Cysteine string proteins (CSPs) are conserved secretory vesicle proteins involved in regulating neurotransmitter and peptide release. While the function of the J-domain has been studied in detail, little is known about other conserved regions. We have constructed mutant genes coding for proteins with modified cysteine string, linker region or C terminus and transformed them into Csp null-mutant Drosophila. In the living animal,mutated CSP lacking all cysteines fails to associate with membranes, does not concentrate in synaptic terminals, and cannot rescue adult temperature-sensitive paralysis and short life span, both prominent null mutant phenotypes. A mutant protein with 5 instead of 11 string cysteines appears to be normally targeted but cannot rescue paralysis at 37°C. We propose that the cysteine string, in addition to its role in targeting, may be essential for a function of CSP that is dependent on the number of cysteines in the string. A deletion in the linker region or the C terminus does not affect CSP targeting, and function in adults is only marginally impaired.

Cysteine string proteins (CSPs) are unique cysteine-rich proteins present on synaptic vesicles and secretory organelles(Buchner and Gundersen, 1997; Chamberlain and Burgoyne, 2000; Zinsmaier and Bronk, 2001). The characteristic and conserved regions in the primary structure of invertebrate and vertebrate CSPs comprise an N-terminal phosphorylation site for protein kinase A (PKA), a J-domain, a cysteine string (CS) consisting of 13–15 cysteines within a stretch of 25 amino acids, a `linker' region connecting J and CS, and a less conserved C-terminal domain.

Phosphorylation of vertebrate CSP at Ser10 reduces its interaction with syntaxin and may thus modulate exocytosis(Evans et al., 2001). The J-domain has been linked to a putative co-chaperone function of CSPs. CSPs bind to and stimulate the ATPase activity of the molecular chaperone HSC70 only if the J-domain is intact (Braun et al., 1996; Chamberlain and Burgoyne, 1997). In Drosophila the Csp and hsc70-4 genes interact (Bronk et al., 2001). Recently, the small glutamine-rich tetratrico-peptide repeat protein (SGT) has been identified as a third component of this chaperone complex (Tobaben et al.,2001). The function of the cysteine string has been controversial. The extensive palmitoylation of the cysteine string suggested a role in membrane attachment (Gundersen et al.,1994); however, depalmitoylation does not displace CSPs from membranes (van de Goor and Kelly,1996; Chamberlain and Burgoyne,1998), indicating that the hydrophobic amino acids associated with the cysteine string are sufficient to keep the protein attached to the membrane (Mastrogiacomo et al.,1998). Replacement of seven of the 14 cysteines in PC12 and HeLa cells demonstrated that the intact string is required for initial membrane targeting in cultured cells (Chamberlain and Burgoyne, 1998). Information on the function of the linker region and the C terminus is sparse so far. The inhibitory effect on regulated exocytosis of overexpressing the mammalian CSP2 isoform in hamster insulinoma cells is reduced by a point mutation in the linker region. The C-terminal difference between the two known vertebrate isoforms appears to be critical for this suppression (Zhang et al.,1999). Linker and/or cysteine string appear to be important for CSP-CSP self-association (Swayne et al.,2003).

Deletion of the Csp gene from the Drosophila genome causes semi-lethality during prolonged development, a severely shortened life span of escapers, temperature-sensitive paralysis, and breakdown of evoked synaptic transmission at elevated temperatures(Umbach et al., 1994; Zinsmaier et al., 1994; Eberle et al., 1998). The complexity of the null phenotype suggests that CSP function may be dissected,i.e. that the integrity of different regions of the protein can be related to different aspects of its function in the wild type.

In the present study we therefore mutated in vitro the regions of the Drosophila Csp cDNA that code for the cysteine string, the linker region and the C terminus, ligated the mutated cDNAs to Cspregulatory sequences, and transformed the modified gene into the germline of wild-type and null-mutant flies. The phenotype of these transgenic flies as well as the distribution and biochemical properties of the mutated proteins are investigated. We show that an intact cysteine string is required for almost all known functional aspects of the protein, whereas deletions in the linker region or the C-terminal domain cause more selective defects.

Materials

Wild-type flies and transgenic mutants were raised on standard food medium at 18°C and kept over balancer TM6,Tb,Sb unless otherwise noted. The CspU1 mutant was generated by unequal crossing over between two P(w+) elements flanking the Csp gene and thus contains a P(w+) element in place of this gene. In the CspU1w mutant this P(w+) element has been removed by remobilization (Eberle et al.,1998). Both strains are in w1118 background. To eliminate genetic modifiers from the CspU1 strain these(red-eyed) flies were out-crossed to w1118 for 12 generations and selected for the red eye color marker of the P(w+) element to generate the strain CspU1oc, which was then maintained over TM6,Sb,Tb balancer.

Antibodies were obtained from the following sources. Anti-Drosophila CSP: DCSP1 is mouse monoclonal antibody (mAb) ab49(Hofbauer, 1991; Buchner et al., 1986), DCSP2(6D6) and DCSP3 (1G12) were kindly provided by K. Zinsmaier. Anti-Drosophila SAP47: mouse mAb nc46(Hofbauer, 1991; Reichmuth et al., 1995). Anti-Drosophila syntaxin: 3D8 was kindly provided by S. Benzer(Caltech, Pasadena, CA, USA) and later obtained from Developmental Studies Hybridoma Bank (DSHB), University of Iowa, USA.

The QuickChange™ site-directed mutagenesis kit was obtained from Stratagene (Cambridge, UK). Restriction enzymes were purchased from Life Technologies (Karlsruhe, Germany) and Fermentas MBI (St Leon-Rot, Germany). Enhanced chemiluminescense antibody binding detection kit was obtained from Amersham Corp. (Slough, UK). Oligonucleotides were synthesized by MWG(Ebersberg, Germany).

