCHIDA et al. 1996 ; RETTIG et al. 1997 ). In addition, 1-subunit interactions with the t-SNARE, syntaxin, may regulate the availability of calcium channels for voltage activation (BEZPROZVANNY et al. 1995 ).

The biochemical interactions of NSF, SNAPs, SNAREs, and calcium channels imply close functional relationships among these components of the neurotransmitter release apparatus. To further investigate the molecular basis of dNSF1 function in neurotransmitter release and to broaden our analysis of synaptic mechanisms to other proteins, we have performed a genetic screen to identify mutations that interact with comt. Here we report identification, genetic characterization, and behavioral analysis of four genetic modifiers of comt. These include intragenic mutations that will contribute to structure-function analysis of dNSF1, as well as extragenic mutations that identify other gene products with related functions in synaptic transmission. Some of this work has been reported in preliminary form (DELLINGER et al. 1999 ; KAWASAKI et al. 1999A ).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila stocks:
All stocks were maintained at a room temperature of 23°–25°. The previously isolated cacophony (cac) alleles, l(1) L13 and cac^S, were generously provided by Jeffrey C. Hall (Brandeis University). comt^ST53 and comt^ST17 were from our laboratory stock collection. X chromosome meiotic mapping was carried out by conventional methods using a y m wy g f chromosome. Deficiency lines were obtained from the Bloomington and Umeå stock centers.

Mutagenesis and screening:
Male flies were isolated from females for 24 hr and then starved for a period of 3–5 hr preceding the mutagenesis. These males were exposed to a solution of 25 mM ethyl methanesulfonate (EMS) in 1% sucrose for 24 hr and then mated in groups of 30 to a similar number of virgin females. Five days after mating, the EMS-treated males were discarded. F₂ progeny were tested for temperature-sensitive paralysis by placing flies in a vial maintained at 36° by immersion in a water bath. Tests were typically 3 min in duration. F₂ progeny were collected twice from each cross, approximately 14 and 21 days after mating. A total of 30–50 progeny were tested from a typical cross.

Behavioral analysis:
Behavioral testing was carried out in vials using the same apparatus described for screening. Two-day-old flies were tested in groups of six, and five groups of flies were tested for each genotype (n = 5). Time for 50% paralysis represents the time at which three flies were no longer able to stand. The test vials were plugged with cotton. In all tests exceeding 5 min in duration, the cotton plug was wet with water after 5 min to prevent dehydration.

Sequencing intragenic modifiers:
Genomic DNA was prepared from males carrying the modifier mutation. This was used as template in PCR reactions amplifying a 3-kb region containing the entire coding sequence of dNSF1. Direct sequencing of gel-purified PCR products was carried out by automated sequencing at the Penn State University nucleic acids facility. Sequences from the mutants were compared directly to the comt^ST53 parent chromosome used in the mutagenesis.

Data analysis:
Graphing and analysis of data were carried out using Microsoft Excel. All data are presented as the mean ± SEM. Using an unpaired Student's t-test, statistical significance was assigned to comparisons with P values 0.05. In the figures, values significantly different from control values are marked with an asterisk.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

As originally reported by SIDDIQUI and BENZER (1976), comt is located on the X chromosome at an approximate meiotic map position of 1-40. The first comt mutation to be identified, a TS paralytic allele referred to as com (SIDDIQI and BENZER 1976 ), is now known as comt^ST53. This and all other reported mutations of comt are recessive. A simple genetic screen was utilized to identify modifiers of TS paralysis in comt. comt^ST53 males were exposed to the chemical mutagen EMS and mated to females carrying a compound X (attached-X) chromosome (Fig 1). This cross produces F₁ males carrying a mutagenized paternal X chromosome, as well as mutagenized paternal autosomes. These F₁ males are comt^ST53 and can be screened for deviations from typical paralytic behavior. While such F₁ screens have been highly productive (SUZUKI et al. 1971 ; GRIGLIATTI et al. 1973 ; SIDDIQI and BENZER 1976 ), we found it advantageous to backcross individual F₁ males to attached-X females and to screen the resulting F₂ generation (Fig 1; see DISCUSSION).

View larger version (23K): In this window In a new window Download PPT slide   Figure 1. An F2 screen for modifiers of comt.

To test for deviations from the comt phenotype, flies were examined for TS paralysis at 36°. At this temperature, comt^ST53 is paralyzed in ~1.5 min, allowing either acceleration or slowing of paralysis to be observed. The progeny of 3196 individual F₁ males were tested, and a total of six candidate modifier mutations were subjected to genetic characterization. Two were found to be TS paralytic mutations exhibiting no apparent interaction with comt [these include one paralyzed (para) allele and one TS allele of Shaker] and are not described further. The genetic and behavioral analysis of the remaining four mutations is described below.

