Every moment of our lives, countless numbers of neurones transmit signals throughout our bodies, communicating between nerves by releasing tiny neurotransmitter-packed vesicles across the synapses that separate them. The mechanics of synaptic transmission fascinates Erich Buchner, and over the years he has identified a membrane associated protein, called the cysteine string protein (CSP), that seems to play several significant roles in communication between neurones. Intrigued by some unusual features in the protein's sequence, Buchner and his team in Würzburg Germany embarked on a tricky set of experiments to identify some of the protein's functions from a physiological perspective, and found that the cysteine string, which gives the protein its name, is absolutely necessary for the protein's function(p. 1313).

Buchner explains that Alois Hofbauer and Konrad Zinsmaier in his group discovered the cysteine string protein during an extensive screen of synapse-related proteins in Drosophila brains. Working with a model organism gave Buchner and Kai Eberle the opportunity to design an insect that lacked the protein. The results were dramatic. The flies developed seriously debilitating spasms early in life before dying prematurely at the age of 15 days. And when Eberle inadvertently warmed the flies above 18°C, they collapsed with paralysis, but revived after cooling. Life without cysteine string protein wasn't particularly long or healthy.

Sure that he was on to something interesting, Buchner and Christine Arnold decided to see if they could cure the CSP-less insects. They designed a genetic construct containing an isoform of the cysteine string protein, which they returned to the genome of the beleaguered insects; all of the disturbing symptoms vanished and the flies longevity was restored. The team were ready to begin taking the protein apart.

Focusing on the string of eleven cysteine residues, Arnold modified the string, removing all or a few of the cysteines, or replacing them with serines, which resemble cysteine. Then the team tested whether the protein was expressed and produced a protein in the flies' brains. They also checked whether the protein was able to integrate into the cell's membranes, or remained in solution. Natascha Reisch found that without cysteines, the protein remained in the soluble cell contents, and was unable to integrate into the cell membrane and function. But it appeared that with only five of the string's cysteines present, the protein was able to integrate into the cell membrane. Could this reduced cysteine string protein still function? The team tested these flies' tolerance to high temperature; again they became paralysed, just as the flies that lacked cysteine string protein had. Christian Leibold also tested the neurofunction of the protein where some of cysteines had been replaced with serines, and found that at elevated temperatures the larvae were unable to transmit signals across the neuromuscular junction. Without cysteine, the protein failed to function; the cysteine string was essential.

Buchner was also curious about the functions of two other domains, the linker domain and C-terminal domain. Deleting regions of both domains from the protein, the team found that the results weren't so clear-cut. The flies without the C terminal domain seemed as healthy as normal flies, while the flies with a modified linker domain seemed to suffer some reduction in the protein's function, although its not clear why.

Arnold, C., Reisch, N., Leibold, C., Becker, S., Prüfert,K., Sautter, K., Palm, D., Jatzke, S., Buchner S. and Buchner, E.(
). Structure–function analysis of the cysteine string protein in Drosophila: cysteine string, linker and C terminus.
J. Exp. Biol.