Towards a temperature-sensitive proteome: developing a Drosophila-friendly degron

Here we propose to develop a new technology that enables biologists to turn activity of genes on and off simply by changing the temperature, by targeting their products for degradation only at higher culture temperatures. This technology is based on well-established biological mechanisms employed by the cell to achieve regulated removal of unwanted proteins. This regulated degradation is mediated by the attachment of degradation signals to the target. Some proteins (degrons), identified in yeast, can induce attachment of these signals to themselves when their structure is disrupted at higher temperatures, thus inducing degradation of themselves and any fusion proteins that they are part of. We and others have successfully shown proof-of-principle that the system can be used in a more complex model organism, Drosophila (fruitfly). However, the temperatures at which degradation of the target is induced in yeast are not well-tolerated by Drosophila, or by other 'cold-blooded' organisms. Therefore, we propose to isolate novel variants of the yeast degron signal, that work better in the normal temperature ranges of Drosophila and of other model organisms widely used by biologists, and to test these in cultured Drosophila cells and in genetically manipulated flies.

The number of sequenced eukaryotic genomes is increasing. However, understanding gene function is a lower throughput undertaking, and generation of mutant or transgenic organisms with inducible phenotypes is a significant bottleneck in this. Inducible phenotypes allow gene function to be investigated at any stage of development, even for genes that are homozygous lethal in early development when mutant. Classical temperature-sensitive alleles are of enormous use, but for most proteins occur only sporadically, and a more systematic approach is desirable. The ubiquitin/proteasomal system has previously been used to obtain target-directed proteolysis in a temperature-dependent manner, in two different model organisms. We aim to make this approach more widely applicable, by making the temperature profile of the switch more suitable to the temperature range of Drosophila and other poikilothermic model organisms. To achieve this, we will mutagenise a domain previously shown to function as a degron, fused to a counterselectable marker in yeast, and screen for mutations that cause loss of activity of the fusion at a high temperature, but not at lower temperature. Suitable mutant domains will be assessed for their ability to mediate temperature dependent degradation by transient transfection in Drosophila cultured cells, and the best candidates will be tested in transgenic flies. A successful outcome will allow the creation of temperature-sensitive protein fusions, and thus temperature-sensitive mutant organisms, either in a directed or in an undirected genome-wide manner, and will facilitate addressing of many questions about the function of proteins that are required at multiple stages of development.