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First published online March 9, 2004
Journal of Experimental Biology 207, 1323-1334 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00898

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Structure–function analysis of the cysteine string protein in Drosophila: cysteine string, linker and C terminus

Christine Arnold^1,*, Natascha Reisch^1,*, Christian Leibold^1,*, Sonja Becker¹, Kristina Prüfert¹, Kerstin Sautter¹, Dieter Palm², Susanne Jatzke¹, Sigrid Buchner¹ and Erich Buchner^1,

¹ Lehrstuhl für Genetik und Neurobiologie, Theodor-Boveri-Institut für Biowissenschaften, Am Hubland, D-97074 Würzburg, Germany
² Lehrstuhl für Physiologische Chemie I, Theodor-Boveri-Institut für Biowissenschaften, Am Hubland, D-97074 Würzburg, Germany

View larger version (40K): [in a new window]   Fig. 1. (A) Primary structure of the three cDNA-derived cysteine string protein isoforms CSP1, CSP3 and CSP4, and the postulated isoform CSP2*. The conserved `J' domain, the cysteine string region (`CS'), and the epitopes recognized by the three available monoclonal antibodies DCSP1, DCSP2 and DCSP3 are boxed, the amino acids deleted in the mutant proteins L8 and C27 are underlined. (B) Schematic representation of the exon-intron structure of the three known Csp transcripts cDNA-1, cDNA-3 and cDNA-4, and the hypothetical cDNA-2*. The white vertical lines in exons 1, 6* and 7 indicate start and stop codons, which delimit the open reading frames. The approximate molecular masses in SDS gels and the recognition (+) of the four isoforms by the monoclonal antibodies DCSP1, DCSP2 and DCSP3 are indicated. (C) Genomic region of the Csp locus and the extent of the deficiency of the CspU1w null mutant compared with wild type (WT). (D) cDNA-1 derived rescue constructs lcDna1 (lcD-1) and scDna1 (scD-1). The constructs consist of genomic fragments of different sizes (compare with C) containing regulatory sequences and exons 1, 2 and part of exon 3, as well as introns 1 and 2, and a common cDNA-1 fragment (hatched) containing the rest of exon 3, and exons 4, 5, 6 and 7. The depicted constructs and their in vitro modified versions (cf. A and E) were cloned into the pW8 P-element vector. Restriction enzymes: A=Asp718, B=BamH I, E=EcoRI, H=HindIII, P=Bsp1407I, S=SalI. (E) Amino acid sequence of the cysteine string region of Drosophila CSP and the cysteine string mutant proteins `short cysteine string protein' (SCSP), `cysteine string-less protein' (CSLP), `serine string protein' (SSP) and `cysteine-less protein' (CLP). Cysteine residues (C) are shown in bold.

View larger version (64K): [in a new window]   Fig. 2. Immunoblots of (white-eyed) wild type (WT), null mutant CspU1w (U1), cDna1-rescued null mutant (cDNA1), and transgenic mutants (serine string protein (SSP), cysteine-less protein (CLP), linker deletion (C8), C-terminal deletion (C27), cysteine stringless protein (CSLP), and short cysteine string protein (SCSP), all except for CSLP in B in CspU1w genetic background. Vertical dotted lines separate lanes of different mutants and vertical solid lines separate different blots. The SAP47-marked signals represent loading controls. Blots were developed with mAbs DCSP1 (DCSP2 when C27 was loaded) and mAb nc46 for detection of SAP47. (A) Heads homogenized in SDS buffer, 1–2 heads per lane. The leftmost WT lane was from a large gel for improved separation and has been graphically compressed for comparison with the small gel lanes. (B) Heads were homogenized in buffer A, a post-nuclear supernatant (S1) was fractionated by ultracentrifugation to separate soluble proteins (S2) from membrane or cytoskeleton associated proteins (P2). Wild-type CSPs (WT, cDNA1), L8, C27 and SCSP are detected exclusively in the membrane fraction, CSLP (analyzed here in WT background as control) is present in both fractions, whereas CLP and SSP are seen only in the soluble fraction. A single WT head homogenized in SDS buffer is shown for comparison (H). (C) Pellets P1 were deacylated with hydroxylamine and ultracentrifuged to obtain supernatant S3 and pellet P3. A smaller protein after deacylation (P3) indicates the loss of palmityl residues in wild-type CSPs (WT, cDNA1), L8, C27 and SCSP. 32, position of the 32 kDa marker protein.

