Structure-function analysis of the cysteine string protein in Drosophila: cysteine string, linker and C terminus.
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 degrees 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.
Arnold
Christine
C
Lehrstuhl für Genetik und Neurobiologie, Theodor-Boveri-Institut für Biowissenschaften, Am Hubland, D-97074 Würzburg, Germany.
Reisch
Natascha
N
Leibold
Christian
C
Becker
Sonja
S
Prüfert
Kristina
K
Sautter
Kerstin
K
Palm
Dieter
D
Jatzke
Susanne
S
Buchner
Sigrid
S
Buchner
Erich
E
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D018604:Epitope Mapping; D040461:Gene Components; D015151:Immunoblotting; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013696:Temperature; D014170:Transformation, Genetic
C069672:Csp protein, Drosophila; D017931:DNA Primers; D018076:DNA, Complementary; D029721:Drosophila Proteins; D003545:Cysteine