Generation of the plasmid constructs

Csp cDNA-1 [`lcz49-9'; Zinsmaier et al.(1990); `type-I'; Zinsmaier et al. (1994)], was cleaved by Bsp1407I and EcoRI and the resulting 1.9 kb fragment containing 3′ sequences of exon 3 and exons 4–7. This fragment was ligated to a HindIII/Bsp1407I (long) or an Asp718/Bsp1407I (short) genomic fragment, which contained (long or short) regulatory sequences of the Csp gene, exon 1, intron 1, exon 2, intron 2, and the 5′ sequences completing exon 3. Thus the two constructs generate a short and a long Csp cDNA-1 rescue construct, scDna1 and lcDna1, respectively (cf. Fig. 1C,D). The fragments were cloned into the pW8 P-element vector(Klemenz et al., 1987) and transformed into w1118 flies by standard procedures(Ashburner, 1989).

For site-directed mutagenesis the type-I cDNA was used as template in the QuickChange™ system. The deletion of 11 cysteines was achieved in two steps. The forward primers (reverse = complement) used were 5′-CACTGGATGCTGTTGTTGCTGCAACTTCTGCTGC-3′ for the first step,resulting in the deletion of six cysteines in the construct Scsp(short cysteine string protein), and 5′-TGTGCCGTGATCACTGGAAACTTCTGCTGCGG-3′ for the second step,leading to the construct Cslp (cysteine string less protein). The conversion of the two flanking pairs of cysteines to serines was again obtained in two steps. The primers for this mutation were 5′-AAGGCGGTGGTTATCTCCTCTGCCGTGATCACTGGA-3′ and its complement for the first step and 5′-TGATCACTGGAAACTTCTCCTCCGGCAAGTTCAAGC-3′ and its complement for the second step, to obtain the construct Clp(cysteine less protein). For the replacement of the cysteine string by a serine string in the Ssp (serine string protein) construct the primers were 5′-CGTGATCACTGGATCCTCTTCTTCCTCCTCCTCCTCCTCCTCTTCCAACTTCTGCTGCGG-3′and its complement. Deletion of the eight amino acids AEQFGEEN in the linker region of the LΔ8 construct was achieved by the primer 5′-CGGCTTGTACATAGTCAACGCATACTTCGTGGTCACTTCACCG-3′ and its complement. In order to eliminate 27 C-terminal amino acids in the CΔ27 construct the condons for the amino acids PVAA were replaced by TAGTAGTAGCT, introducing three inframe stop codons and a frame shift.

To confirm the fidelity of the introduced mutations the DNA of all mutant clones was sequenced prior to transformation. For transformation into the genome of Drosophila the modified cDNA was cleaved and ligated to the putative genomic promoter/enhancer region of Csp gene as described above for the rescue vector lcDna1.

Preparation and chemical treatment of cell membranes

Heads of wild-type and mutant Drosophila were either directly homogenized in 1 μl head–1 of sample buffer(Laemmli, 1970) or collected on ice and homogenized with 5 μl head–1 buffer A (150 mmol l–1 NaCl, 10 mmol l–1 Hepes, pH 7.4, 1 mmol l–1 EGTA, 0.1 mmol l–1 MgCl2). A post-nuclear supernatant was obtained by 1000 g centrifugation for 10 min at 4°C. The supernatant was subjected to a 100 000 g centrifugation for 60 min at 4°C to separate soluble proteins from proteins attached to membranes or cytoskeleton. For deacylation pellets were resuspended in 1 mol l–1 hydroxylamine (pH 7)and incubated for 20 h at room temperature. After incubation, an additional 60 min centrifugation step at 100 000 g and 4°C followed in order to separate proteins solubilized by the treatment (S3) from the membrane fraction (P3). Pellets were finally dissolved in equivalent volumes of sample buffer, and proteins from equal volumes of supernatants and dissolved pellets were separated on 12.5% gels by SDS-PAGE followed by western blot analysis using monoclonal antibodies. Separation of different membrane fractions was achieved by glycerol gradient velocity sedimentation as described by van de Goor et al. (1995). All experiments were done at least twice.

Immunochemistry

Immunohistochemistry was carried out as described earlier(Buchner et al., 1986; Ashburner, 1989). The mouse monoclonal supernatants were applied at dilutions of 1:10 to 1:100. For visualizing antibody binding the ABC detection system (Vector Labs,Burlingame, USA) was used. Western blots were incubated in monoclonal supernatants at 1:100 dilution and developed using the ECL detection system as recommended by the manufacturer.

Epitope mapping

The epitopes of the three anti-CSP monoclonal antibodies used in this study(DCSP1-3) were mapped by spotting sequential decapeptides on nitrocellulose membranes according to a procedure described in detail elsewhere(Munch et al., 1999), followed by spot detection using ECL as in western blots. Amino acids common to decapeptides binding the antibody are defining the epitope.

Behavioral analysis

Temperature sensitivity of the various genotypes was scored by transferring groups of 10–15 1-day-old flies in empty food vials to a water bath of defined temperature and counting fully paralyzed animals after 5 min. To prevent interference of adaptive phenomena all flies were tested only once. Effects of variations in genetic background were minimized by using flies that had recently been crossed into the out-crossed null mutant CspU1oc.

Survival analysis

Normalized life span was determined by transferring groups of 10 F1 siblings from heterozygote crosses to fresh food vials at 25°C and counting dead flies daily.