Intragenic modifiers of comt:
comt^Su1 is a dominant, X-linked suppressor of comt. Thus flies homozygous for comt^ST53 and heterozygous for comt^Su1 exhibit the suppressed phenotype (Table 1). This mutation could not be separated from comt^ST53 by recombination, suggesting that it may be an intragenic modifier. A second mutation, comt^e1, is a recessive, X-linked enhancer (Table 1). comt^e1 appeared to be an intragenic modifier on the basis that its recessive phenotype was not complemented by a comt^- deficiency, Df(1)wy26.

To further characterize these putative intragenic modifiers, the dNSF1 open reading frame was sequenced in each mutant. The coding sequence of dNSF1 is interrupted by only a few small introns and thus direct sequencing of PCR products was performed using genomic DNA as template. Sequence from each of the mutants was compared directly to the comt^ST53 parent chromosome used in the mutagenesis. Consistent with the use of EMS as a mutagen (ASHBURNER 1989 ), comt^Su1 differed from the parent chromosome sequence by only a single G A transition. This mutation occurred at position 1114 in the dNSF1 cDNA sequence, converting an invariant alanine to threonine at position 279 in the D1 domain (Fig 2A). comt^e1 contained a single G A transition at position 1930 of the dNSF1 cDNA sequence. This mutation changes an invariant alanine to threonine at position 551 within the D2 domain (Fig 2B). The presence of the comt^ST53 mutation was confirmed in each case. As reported earlier (PALLANCK et al. 1995 ), comt^ST53 occurs at position 1727 of the dNSF1 cDNA sequence and converts a serine to leucine near the C-terminal end of the D1 domain (Ser 483).

View larger version (35K): In this window In a new window Download PPT slide   Figure 2. comtSu1 and comte1 are missense mutations in dNSF1. (A and B) Aligned sequences representing the ATP-binding regions of the D1 (A) and D2 (B) binding domains. The dNSF1 sequence is aligned with CHO cell NSF and the yeast NSF protein, SEC18. Identical residues are shaded. A black bar marks the homologous ATP-binding regions of D1 and D2, including the "p-loop" (LENZEN et al. 1998 ) and other key residues. Note that the comtST53 mutation (S483L) is located near the C-terminal end of the D1 domain. (C) Residues involved in ATP binding by D2 are shown in contact with ATP. This dNSF1 structure is inferred from the positions of identical residues within the structure of the CHO NSF D2 domain (LENZEN et al. 1998 ). Note Ala551, the residue altered by comte1.

Both intragenic modifiers change amino acids close to or within an ATP-binding site. In the case of comt^Su1, the mutation is located near the ATP-binding region of the D1 domain (Fig 2A). The second intragenic modifier mutation, comt^e1, occurs within the D2 ATP-binding site. The latter mutation is of particular interest to structure-function analysis because alanine 551 (Fig 2B) plays a key role in ATP binding by the D2 domain. In the crystal structure of the D2 domain of Chinese hamster ovary (CHO) cell NSF (LENZEN et al. 1998 ), the analogous alanine contacts ATP at both the ribose ring and the -phosphate (Fig 2C). Thus comt^e1 is likely to be defective in ATP binding by the second repeat.

To further characterize the comt^Su1 and comt^e1 phenotypes, behavioral analysis was performed for each of these mutations in a comt^ST53 genetic background. comt^Su1 comt^ST53 double mutants exhibited slower paralysis than did comt^ST53 alone (Fig 3). At 36°, time for 50% paralysis was increased from 1.53 ± 0.05 min (n = 5) in comt^ST53 to 3.01 ± 0.13 min (n = 5) in the double mutant. The other intragenic modifier, comt^e1, accelerated comt^ST53 paralysis (Fig 3). In comt^ST53 comt^e1 double mutants, the time for 50% paralysis at 36° was reduced to 0.81 ± 0.05 min (n = 5).

View larger version (18K): In this window In a new window Download PPT slide   Figure 3. Behavioral analysis of the intragenic modifiers, comtSu1 and comte1. For all behavioral tests reported in this study, five groups of flies were tested (n = 5). Asterisks mark values significantly different from control values (see also MATERIALS AND METHODS).