View larger version (76K): [in a new window]   Fig. 3. Glycerol gradient velocity sedimentation of wild-type CSPs (cDNA1), CLP, SSP, CSLP, L8 and C27 in null-mutant (U1) and wild-type (WT) backgrounds. S, supernatant; P, pellet, SP soluble proteins; SV, synaptic vesicles; PM, plasma membrane. The gradient was allowed to develop overnight from 5% to 25% in 5% steps. Wild-type CSPs migrate in the synaptic vesicle fractions whereas SSP and CLP comigrate with the soluble control protein SAP47. A significant portion of CSLP, L8 and C27 appears to comigrate with the plasma membrane fraction identified by the PM protein syntaxin (SYX).

View larger version (112K): [in a new window]   Fig. 4. Distribution of wild-type or mutated CSP forms in wild-type (w1118; A,C), and in cDna1–, Ssp–, Cslp–, Clp– and Scsp-transformed flies in CspU1 null mutant background (B,D–G), and in hypomorphic mutant CspU2 (H). Horizontal frozen head sections. Sections A+B, C+D, were stained on the same microscope slides and processed identically for better comparison. There are no clear differences in staining between wild type (A) and cDna1; CspU1 rescue (B). SSP (D) and CLP (F) distribute homogeneously throughout cellular rind (CR) or retina (R), fiber tracts such as the optic chiasms (OC) and neuropil (NP). CSLP (E) apparently is targeted to synaptic neuropil less efficiently than WT CSPs. Scsp;CspU1 preparations (G) demonstrate low abundance of SCSP but normal targeting of this isoform to synaptic neuropil. Specificity of staining is demonstrated by comparison with the null mutant CspU1 (I). Scale bar, 100 µm.

View larger version (33K): [in a new window]   Fig. 5. (A) Survival rates of homozygous CspU1oc mutants (U1oc/U1oc), balanced siblings (U1oc/TM) and cDna1–, L8–, C27 or Ssp-transformed flies in null mutant background at 25°C culture temperature. In contrast to the wild-type CSP, L8 or C27, transgenic expression of SSP cannot rescue the short-life phenotype of null mutants and extends their life time only by ca. 20%. (B) Survival time for Clp– and Ssp– transformed flies are similar. Note the slightly increased survival time of CspU1/CspU1oc compared to CspU1oc/CspU1oc in A.

View larger version (20K): [in a new window]   Fig. 6. Temperature-sensitive paralysis of adult wild type (WT), null mutant (CspU1), and cDna1–, L8–, C27–, Ssp–, or Clp– transformed flies in null mutant background. Note again the difference between CspU1 and CspU1oc.

View larger version (21K): [in a new window]   Fig. 7. (A) Evoked excitatory junction potentials (EJPs) recorded from larval nerve–muscle preparations of WT, null mutant CspU1oc, and cDna1–, Ssp–, Clp–, L8–, C27 transformed animals in CspU1oc null mutant background. Data were pooled for at least two independent transgene insertions except for L81 and L82, which differed significantly (see Results). The two independent C27 transformants display normal transgene expression, indicating that this construct does not fully rescue the larval temperature-sensitive phenotype, whereas adults of these lines show no obvious defects. (A) Sample traces at permissive (18°C) and non-permissive (32°C) temperatures. (B) Evoked EJP amplitudes (mean ± S.E.M.) of (N) preparations.

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