Electrophysiology

For electrophysiological recordings climbing unbalanced third instar larvae were immobilized for 20 min on ice. Preparation and recording essentially followed the procedure described by Stewart et al.(1994). Larvae were dissected in an Elastosil® coated Petri dish under modified HL3 Ringer containing 1 mmol l–1 Ca2+ starting with mid-dorsal incision.`Filets' were pinned out flat and internal organs were removed carefully. After transfer to a temperature-controlled fixed microscope stage,intracellular recordings were made from ventral longitudinal muscle 6 of abdominal half-segment 3 using quartz glass microelectrodes of 20–30 MΩ resistance, prepared by a laser-based microelectrode puller (P-2000,Sutter Instrument Co., Novato, CA, USA) and filled with 3 mol l–1 KCl. Excitatory junctional potentials (EJPs) were evoked by stimulating the distal portion of the motor nerve taken up into a fire-polished borosilicate suction electrode. Stimulus amplitude of 0.1 ms pulses was adjusted to a value 2.5-fold of the initial threshold. Signals were amplified with Neuroprobe Amplifier (Model 1600, A-M Systems, Inc., Carlsborg,WA, USA), low-pass filtered at 50 kHz, and digitized via an A/D interface (Data Acquisition Processor DAP 3200e/315, Microstar Laboratories,Inc., Bellevue, WA, USA). Stimuli, signals and temperature were digitized at 10 kHz, data were analyzed with DASYLab Ver. 5.03.30 (measX, GmbH and Co. KG,Mönchenglattbach, Germany). Temperature was measured by a temperature sensor (Newport Electronics GmbH, Deckenpfronn, Germany), positioned in the Ringer solution near the larval filet. For each preparation six evoked EJPs were averaged 5–7 min after reaching the set temperature. If more than six stimuli produced no response the amplitude was registered as `zero'. Each experiment was performed with at least four larvae. Statistical analysis was performed using GraphPad Instat™ Ver. 2.04a (GraphPad Software, San Diego, CA, USA) and Statistica Ver. 6 (StatSoft, Inc., Tulsa, OK, USA).

In Drosophila four different CSP isoforms (denoted as CSP1–CSP4 in Fig. 1) are observed in western blots of head homogenates(Zinsmaier et al., 1994),while only the largest isoform (CSP1) is detected in non-neural organs(Eberle et al., 1998). No information is presently available on the functional differentiation of the various isoforms or the relevance of their selective distribution in the brain(Eberle et al., 1998; Nie et al., 1999). Three different cDNAs have been isolated so far (Zinsmaier et al., 1990, 1994). In order to assign these cDNAs to the isoforms detected in western blots we mapped the epitopes of the three available monoclonal antibodies against Drosophila CSPs,mAbs DCSP1, DCSP2 and DCSP3, by oligopeptide spotting. The boxes marked`DCSP1', `DCSP2', and `DCSP3' in Fig. 1A comprise those amino acids that are common to all decapeptide spots recognized by the respective antibody. The data demonstrate that mAbs DCSP1 and DCSP2 recognize all cDNA-encoded proteins while mAb DCSP3 binds only to the isoforms encoded by cDNA-1 and cDNA-3(Zinsmaier et al., 1994; Eberle et al., 1998). We speculate that a hitherto undetected fourth splice variant (cDNA-2*) may code for the second largest isoform recognized in western blots, as indicated in Fig. 1B, which summarizes cDNA structures, approximate isoform sizes, and antibody recognition in western blots.

The main aim of the present work was to determine the involvement of the cysteine string, the linker region, and the C terminus of DrosophilaCSP in membrane association, synaptic vesicle targeting and function. We first tested whether a single isoform (CSP1) expressed in Csp null mutants can `rescue' the mutant phenotype. To this end we designed two different wild-type Csp cDNA `rescue' gene constructs, scDna1 and lcDna1. The various transgenic mutants generated by transforming in vitro mutagenized versions of these cDNA constructs into Csp-null background can then be compared to the `rescue'transformants in order to assess the effect of the mutations on CSP function.

cDNA-1 rescues all major phenotypes of Csp null mutants

We used cDNA-1, which contains the largest open reading frame. Since the regulatory region of the Csp gene has not yet been characterized we tested two genomic fragments of different lengths upstream of the gene as depicted in Fig. 1D for their capacity to direct wild-type expression of the cDNA. Because there was evidence that the first intron may contain important Csp regulatory sequences (K. E. Zinsmaier, personal communication), fragments were chosen that extend from 2.3 kb (scDna1) or 4.5 kb (lcDna1) upstream of the transcription start to a Bsp1407I site in the third exon and thus include the first two introns. These genomic fragments were ligated to the cDNA-1 fragment downstream of the Bsp1407I site (hatched in Fig. 1D). The constructs were transformed into the germ line of Drosophila wild type. Since no difference has been observed in the expression or function of the two constructs, both are referred to below as construct cDna1. In western blots of head homogenates from cDna1 transgenic flies the monoclonal antibody DCSP-1 detects a highly overexpressed largest CSP isoform in addition to the three other wild-type isoforms (data not shown). Surprisingly, we do not observe any of the phenotypes described for flies overexpressing CSP by use of the UAS/GAL4 system (cf. Discussion). Head homogenates of flies homozygous for the cDna1 transgene and the null allele CspU1w produce only a single very strong signal on western blots. This signal matches in size the largest isoform of wild-type homogenates, and in fractionation and deacylation experiments behaves like all four isoforms of wild-type flies (Figs 2, 3, and data not shown). Immunohistochemistry employing mAb DCSP1 on head and body sections of these flies shows an expression pattern of CSP1 both in nervous and non-nervous tissues that is indistinguishable from wild-type stainings(Eberle et al., 1998), with high concentrations of CSP1 in synaptic terminals on muscles and in the neuropil of brain and thoracic ganglia(Fig. 4 and data not shown). All tested cDna1 transformants rescue the premature death(Fig. 5A) and the temperature-sensitive paralytic phenotype(Fig. 6) of the null mutant CspU1, demonstrating that the 2.3·kb upstream genomic region and the first two introns contain all essential regulatory regions of the Csp gene and that the CSP1 isoform is sufficient to maintain all known functions of the four natural isoforms. Thus cDna1constructs can be used as targets for introducing site-directed mutations into the Csp gene in vitro in order to study the effects of such mutations in vivo after transformation of the modified gene into CspU1 flies. cDna1 transformants and CspU1 null mutants serve as positive and negative controls, respectively.