Extragenic modifiers of comt:
A third mutation was also X linked, but could be separated from comt by recombination and exhibited a TS behavioral phenotype by itself. At 36° or 38°, this mutant typically stands motionless and only infrequently walks a short distance (typically 2–3 body lengths). In contrast, wild-type flies typically exhibit fast, uninterrupted walking and a generally high level of activity under these conditions. Because this mutant appears to stall at high temperature, we have named it overheated (ovr). As shown in Table 2, ovr is recessive. Recombinational mapping indicated that ovr lies ~7 map units to the right of forked. Deficiency mapping showed that it is included within Df(1) HF396, but not within Df(1)mal3, thus localizing the mutation to cytological region 18E1-19A2.

Although ovr by itself does not exhibit paralysis at 36°, it accelerates the paralysis observed in comt^ST53 (Fig 4A). ovr alone is paralyzed after a long exposure to 38°, exhibiting 50% paralysis at 22.04 ± 0.85 min (n = 5). To examine whether the genetic interaction between comt and ovr is specific to the comt^ST53 allele, comt^ST17 ovr double mutants were generated and subjected to behavioral analysis. The ovr mutation enhanced the TS paralytic phenotype of comt^ST17 as well (Fig 4B), indicating a general interaction between the ovr and dNSF1 gene products.

View larger version (15K): In this window In a new window Download PPT slide   Figure 4. ovr is an enhancer of both comtST53 (A) and comtST17 (B). Note that ovr behavioral tests were truncated after 50 min.

The final mutation is also an X-linked recessive enhancer. This mutation could be separated from comt and by itself exhibited rapid TS paralysis at 38° (Table 2). Meiotic mapping placed this mutation ~2 map units to the left of comt. Deficiency mapping refined this position to the cytological interval 11A1-2, as defined by the left limit of Df(1) KA10 and the right limit of Df (1) HA85. This region includes the cac gene, which encodes a homolog of voltage-gated calcium channel 1-subunits (SMITH et al. 1996 , SMITH et al. 1998 ). Failure of complementation was observed between the new TS mutation and previously isolated alleles of cac, including cac^S and l(1) L13 (Table 2). These results indicate that this new mutation represents a TS paralytic allele of cac, and we have designated it cac^TS2. Similarities were observed in the behavior of cac^TS2 and the original cac allele, cac^S. At an elevated temperature of 36°, both mutants exhibit spinning behavior and a lack of motor coordination as described previously for cac^S (PEIXOTO and HALL 1998 ). Only cac^TS2 exhibited rapid TS paralysis at 38°.

The cac locus was originally identified in a screen for mutations altering courtship song (VON SCHILCHER 1976 , VON SCHILCHER 1977 ) and was subsequently found to be allelic to the nightblind A (nbA) locus (SMITH et al. 1998 ). A synaptic function for cac-encoded calcium channels is suggested by electroretinogram recordings consistent with a defect in synaptic transmission (HEISENBERG and GOTZ 1975 ; SMITH et al. 1998 ), by the similarity of cac to calcium channel 1-subunits implicated in neurotransmitter release (SMITH et al. 1996 ), and by the identification of cac^TS2 as a modifier of comt. Our recent analysis of synaptic function in cac^TS2 and cac^S indicates that cac encodes a primary calcium channel 1-subunit responsible for neurotransmitter release at neuromuscular synapses (KAWASAKI et al. 2000 ).

Analysis of TS paralysis in cac^TS2 comt^ST53 double mutants confirmed the interaction between these mutations (Fig 5). Although cac^TS2 alone is not paralyzed at 36°, cac^TS2 comt^ST53 double mutants exhibited faster paralysis at this temperature than did comt^ST53 alone. At 38°, rapid paralysis in the double mutant was also faster than in cac^TS2 alone, indicating that comt^ST53 is an enhancer of cac^TS2.

View larger version (13K): In this window In a new window Download PPT slide   Figure 5. cacTS2 is both an enhancer of comtST53 and a TS paralytic mutation itself. Note that cacTS2 behavioral tests at 36° were truncated after 50 min. Paralysis of cacTS2 comtST53 double mutants is significantly faster than paralysis in either comtST53 or cacTS2 alone.

To examine whether the interaction between cac^TS2 and comt was specific to the comt^ST53 allele, cac^TS2 comt^ST17 double mutants were generated and subjected to similar behavioral tests. As was the case with comt^ST53, cac^TS2 enhanced paralysis of comt^ST17 (Fig 6A), indicating a general interaction between cac^TS2 and dNSF1 loss-of-function mutations. The allele specificity of this interaction was further explored by generating cac^S comt^ST17 double mutants. These double mutants exhibited faster TS paralysis than did comt^ST17 alone at both 36° and 38° (Fig 6B). As described previously (PEIXOTO and HALL 1998 ), cac^S alone produced slow TS paralysis at 36° and 38°, exhibiting 50% paralysis in 39.57 ± 1.25 min (n = 5) and 19.97 ± 1.78 min (n = 5), respectively (Fig 6B). Taken together, the above results demonstrate a lack of allele specificity in the interactions of cac and comt, suggesting a close relationship between the wild-type functions of these gene products.

