Alternative tasks of Drosophila tan in neurotransmitter recycling versus cuticle sclerotization disclosed by kinetic properties.
Upon a stimulus of light, histamine is released from Drosophila photoreceptor axonal endings. It is taken up into glia where Ebony converts it into beta-alanyl-histamine (carcinine). Carcinine moves into photoreceptor cells and is there cleaved into beta-alanine and histamine by Tan activity. Tan thus provides a key function in the recycling pathway of the neurotransmitter histamine. It is also involved in the process of cuticle formation. There, it cleaves beta-alanyl-dopamine, a major component in cuticle sclerotization. Active Tan enzyme is generated by a self-processing proteolytic cleavage from a pre-protein at a conserved Gly-Cys sequence motif. We confirmed the dependence on the Gly-Cys motif by in vitro mutagenesis. Processing time delays the rise to full Tan activity up to 3 h behind its putative circadian RNA expression in head. To investigate its pleiotropic functions, we have expressed Tan as a His(6) fusion protein in Escherichia coli and have purified it to homogeneity. We found wild type and mutant His(6)-Tan protein co-migrating in size exclusion chromatography with a molecular weight compatible with homodimer formation. We conclude that dimer formation is preceding pre-protein processing. Drosophila tan(1) null mutant analysis revealed that amino acid Arg(217) is absolutely required for processing. Substitution of Met(256) in tan(5), on the contrary, does not affect processing extensively but renders it prone to degradation. This also leads to a strong tan phenotype although His(6)-Tan(5) retains activity. Kinetic parameters of Tan reveal characteristic differences in K(m) and k(cat) values of carcinine and beta-alanyl-dopamine cleavage, which conclusively illustrate the divergent tasks met by Tan.
Aust
Silvia
S
Faculty of Chemistry and Biochemistry, AG Molekulare Zellbiochemie, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Brüsselbach
Florian
F
Pütz
Stefanie
S
Hovemann
Bernhard T
BT
D000818:Animals; D001369:Axons / Q000502:physiology; D002336:Carnosine / Q000031:analogs & derivatives / Q000378:metabolism; D002850:Chromatography, Gel; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D019281:Dimerization; D029721:Drosophila Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D004331:Drosophila melanogaster / Q000235:genetics* / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D017344:Genes, Insect; D005998:Glycine / Q000235:genetics; D006867:Hydrolases / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000502:physiology*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D015091:beta-Alanine / Q000235:genetics / Q000378:metabolism
D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D018377:Neurotransmitter Agents; D011993:Recombinant Fusion Proteins; C116191:e protein, Drosophila; C431070:t protein, Drosophila; D015091:beta-Alanine; D012313:RNA; D002336:Carnosine; D006867:Hydrolases; D003545:Cysteine; D005998:Glycine; C015055:carcinine