Generation of transgenic flies with deletions in the linker, cysteine string and C-terminal regions of CSP

Mutation of conserved amino acids in a protein is likely to modify its function. We therefore designed Csp transgene constructs coding for the following modified proteins: a variant with a deletion of eight amino acids (underlined in Fig. 1A)in the linker region (LΔ8), four different variants with mutated cysteine string (Fig. 1E), and a variant with a deletion of the 27 C-terminal amino acids (underlined in Fig. 1A; CΔ27). In the`short cysteine string protein' (SCSP)variant, six amino acids of the cysteine string are deleted while the entire string of 11 cysteines is removed in the `cysteine string-less protein' (CSLP) variant. In the variant`serine string protein' (SSP), these 11 cysteines are replaced by 11 serines. Serine is a small, uncharged amino acid with structural similarity to cysteine. Replacement of the cysteine string by a serine string possibly produces smaller overall structural changes in the protein than its deletion. In the variant `cysteine-less protein' (CLP) the 11 string cysteines have been deleted and the two flanking pairs of cysteines mutated to serines(Fig. 1E) such that the encoded protein contains no cysteines. Transgenic flies with these constructs were generated as described above for the lcDna1 construct and were crossed into CspU1w and CspU1oc mutant background (cf. Materials and methods for difference between these two mutant strains).

Analysis of the mutated proteins

Head homogenates of wild-type and transgenic flies in CspU1w null mutant or wild-type background were analyzed on immunoblots stained with DCSP1 and DCSP2 antibody (Figs 2, 3). No qualitative differences were observed between blots stained with these two antibodies except for the CΔ27 isoform, which is not recognized by DCSP1. Apparently, the epitope at the very C-terminal end of the truncated protein cannot bind the antibody(Fig. 1A). The calculated molecular masses of the serine string protein (SSP), the short cysteine string protein (SCSP), the cysteine string-less protein (CSLP) and the cysteine-less protein (CLP) differ from the largest wild-type isoform CSP1 only by 0.176,0.726, 1.331 and 1.395 kDa, respectively. The apparent molecular mass as seen in the SDS gel, however, is approximately 6, 4, 7 and 7 kDa smaller,respectively, compared to the 36 kDa CSP1 isoform. This discrepancy is presumably due to the fact that most cysteines of wild-type CSP are palmitoylated. Indeed, the untreated SSP migrates at about the same position as the deacylated 36 kDa CSP1 isoform or the largest deacylated isoform of wild-type head homogenate (Fig. 2C). Hydroxylamine treatment of S2 supernatants of SSP and CLP did not alter apparent molecular masses demonstrating that these proteins are not significantly acylated (data not shown). The C-terminal deletion of 27 amino acids leads to a shift in western blots of about 5 kDa, somewhat larger than the calculated loss of 3.868 kDa. In contrast, the protein with a deletion in the linker region (LΔ8) shows the expected approximate molecular mass difference of 1.048 to the 36 kDa wild-type isoform(Fig. 2A). Note that the strength of the western signals of all mutated proteins except CΔ27 is strongly reduced (Fig. 2A; cf. Discussion).

In order to test if the mutated proteins associate with membranes,homogenates of heads from transformants and wild type were separated into cytosolic and membrane fractions and analyzed by immunoblotting(Fig. 2B). In contrast to the wild-type CSP isoforms from wild-type flies or cDna1 transformants,the CLP and SSP mutant forms can be detected only in the soluble fraction. This is confirmed by glycerol gradient velocity sedimentation experiments using cDna1, Ssp and Clp flies in CspU1wand wild-type background (Fig. 3). Endogenous or transgenic wild-type CSPs migrate predominantly in the synaptic vesicle fractions (van de Goor et al., 1995) whereas SSP and CLP comigrate with the loading control protein SAP47, which is soluble(Reichmuth et al., 1995). Transgenic cysteine string-less protein CSLP, on the other hand, is found in similar concentrations in soluble and membrane fractions(Fig. 2B). The protein with the short cysteine string (SCSP) is found in the pellet, similar to the wild-type isoforms. The fractionation of wild-type CSPs is apparently not significantly influenced by the overexpression in the same cells of the CSP1 isoform in a wild-type background. The proteins with intact cysteine string but deletions in the linker or C-terminal domains, LΔ8 and CΔ27, are found in the synaptic vesicle fractions and, compared to wild-type CSPs, appear to be somewhat enriched in the plasma membrane fractions (Figs 2, 3). Fractions containing plasma membranes are identified in the gradient by use of an antibody against syntaxin (mAb 8C3, SYX in Fig. 3).

Tissue distribution of mutated proteins

In wild-type flies, CSPs are highly concentrated in synaptic neuropil,synaptic terminals on muscles, and various non-nervous structures(Eberle et al., 1998). No differences in the immunohistochemical staining patterns have been observed between wild-type flies and cDna1;CspU1w transformants(Fig. 4A,B, and data not shown). In these experiments head sections of wild-type and transgenic flies were stained with mAb DCSP1 on the same microscope slide to ensure identical immunohistochemical treatment. Comparison of Fig. 4C with D and F reveals the selective disruption of synaptic terminal targeting of the cysteine string mutant proteins SSP and CLP in the respective transformants in null mutant background. In Ssp;CspU1w or Clp;CspU1w the modified proteins distribute rather homogeneously throughout all parts of the brain, including cellular rind,axonal bundles (e.g. in the optic chiasms) and neuropil(Fig. 4D,F). In the Cslp;CspU1w transformant a certain fraction of the modified protein is apparently targeted to the synaptic terminals while the remaining fraction distributes to other parts of the neurons(Fig. 4E). The deletion of six cysteines of the string (SCSP) (Fig. 4G) or of eight amino acids in the linker region (LΔ8) (not shown) strongly reduces staining intensity but does not alter the distribution of the protein. Deletion of the C terminus (CΔ27) (not shown) does not affect immunohistochemical staining patterns. For comparison we show stainings of two mutants. The CspU2 mutation(Eberle et al., 1998) leaves the coding region intact but carries a B104 transposon insertion in intron 4 of the Csp gene. This mutant shows selective CSP accumulation in mushroom body neuropil (not shown) and in synaptic terminals of photoreceptors and visual interneurons (Fig. 4H), but CSP expression in the rest of the brain is drastically reduced. The CspU1 null mutant serves as a negative control (Fig. 4I),demonstrating that fat body staining is unspecific.