View larger version (25K): In this window In a new window Download PPT slide   Figure 6. The genetic interaction between cac and comt is not allele specific. (A) Both cacTS2 and comtST17 TS paralysis are enhanced in cacTS2 comtST17 double mutants. Note that cacTS2 behavioral tests at 36° were truncated after 50 min. At 38°, paralysis of cacTS2 comtST17 double mutants is significantly faster than paralysis in either comtST17 or cacTS2 alone. (B) cacS enhances rapid paralysis in comtST17.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Here we report the isolation and characterization of four genetic modifiers of comt, including an intragenic suppressor, an intragenic enhancer, and two extragenic enhancers. Molecular analysis of the intragenic modifiers, characterization of the interacting extragenic loci, and analysis of the allele specificity of these interactions is described. The results reported here may further define the structure-function relationship of dNSF1, as well as the interactions of dNSF1 with other gene products in the neurotransmitter release process.

A landmark screen carried out by the Suzuki laboratory (GRIGLIATTI et al. 1973 ) recovered 10 TS behavioral mutant lines by screening 1.1 million F₁ flies at 29°. These mutations fell into three X-linked complementation groups, including six alleles of shibire (shi), two alleles of para, and two alleles of stoned (stn). In a subsequent F₁ screen (SIDDIQI and BENZER 1976 ), an unreported number of progeny were tested at 38°. This screen recovered multiple alleles in each of three X-linked complementation groups: shi, para, and comt. In comparison, the F₂ screen reported here recovered 6 TS behavioral mutant lines by screening the progeny of 3196 flies. Four of these were modifiers of comt, including two intragenic mutations and two extragenic mutations mapping to other X chromosome loci. Although both extragenic enhancers exhibit TS phenotypes when separated from comt, neither had been identified in previous screens for TS behavioral mutants. Differences between the present results and those of previous screens may be attributed to differences in the screening temperature, to the comt mutant background, and to differences between F₁ and F₂ screening. Although the reduced number of chromosomes examined is a major disadvantage of an F₂ screen, the ability to observe a modified phenotype in multiple F₂ progeny may enhance detection of subtle phenotypes. We also performed a pilot F₁ screen, in which screening more than 10,000 chromosomes did not recover any heritable modifiers of comt.

Intragenic modifiers:
comt^Su1 is an alanine to threonine missense mutation near the ATP-binding site of D1 (Ala 279). This mutation is a dominant suppressor of TS paralysis in comt^ST53, which is a serine to leucine missense mutation near the C-terminal end of D1 (Ser 483; PALLANCK et al. 1995 ). One possible mechanism underlying comt^Su1 suppression is a direct physical interaction between Ser 483 and Ala 279. In this case, the two mutations would be complementary and thus partially restore an intramolecular interaction disrupted by comt^ST53. Because both mutations are located in D1, the structure of this domain will be required to definitively address this possibility. However, the analogous residues are conserved between the D1 and D2 domains, and thus the D2 domain structure may be instructive. The D2 residues analogous to Ser 483 and Ala 279 are well separated in this structure (LENZEN et al. 1998 ; YU et al. 1998 ), suggesting that a direct physical interaction may be unlikely.

Given the proximity of comt^Su1 to the D1 ATP-binding site, another possibility is that suppression results from an alteration in ATP binding. In vitro structure-function analysis indicates that ATP binding and hydrolysis by D1 are essential for NSF activity (WHITEHEART et al. 1994 ) and that stimulation of NSF ATPase activity causes increased disassembly of the SNARE complex (BARNARD et al. 1997 ). Thus the dominant comt^Su1 mutation might suppress comt paralysis by stimulating D1 ATPase activity.

An important step in addressing these mechanisms will be analysis of comt^Su1 alone, as well as its interactions with other comt alleles. Although it may be difficult to separate the comt^ST53 and comt^Su1 mutations by recombination (they are separated by 613 nucleotides), these interactions may be investigated by transgenic rescue of comt (PALLANCK et al. 1995 ; KAWASAKI et al. 1998 ; KAWASAKI and ORDWAY 1999A ), following site-directed mutagenesis of dNSF1 transgenes.