Phenotypes of cysteine-string mutant transformants

Longevity

A severely reduced life span of adult flies has been observed previously as a major phenotype of Csp null mutants. We noted that quantitative aspects of this and other phenotypic traits of CspU1flies, such as the degree of developmental delay and semi-lethality, the critical temperature for the failure of larval synaptic transmission or adult paralysis, all significantly depend on genetic background. The originally semilethal CspU1 and CspU1w stocks(Eberle et al., 1998) that had been kept at 18°C in the homozygous condition could after several years be maintained at 25°C, larvae no longer showed reversible synaptic failure in nerve muscle preparations at 32°C, and adults lived significantly longer than observed previously. We therefore out-crossed for 12 generations the CspU1 stock against white(w1118;Csp+ in Canton-S background) until we noted that homozygous flies again showed the strong phenotype described earlier (Umbach et al., 1994; Zinsmaier et al., 1994). To further reduce the influence of genetic background we performed the experiments analyzing phenotypes of the transformants on siblings whenever possible. With P(tg) transgene insertions on the X chromosome (tg=cDna1, Ssp, LΔ8 or CΔ27), crosses of TM3 balanced flies heterozygous for the out-crossed CspU1oc mutation and the P insertion of the transformation (all in wbackground) were used:
\[\frac{w,\mathrm{P(tg)}}{w};;\frac{Csp^{Uloc}}{\mathrm{TM}3}{\times}\frac{w,\mathrm{P(tg)}}{\mathrm{Y}};;\frac{Csp^{Uloc}}{\mathrm{TM}3}.\]
where TM3 denotes the balancer chromosome and Y the Y-chromosome.
The survival functions at 25°C for the balanced flies (one copy of the Csp+ gene), the homozygous CspU1ocflies and the four P transgenics in homozygous CspU1ocbackground are shown in Fig. 5A. Obviously, the dramatic life-shortening phenotype of the CspU1oc mutation is rescued by the wild-type cDna1 construct and by the constructs with a deletion in the linker region and in the C terminus, LΔ8 and CΔ27. Flies with only the Ssp construct, on the other hand, live merely 1 day longer than the null mutants. Life expectancy (50% survival time) of white flies at 25°C is about 35 days under these conditions. For characterization of the Clptransformant, which mapped to the third chromosome and was recombined with the original CspU1w mutation, siblings from the following crosses were analyzed:
\[\begin{array}{rl}&\frac{w}{w};;\frac{\mathrm{P(Clp)},Csp^{Ulw}}{\mathrm{P(Clp)},Csp^{Ulw}}{\times}\frac{w}{\mathrm{Y}};;\frac{Csp^{Uloc}}{\mathrm{TM}6},\\&\frac{w}{w};;\frac{Csp^{Ulw}}{Csp^{Ulw}}{\times}\frac{w}{\mathrm{Y}};;\frac{Csp^{Uloc}}{\mathrm{TM}6}.\end{array}\]

In the F1 generation we can compare longevity of Csp+ (balanced) flies and Csp-null flies of mixed genetic background (CspU1w/CspU1oc),with or without the Clp transgene. The survival curves for these four genotypes are shown in Fig. 5B. Again, the construct without cysteines (Clp) cannot rescue the short life phenotype and increases life expectancy of the null mutant only by 10–20%. Transformants with Cslp and Scsp constructs in CspU1 background could not be tested for longevity because several independent transformant lines were difficult to maintain, indicating that these constructs may produce a dominant negative effect (cf. Discussion). Life expectancy of the hypomorphic CspU2 mutant at 18°C is about 44 days, compared to 12 days for CspU1and 88 days for wild type under identical conditions. This demonstrates that small amounts of intact CSP can sustain much of its vital function whereas relatively normal expression of CLP or SSP is almost ineffective for sustaining the life of adults.

Temperature-sensitive paralysis of adult flies

Paralysis at elevated temperatures represents a second prominent phenotypic aspect of Csp null mutants. Temperature sensitivity was quantified as shown in Fig. 6 for wild type(w1118;;Csp+), two null mutant lines(CspU1w and CspU1oc), and five transgenic lines in CspU1oc background (cDna1, LΔ8,CΔ27, Clp, Ssp). For the transgenic lines, each curve represents the average of two independent insertion lines that did not differ significantly. The difference between the two null mutants reflects the influence of genetic background mentioned above (cf. Discussion). This phenomenon makes it rather difficult to interpret the small difference between the transgenic lines Clp and Ssp or CΔ27 and LΔ8. Yet it is clear that the CΔ27 and LΔ8constructs essentially rescue temperature-sensitive paralysis whereas constructs with mutations in the cysteine string do not. A weak dominant negative effect is apparent in the two independent insertions of the Clp construct in the Clp;CspU1oc lines. Qualitative tests of the few surviving adult Cslp;CspU1wand Scsp;CspU1w transformants reveal temperature-sensitive paralysis similar to the null mutant. Again the hypomorphic CspU2 mutant displays a much weaker phenotype than the string mutants as it paralyzes at 37°C only after 6–10 min.