The comt^e1 mutation, Ala551Thr, is a recessive, intragenic enhancer of comt. Ala 551 is an invariant residue within the ATP-binding site of the D2 domain and contacts both the ribose ring and the -phosphate of bound ATP (Fig 2). In vitro structure-function analysis of NSF suggests that the D2 domain plays an important role in oligomerization of NSF, and that ATP binding (but not hydrolysis) by D2 is critical for NSF activity (WHITEHEART et al. 1994 ). comt^e1 comt^ST53 double mutants are viable and fertile, whereas severe loss of function alleles of comt are lethal. Thus, somewhat surprisingly, changing a key residue in the conserved ATP-binding site of D2 does not greatly disrupt dNSF1 activity at permissive temperature. Further analysis of the comt^e1 phenotype may begin to address the in vivo role of the D2 domain in synaptic transmission.

Extragenic modifiers:
The genetic interaction between ovr and comt^ST53 suggests that the ovr gene product may function at synapses, and thus further characterization of ovr may provide new information about the molecular mechanisms of synaptic transmission. The ability of ovr flies to stand at restrictive temperatures suggests a more subtle neuromuscular synaptic phenotype than has been observed in comt (KAWASAKI et al. 1998 , KAWASAKI et al. 1999B ; KAWASAKI and ORDWAY 1999A ).

Identification of cac^TS2 as a modifier of comt raises a number of interesting issues. The original cacophony mutant (now known as cac^S) was named on the basis of an aberrant male courtship song (VON SCHILCHER 1976 , VON SCHILCHER 1977 ). The courtship song is produced by a patterned beating of the wings, and this pattern as well as the wing beat amplitude are altered in cac^S mutants (VON SCHILCHER 1976 , VON SCHILCHER 1977 ; SMITH et al. 1998 ). Our finding that cac-encoded calcium channels function in neurotransmitter release (KAWASAKI et al. 1999A , KAWASAKI et al. 2000 ) suggests that impairment of central synapses may contribute to altered song patterning in cac^S. Given that cac-encoded 1-subunits function at flight muscle neuromuscular synapses (KAWASAKI et al. 1999A , KAWASAKI et al. 2000 ), peripheral synaptic defects may contribute to the song phenotype as well.

A second issue is whether the genetic interaction of cac^TS2 and comt reflects direct or indirect interactions of the encoded gene products. Electrophysiological analysis indicates that the cac-encoded 1-subunit mediates fast neurotransmitter release (KAWASAKI et al. 2000 ) and that dNSF1 functions in maintaining the readily releasable pool of synaptic vesicles (KAWASAKI et al. 1998 , KAWASAKI et al. 1999B ; KAWASAKI and ORDWAY 1999B ). Thus the observed genetic interaction may reflect simply that both the comt and cac gene products function in neurotransmitter release. Alternatively, the genetic interaction may result from the well-characterized biochemical interactions of SNAREs with both NSF and calcium channels. While this issue remains unresolved, two observations favor the former possibility. First, no sequence homology has been detected between the cac-encoded 1-subunit and SYNPRINT sequences (PEIXOTO et al. 1997 ; SHENG et al. 1998 ; WISER et al. 1999 ) thought to mediate direct interactions with other synaptic proteins. Second, preliminary synaptic electrophysiology in cac^TS2 comt^ST17 double mutants (F. KAWASAKI and R. W. ORDWAY, unpublished observations) is consistent with independent actions of comt and cac mutations in neurotransmitter release.

The results presented here identify four mutations that modify the comt TS paralytic phenotype. Two intragenic modifiers, comt^Su1 and comt^e1, are of interest to those investigating the structural basis of dNSF1 activity in vivo. Two extragenic modifiers include a TS allele of cac and a novel TS behavioral mutation. Analysis of synaptic vesicle trafficking in these mutants alone and in combination is expected to further define the in vivo functions and interactions of specific gene products in synaptic transmission.

	ACKNOWLEDGMENTS

We thank several members of the lab, Kamal Tilakaratne, Christian McKinney, Missy Hazen, and Fumiko Kawasaki, who participated in the isolation and/or analysis of the mutants described here. We thank Jeffrey C. Hall (Brandeis University) for generously providing several cac stocks. Deficiency stocks were provided by the Bloomington and Umeå stock centers. This work was supported by National Science Foundation grant IBN-9514485.

Manuscript received September 7, 1999; Accepted for publication February 2, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ASHBURNER, M., 1989 Drosophila. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

BANERJEE, A., V. A. BARRY, B. R. DASGUPTA, and T. F. J. MARTIN, 1996  N-ethylmaleimide-sensitive factor acts at a prefusion ATP-dependent step in Ca²⁺-activated exocytosis. J. Biol. Chem. 271:20223-20226[Abstract/Free Full Text].

BARNARD, R. J. O., A. MORGAN, and R. D. BURGOYNE, 1997  Stimulation of NSF ATPase activity by -SNAP is required for SNARE complex disassembly and exocytosis. J. Cell Biol. 139:875-883[Abstract/Free Full Text].

BEZPROZVANNY, I., R. H. SCHELLER, and R. W. TSIEN, 1995  Functional impact of syntaxin on gating of N-type and Q-type calcium channels. Nature 378:623-626[Medline].

CATTERALL, W. A., 1998  Structure and function of neuronal Ca²⁺ channels and their role in neurotransmitter release. Cell Calcium 24:307-323[Medline].

CHEN, Y. A., S. J. SCALES, S. M. PATEL, Y.-C. DOUNG, and R. H. SCHELLER, 1999  SNARE complex formation is triggered by Ca²⁺ and drives membrane fusion. Cell 97:165-174[Medline].

COORSSEN, J. R., P. S. BLANK, M. TAHARA, and J. ZIMMERBERG, 1998  Biochemical and functional studies of cortical vesicle fusion: the SNARE complex and Ca²⁺ sensitivity. J. Cell Biol. 143:1845-1857[Abstract/Free Full Text].

DELLINGER, B. B., R. FELLING, C. MCKINNEY, F. KAWASAKI and R. W. ORDWAY, 1999 Temperature-sensitive paralytic mutants reveal in vivo functional interactions between NSF and calcium channel proteins in synaptic transmission. Abstracts of the 40th Annual Drosophila Research Conference, March 1999, Seattle.

GRIGLIATTI, T. A., L. HALL, R. ROSENBLUTH, and D. T. SUZUKI, 1973  Temperature-sensitive mutations in Drosophila melanogaster. XIV. A selection of immobile adults. Mol. Gen. Genet. 120:107-114[Medline].

HANSON, P. I., J. E. HEUSER, and R. JAHN, 1997a  Neurotransmitter release—four years of SNARE complexes. Curr. Opin. Cell Biol. 7:310-315.

HANSON, P. I., R. ROTH, H. MORISAKI, R. JAHN, and J. E. HEUSER, 1997b  Structure and conformational changes in NSF and its membrane receptor complexes visualized by quick-freeze/deep-etch electron microscopy. Cell 90:523-535[Medline].

HAY, J. C. and R. H. SCHELLER, 1997  SNAREs and NSF in targeted membrane fusion. Curr. Opin. Cell Biol. 9:505-512[Medline].

HEISENBERG, M. and K. G. GÖTZ, 1975  The use of mutations for the partial degradation of vision in Drosophila melanogaster.. J. Comp. Physiol. 98:217-241.

KAWASAKI, F. and R. W. ORDWAY, 1999a  The Drosophila NSF protein, dNSF1, plays a similar role at neuromuscular and some central synapses. J. Neurophysiol. 82:123-130[Abstract/Free Full Text].

KAWASAKI, F., and R. W. ORDWAY, 1999b A new model synapse preparation allows direct demonstration of a strictly presynaptic role for the Drosophila NSF protein, dNSF1. Abstracts of the 40th Annual Drosophila Research Conference, March 1999, Seattle.

KAWASAKI, F., A. M. MATTIUZ, and R. W. ORDWAY, 1998  Synaptic physiology and ultrastructure in comatose mutants define an in vivo role for NSF in neurotransmitter release. J. Neurosci. 18:10241-10249[Abstract/Free Full Text].

KAWASAKI, F., R. FELLING and R. W. ORDWAY, 1999a A temperature-sensitive paralytic mutant defines a primary synaptic calcium channel in Drosophila. Abstracts of the Cold Spring Harbor Meeting on Neurobiology of Drosophila, October, 1999, Cold Spring Harbor, NY.

KAWASAKI, F., M. HAZEN and R. W. ORDWAY, 1999b A strictly presynaptic function for the Drosophila NSF protein, dNSF1, includes a role in fast recycling of synaptic vesicle membranes. Abstracts of the Cold Spring Harbor Meeting on Neurobiology of Drosophila, October, 1999, Cold Spring Harbor, NY.

KAWASAKI, F., R. FELLING, and R. W. ORDWAY, 2000  A temperature-sensitive paralytic mutant defines a primary synaptic calcium channel in Drosophila. J. Neurosci. in press.

LENZEN, C. U., D. STEINMANN, S. W. WHITEHEART, and W. I. WEIS, 1998  Crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein. Cell 94:525-536[Medline].

MOCHIDA, S., Z.-H. SHENG, B. CARL, H. KOBAYASHI, and W. A. CATTERALL, 1996  Inhibition of neurotransmission by peptides containing the synaptic protein interaction site of N-type Ca²⁺ channels. Neuron 17:781-788[Medline].