Electrophysiology

The influence of the various mutations in the Csp transgene on neuromuscular transmission has been studied by intracellular recordings from body wall muscle 6 of third instar larvae. The motor neurons innervating this muscle were cut and stimulated by a suction electrode. All transgenes were analyzed in homozygous CspU1oc genetic background. The results in Fig. 7 demonstrate that replacement of the cysteine string in Ssp;CspU1octransgenic mutants causes significantly reduced excitatory junction potential(EJP) amplitude at 18°C and break-down of synaptic transmission at 32°C, similar to the null phenotype(Umbach et al., 1994).(Control experiments with wild type and null mutant had shown that responses were similar at 0.2 and 1.0 Hz). Contrary to expectations on the basis of the biochemical data and adult phenotypes, larval neuromuscular transmission in Clp;CspU1oc larvae persisted in four out of five preparations while the fifth preparation showed the expected null mutant phenotype. Behavioral observation verified the paralysis of intact Ssp;CspU1oc transgenic larvae at 32°C while Clp;CspU1oc larvae appeared almost unaffected by this temperature. Again unexpectedly, synaptic transmission failed at 32°C in three out of seven transgenic CΔ27;CspU1oclarvae. Equivocal results were obtained with two independent insertion lines of the LΔ8 construct. LΔ81 responses were similar to those of the null mutant, LΔ82 showed wild-type-like EJPs. It was noted that expression of the transgene was strongly reduced in LΔ81 larvae compared to LΔ82 larvae but similar in adults of the two lines. It is therefore assumed that synaptic failure in LΔ81 larvae is due to low expression levels rather than the deletion in the linker region.

The Csp locus

Sequence annotation of the Drosophila genome project has identified two genes in close proximity upstream (CG11523) and downstream(CG11489) of the Csp gene. Possible influences of these genes on the data presented here have to be considered. Based on the gene prediction data,the CspU1 but not the CspX1 deletion(Eberle et al., 1998) could affect the downstream gene. However, northern blots using EST clones of both genes as probes (Reisch, 2003)did not reveal any differences between w1118,CspU1 or CspX1, and no phenotypic differences between CspU1 and CspX1have so far been described (disregarding some weak immunohistochemical staining in the subesophageal ganglion in CspX1; Eberle et al., 1998). We conclude that if transcripts of the adjacent genes are affected by the CspU1 mutation they must be of low abundance and have no obvious influence on adult nervous system function.

Linker region and C terminus are not involved in membrane binding

CSPs from the electric organ of Torpedo were shown to be membrane associated, and the demonstration that at least 11 residues of the cysteine string were thioester-linked with palmitoic acids suggested that this highly hydrophobic region was responsible for the observed properties of CSPs as integral membrane proteins (Gundersen et al., 1994; Mastrogiacomo et al., 1994). Injection of unmodified CSPs into frog oocytes supported this notion, but deacylation of membrane proteins in experiments using PC12 cells, HeLa cells or Drosophila heads failed to release CSPs into the supernatant (van de Goor and Kelly 1996; Chamberlain and Burgoyne 1998). These findings suggested that the palmitoylated cysteine string is not required for stable membrane attachment of CSPs and it was proposed that a different domain might be responsible for membrane association (Chamberlain and Burgoyne 1998; van de Goor and Kelly 1996). None of the mutations introduced in the present work outside the cysteine string resulted in a release of membrane-bound CSP variants into the supernatant during a deacylation experiment. Thus neither the linker nor the C-terminal domain are relevant for membrane attachment of deacylated CSP.

A cDNA transgene construct coding for the largest CSP isoform rescues the null mutant

In the present study two wild-type Csp cDNA gene constructs were designed and transformed into Csp null-mutant flies. Both constructs express the 36 kDa CSP1 isoform. In the wild type this isoform represents the sole CSP in non-neural tissues, whereas in the brain three additional smaller isoforms of 34, 33 and 32 kDa are expressed more abundantly(Eberle et al., 1998). On sections of cDna1-rescued null mutants the distribution of CSP as detected by mAb DCSP1 in neuronal (and non-neuronal, data not shown) tissues matches the stainings described for wild-type flies. We conclude that the essential sequences for regulation of CSP expression are contained in a region from 2.3 kb upstream of the transcription start to the end of the second intron of the Csp gene. Both cDna1 constructs rescue the salient features of the null-mutant phenotype, i.e. temperature-sensitive failure of synaptic transmission and early death. Thus expression of the 36 kDa isoform is sufficient to rescue the major cellular functions of the Csp gene. The cell-specific expression of alternatively spliced CSP isoforms in the brain (Zinsmaier et al.,1994; Eberle et al.,1998) may fine-tune synaptic transmission to achieve yet unknown functional specializations. The strong overexpression of the 36 kDa isoform in head homogenates of cDna1 transformants is likely to result predominantly from the absence of alternatively spliced introns such that all transcripts translate into the same protein isoform. This overexpression of the 36 kDa isoform has been analyzed in 12 independent strains with different insertion sites and does not cause any obvious new phenotypes, in either null-mutant background or when co-expressed with the three smaller isoforms in the wild type (Fig. 2B and data not shown). Nie et al. (1999)demonstrate rescue of the attenuated excitatory junction potential (EJP)amplitude of CspU1 larval nerve muscle preparations by expressing any of the three known cDNAs under control of the elavpromotor (mediated by the GAL4-UAS system), but find dramatic effects on eye and wing development and on survival rate by overexpressing these cDNAs. Since CSP overexpression from the Csp promotor in genomic rescue transformants (Zinsmaier et al.,1994; Nie et al.,1999) or our cDna1 transformants does not produce these effects, we propose that they may be caused by higher levels of overexpression or temporal and/or spatial misexpression due to the elav GAL4 driver.

Phenotypic analysis of mutants or transgenic animals has to take into consideration the influence of genetic background effects. The loss of both semi-lethality and larval synaptic failure at 32°C in the CspU1 null mutant cultured in the homozygous condition,indicates that genetic modifiers suppressing Csp-related phenotypes have been selected for. Extensive out-crossing against wild type was required to reestablish a line that showed phenotypic characteristics similar to the original null mutant. This suggests that important genetic modifiers may be present on the third chromosome in the vicinity of the Csp gene. All electrophysiological experiments and most behavioral tests were therefore performed on flies in the out-crossed CspU1oc null mutant background.