ORDWAY, R. W., L. PALLANCK, and B. GANETZKY, 1994  Neurally expressed Drosophila genes encoding homologs of the NSF and SNAP secretory proteins. Proc. Natl. Acad. Sci. USA 91:5715-5719[Abstract/Free Full Text].

PALLANCK, L., R. W. ORDWAY, and B. GANETZKY, 1995  A Drosophila NSF mutant. Nature 376:25[Medline].

PEIXOTO, A. A. and J. C. HALL, 1998  Analysis of temperature-sensitive mutants reveals new genes involved in the courtship song of Drosophila.. Genetics 148:827-838[Abstract/Free Full Text].

PEIXOTO, A. A., L. A. SMITH, and J. C. HALL, 1997  Genomic organization and evolution of alternative exons in a Drosophila calcium channel gene. Genetics 145:1003-1013[Abstract].

RETTIG, J., C. HEINEMANN, U. ASHERY, Z.-H. SHENG, and C. T. YOKOYAMA et al., 1997  Alteration of Ca²⁺ dependence of neurotransmitter release by disruption of Ca²⁺ channel/syntaxin interaction. J. Neurosci. 17:6647-6656[Abstract/Free Full Text].

ROTHMAN, J. E. and F. T. WIELAND, 1996  Protein sorting by transport vesicles. Science 272:227-234[Abstract].

SCHWEIZER, F. E., T. DRESBACH, W. M. DEBELLO, V. O'CONNOR, and G. J. AUGUSTINE et al., 1998  Regulation of neurotransmitter release kinetics by NSF. Science 279:1203-1206[Abstract/Free Full Text].

SHENG, Z.-H., R. E. WESTENBROEK, and W. A. CATTERALL, 1998  Physical link and functional coupling of presynaptic calcium channels and the synaptic vesicle docking/fusion machinery. J. Bioenerg. Biomembr. 30:335-345[Medline].

SIDDIQI, O. and S. BENZER, 1976  Neurophysiological defects in temperature-sensitive paralytic mutants of Drosophila melanogaster.. Proc. Natl. Acad. Sci. USA 73:3253-3257[Abstract/Free Full Text].

SMITH, L. A., X. WANG, A. A. PEIXOTO, E. K. NEUMANN, and L. M. HALL et al., 1996  A Drosophila calcium channel 1 subunit gene maps to a genetic locus associated with behavioral and visual defects. J. Neurosci. 16:7868-7879[Abstract/Free Full Text].

SMITH, L. A., A. A. PEIXOTO, E. M. KRAMER, A. VILLELLA, and J. C. HALL, 1998  Courtship and visual defects of cacophony mutants reveal functional complexity of a calcium-channel 1 subunit in Drosophila.. Genetics 149:1407-1426[Abstract/Free Full Text].

SÖLLNER, T., M. K. BENNETT, S. W. WHITEHEART, R. H. SCHELLER, and J. E. ROTHMAN, 1993  A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75:409-418[Medline].

SUZUKI, D. T., T. GRIGLIATTI, and R. WILLIAMSON, 1971  Temperature-sensitive mutations in Drosophila melanogaster. VII. A mutation (para^TS) causing reversible adult paralysis. Proc. Natl. Acad. Sci. USA 68:890-893[Abstract/Free Full Text].

TOLAR, L. A. and L. PALLANCK, 1998  NSF function in neurotransmitter release involves rearrangement of the SNARE complex downstream of synaptic vesicle docking. J. Neurosci. 18:10250-10256[Abstract/Free Full Text].

UNGERMANN, C., K. SATO, and W. WICKNER, 1998  Defining the functions of trans-SNARE pairs. Nature 396:543-548[Medline].

VON SCHILCHER, F., 1976  The behavior of cacophony, a courtship song mutant in Drosophila melanogaster. Behav. Biol. 17:187-196[Medline].

VON SCHILCHER, F., 1977  A mutation which changes courtship song in Drosophila melanogaster. Behav. Genet. 7:251-259[Medline].

WEBER, T., B. V. ZEMELMAN, J. A. MCNEW, B. WESTERMANN, and M. GMACHL et al., 1998  SNAREpins: minimal machinery for membrane fusion. Cell 92:759-772[Medline].

WHEELER, D. B., A. RANDALL, W. A. SATHER, and R. W. TSIEN, 1995  Neuronal calcium channels encoded by the 1A subunit and their contribution to excitatory synaptic transmission in the CNS. Progr. Brain Res. 105:65-78[Medline].