Mutated CSPs cannot fully restore normal function

To elucidate the role of the linker region, the cysteine string (CS), and the C-terminal domain in CSP function, we analyzed expression and membrane association of six mutant isoforms in transgenic flies as well as the associated phenotypes. For each construct several independent transformants were obtained, and at least two were tested in each experiment in order to detect possible effects of the site of transgene insertion. Deletion of the entire cysteine string (11 out of 15 cysteines of the CS region) results in a protein (CSLP) that is found both in the soluble and membrane fractions. Replacement of the 11 cysteines by serines in SSP, however, eliminates membrane targeting. These results suggest that the relative positions of the remaining two pairs of cysteines may be critical for the residual targeting of CSLP. The SSP and the CLP are detected only in the soluble fraction and are not enriched in synaptic terminals. An earlier study used a mammalian mutant CSP construct in which 7 of the 14 cysteines of the string region were replaced by serines. When this mutant protein was expressed in PC12 or HeLa cells, it was not palmitoylated and was not associated with membranes(Chamberlain and Burgoyne,1998). Our data make clear that the cysteine string is required for correct targeting of Drosophila CSPs to synaptic vesicles in an intact organism. In Drosophila the short cysteine string mutant protein (SCSP), in which 6 out of 15 cysteines in the string region have been deleted, appears to be efficiently targeted to membranes. A small increase in electrophoretic mobility is observed after depalmitoylation of SCSP. In spite of partial (CSLP) or normal (SCSP) targeting to membranes, transgenic flies expressing these proteins in null mutant background display paralysis similar to the null mutant, strongly indicating that an intact cysteine string region is necessary for cellular functions of CSPs beyond protein targeting. Such functions may, for example, depend on the tightness of membrane attachment by the palmitoylated cysteine residues. The fact that several transgenic lines containing the Cslp or Scsp constructs were difficult or impossible to maintain in homozygous CspU1w genetic background indicates that mutant CSPs with intact or partially intact targeting but modified or deleted cysteine string disturb cellular processes that can be maintained in the absence of CSPs. SSP and CLP, on the other hand,are not targeted but slightly improve viability.

The present experiments demonstrate that for the most visible consequences of CSP function in adult Drosophila an intact linker region and the C terminus are not essential. Both regions contain amino acids that are highly conserved in evolution, demonstrating fitness-related function. The temperature sensitivity of neuromuscular transmission in some CΔ27;CspU1oc larvae could relate to such a function and indicates that in larval synapses CSPs may function differently compared to adult synapses. The unexpected `rescue' of synaptic transmission and prevention of paralysis at 32°C by CLP in most larvae but not in adults also points in this direction. The intra-strain variability of some of the electrophysiological data might be due to polymorphic genetic background effects, which can never be entirely eliminated. The only defect noticed in the LΔ82;CspU1oc line concerns a moderate temperature sensitivity of adults. The synaptic failure in LΔ81;CspU1oc larvae is assumed to be caused by the low larval expression of the transgene in this strain due to its specific insertion site.

Western blot signals from head homogenates of all mutated proteins except CΔ27 are significantly weaker. Since this effect is independent of transgene insertion sites it is assumed that it reflects reduced stability of the mutated proteins. Instability of mutated or truncated proteins is a general phenomenon. The normal stability of the CΔ27 protein is not unexpected because a natural mammalian CSP isoform lacks the conserved C-terminal domain (31 amino acids; Chamberlain and Burgoyne, 1996; Coppola and Gundersen, 1996). We asked whether the reduced abundance of the mutated proteins might simply be responsible for the phenotype of the transgenic flies in Csp-null background. However, the hypomorphic mutation CspU2, which reduces CSP abundance about tenfold but leaves the coding region intact,displays strongly attenuated phenotypes compared to null mutants, such as viability at 25°C, longevity reduced at this temperature only to about 50%, delayed temperature-sensitive paralysis and fast recovery(Eberle, 1995). These observations support the above proposal, that the strong phenotype shown by the cysteine string mutants is not mainly due to incomplete or incorrect targeting of the mutated protein but that the cysteine string is important for CSP function beyond its demonstrated role in targeting of the protein to the synaptic vesicle membrane. This hypothesis can possibly be tested by replacing the cysteine string with other synaptic vesicle membrane-targeting signals.

This work was supported by grants Bu566/6, GRK 200 and SFB 581/B6 from the Deutsche Forschungsgemeinschaft. We thank Dr K. E. Zinsmaier for kind gifts of DCSP2 (6D6) and DCSP3 (1G12) antibodies, Dr S. Benzer for 8C3 antibody, and Drs A. Fiala and H. Scholz for carefully reading and discussing the manuscript.

Ashburner, M. (
1989
). Drosophila.
A Laboratory Manual.
Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Braun, J. E. A., Wilbanks, S. M. and Scheller, R. H.(
1996
). The cysteine string secretory vesicle protein activates Hsc70 ATPase.
J. Biol. Chem.
271
,
25989
-25993.
Bronk, P., Wenniger, J. J., Dawson-Scully, K., Guo, X., Hong,S., Atwood, H. L. and Zinsmaier, K. E. (
2001
). Drosophila Hsc70-4 is critical for neurotransmitter exocytosis in vivo.
Neuron
30
,
475
-488.
Buchner, E. and Gundersen, C. B. (
1997
). The DnaJ-like cysteine string protein and exocytotic neurotransmitter release.
Trends Neurosci.
20
,
223
-227.
Buchner, E., Buchner, S., Crawford, G., Mason, W. T.,Salvaterra, P. M. and Sattelle, D. B. (
1986
). Choline acetyltransferase-like immunoreactivity in the brain of Drosophilamelanogaster.
Cell Tissue Res.
246
,
57
-62.
Chamberlain, L. H. and Burgoyne, R. D. (
1997
). Activation of the ATPase activity of heat-shock proteins Hsc70/Hsp70 by cysteine-string protein.
Biochem. J.
322
,
853
-858.
Chamberlain, L. H. and Burgoyne, R. D. (
1998
). The cysteine-string domain of the secretory vesicle cysteine-string protein is required for membrane targeting.
Biochem. J.
335
,
205
-209.
Chamberlain, L. H. and Burgoyne, R. D. (
2000
). Cysteine string protein, the chaperone at the synapse.
J. Neurochem.
74
,
1781
-1789.
Coppola, T. and Gundersen, C. (
1996
). Widespread expression of human cysteine string proteins.
FEBS Lett.
391
,
269
-272.
Eberle, K. K. (
1995
).
Mutagenese und Funktionsanalyse des csp-Gens von Drosophila melanogaster
. PhD thesis, University of Würzburg, Germany.
Eberle, K. K., Zinsmaier, K. E., Buchner, S., Gruhn, M., Jenni,M., Arnold, C., Leibold, C., Reisch, D., Walter, N., Hafen, E. et al. (
1998
). Wide distribution of the cysteine string proteins in Drosophila tissues revealed by targeted mutagenesis.
Cell Tissue Res.
294
,
203
-217.
Evans, G. J., Wilkinson, M. C., Graham, M. E., Turner, K. M., Chamberlain, L. H., Burgoyne, R. D. and Morgan, A.(
2001
). Phosphorylation of cysteine string protein by protein kinase A. Implications for the modulation of exocytosis.
J. Biol. Chem.
276
,
47877
-47885.
Gundersen, C. B., Mastrogiacomo, A., Faull, K. and Umbach, J. A. (
1994
). Extensive lipidation of a Torpedo cysteine string protein.
J. Biol. Chem.
269
,
19197
-19199.
Hofbauer, A. (
1991
).
Eine Bibliothek monoklonaler Antikörper gegen das Gehirn von Drosophilamelanogaster
. Habilitationsschrift, Universität Würzburg,Germany.
Klemenz, R., Weber, U. and Gehring, W. J.(
1987
). The white gene as a marker in a new P-element vector for gene transfer in Drosophila.
Nucleic Acids Res.
15
,
3947
-3959.
Laemmli, U. K. (
1970
). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
,
680
-685.
Mastrogiacomo, A., Parsons, S. M., Zampighi, G. A., Jenden, D. J., Umbach, J. A. and Gundersen, C. B. (
1994
). Cysteine string proteins, a potential link between synaptic vesicles and presynaptic Ca2+ channels.
Science
263
,
981
-982.
Mastrogiacomo, A., Kohan, S. A., Whitelegge, J. P. and Gundersen, C. B. (
1998
). Intrinsic membrane association of Drosophila cysteine string proteins.
FEBS Lett.
436
,
85
-91.
Munch, G., Schicktanz, D., Behme, A., Gerlach, M., Riederer, P.,Palm, D. and Schinzel, R. (
1999
). Amino acid specificity of glycation and protein-AGE crosslinking reactivities determined with a dipeptide SPOT library.
Nat. Biotechnol.
17
,
1006
-1010.
Nie, Z., Ranjan, R., Wenniger, J. J., Hong, S. N., Bronk, P. and Zinsmaier, K. E. (
1999
). Overexpression of cysteine-string proteins in Drosophila reveals interactions with syntaxin.
J. Neurosci.
19
,
10270
-10279.
Reichmuth, C., Becker, S., Benz, M., Debel, K., Reisch, D.,Heimbeck, G., Hofbauer, A., Klagges, B., Pflugfelder, G. O. et al.(
1995
). The sap47 gene of Drosophila melanogaster codes for a novel conserved neuronal protein associated with synaptic terminals.
Brain Res. Molec. Brain Res.
32
,
45
-54.
Reisch, N. (
2003
).
Das Cysteine String Protein in Drosophila melanogaster
. PhD thesis, University of Würzburg, Germany.
Stewart, B. A., Atwood, H. L., Renger, J. J., Wang, J. and Wu,C.-F. (
1994
). Improved stability of Drosophilalarval neuromuscular preparations in haemolymph-like physiological solutions.
J. Comp. Physiol.
175
,
179
-191.
Swayne, L. A., Blattler, C., Kay, J. G. and Braun, J. E. A.(
2003
). Oligomerization characteristics of cysteine string protein.
Biochem. Biophys. Res Commun.
300
,
921
-926.
Tobaben, S., Thakur, P., Fernández-Chacón, R.,Südhof, T. C., Rettig, J. and Stahl, B. (
2001
). A trimeric protein complex functions as a synaptic chaperone machine.
Neuron
31
,
987
-999.
Umbach, J. A., Zinsmaier, K. E., Eberle, K. K., Buchner, E.,Benzer, S. and Gundersen, C. B. (
1994
). Presynaptic dysfunction in Drosophila Csp mutants.
Neuron
13
,
899
-907.
van de Goor, J. and Kelly, R. B. (
1996
). Association of Drosophila cysteine string proteins with membranes.
FEBS Lett.
380
,
251
-256.
van de Goor, J., Ramaswami, M. and Kelly, R.(
1995
). Redistribution of synaptic vesicles and their proteins in temperature-sensitive Shibirets1. mutant Drosophila.
Proc. Natl. Acad. Sci. USA
92
,
5739
-5743.
Zhang, H., Kelley, W. L., Chamberlain, L. H., Burgoyne, R. D. and Lang, J. (
1999
). Mutational analysis of cysteine-string protein function in insulin exocytosis.
J. Cell Sci.
112
,
1345
-1351.
Zinsmaier, K. E. and Bronk, P. (
2001
). Molecular chaperones and the regulation of neurotransmitter exocytosis.
Biochem. Pharmacol.
62
,
1
-11.
Zinsmaier, K. E., Eberle, K. K., Buchner, E., Walter, N. and Benzer, S. (
1994
). Paralysis and early death in cysteine string protein mutants of Drosophila.
Science
263
,
977
-980.
Zinsmaier, K. E., Hofbauer, A., Heimbeck, G., Pflugfelder, G. O., Buchner, S. and Buchner, E. (
1990
). A cysteine-string protein is expressed in retina and brain of Drosophila.
J. Neurogenet.
7
,
15
-29.