WHITEHEART, S. W., K. ROSSNAGEL, S. A. BUHROW, M. BRUNNER, and R. JAENICKE et al., 1994  N-ethylmaleimide-sensitive fusion protein: a trimeric ATPase whose hydrolysis of ATP is required for membrane fusion. J. Cell Biol. 126:945-954[Abstract/Free Full Text].

WISER, O., M. TRUS, A. HERNÁNDEZ, E. RENSTRÖM, and S. BARG et al., 1999  The voltage sensitive Lc-type Ca²⁺ channel is functionally coupled to the exocytotic machinery. Proc. Natl. Acad. Sci. USA 96:248-253[Abstract/Free Full Text].

XU, T., U. ASHERY, R. D. BURGOYNE, and E. NEHER, 1999  Early requirement for -SNAP and NSF in the secretory cascade in chromaffin cells. EMBO J. 18:3293-3304[Medline].

YU, R. C., P. I. HANSON, R. JAHN, and A. T. BRÜNGER, 1998  Structure of the ATP-dependent oligomerization domain of N-ethylmaleimide sensitive factor complexed with ATP. Nat. Struct. Biol. 5:803-811[Medline].

YU, R. C., R. JAHN, and A. T. BRUNGER, 1999  NSF N-terminal domain crystal structure: models of NSF function. Mol. Cell 4:97-107[Medline].

This article has been cited by other articles:

				H. A. Lindsay, R. Baines, R. ffrench-Constant, K. Lilley, H. T. Jacobs, and K. M. C. O'Dell The Dominant Cold-Sensitive Out-Cold Mutants of Drosophila melanogaster Have Novel Missense Mutations in the Voltage-Gated Sodium Channel Gene paralytic Genetics, October 1, 2008; 180(2): 873 - 884. [Abstract] [Full Text] [PDF]

				D. K. Dickman, P. T. Kurshan, and T. L. Schwarz Mutations in a Drosophila {alpha}2{delta} Voltage-Gated Calcium Channel Subunit Reveal a Crucial Synaptic Function J. Neurosci., January 2, 2008; 28(1): 31 - 38. [Abstract] [Full Text] [PDF]

				F.-D. Huang, E. Woodruff, R. Mohrmann, and K. Broadie Rolling Blackout Is Required for Synaptic Vesicle Exocytosis J. Neurosci., March 1, 2006; 26(9): 2369 - 2379. [Abstract] [Full Text] [PDF]

				Y. Wu, F. Kawasaki, and R. W. Ordway Properties of Short-Term Synaptic Depression at Larval Neuromuscular Synapses in Wild-Type and Temperature-Sensitive Paralytic Mutants of Drosophila J Neurophysiol, May 1, 2005; 93(5): 2396 - 2405. [Abstract] [Full Text] [PDF]

				F. Kawasaki, B. Zou, X. Xu, and R. W. Ordway Active Zone Localization of Presynaptic Calcium Channels Encoded by the cacophony Locus of Drosophila J. Neurosci., January 7, 2004; 24(1): 282 - 285. [Abstract] [Full Text] [PDF]

				R. Rikhy, M. Ramaswami, and K. S. Krishnan A Temperature-Sensitive Allele of Drosophila sesB Reveals Acute Functions for the Mitochondrial Adenine Nucleotide Translocase in Synaptic Transmission and Dynamin Regulation Genetics, November 1, 2003; 165(3): 1243 - 1253. [Abstract] [Full Text] [PDF]

				I. M. Brooks, R. Felling, F. Kawasaki, and R. W. Ordway Genetic Analysis of a Synaptic Calcium Channel in Drosophila: Intragenic Modifiers of a Temperature-Sensitive Paralytic Mutant of cacophony Genetics, May 1, 2003; 164(1): 163 - 171. [Abstract] [Full Text] [PDF]

				B. Chan, A. Villella, P. Funes, and J. C. Hall Courtship and Other Behaviors Affected by a Heat-Sensitive, Molecularly Novel Mutation in the cacophony Calcium-Channel Gene of Drosophila Genetics, September 1, 2002; 162(1): 135 - 153. [Abstract] [Full Text] [PDF]

				F. Kawasaki, S. C. Collins, and R. W. Ordway Synaptic Calcium-Channel Function in Drosophila: Analysis and Transformation Rescue of Temperature-Sensitive Paralytic and Lethal Mutations of Cacophony J. Neurosci., July 15, 2002; 22(14): 5856 - 5864. [Abstract] [Full Text] [PDF]

				F. Kawasaki, R. Felling, and R. W. Ordway A Temperature-Sensitive Paralytic Mutant Defines a Primary Synaptic Calcium Channel in Drosophila J. Neurosci., July 1, 2000; 20(13): 4885 - 4889. [Abstract] [Full Text] [PDF]