biochemical grounds but in which a genetic approach provided fundamental insight as to their functional requirement. Examples are the cyclophilin homologue ninaA and its role in rhodopsin biogenesis (14–17), an eye-specific protein kinase C (PKC) required for deactivation and calcium feedback regulation (18,19), the role of Gβ in the termination of the light response (20), and a number of enzymes involved in inositol phospholipid metabolism and shown to be required for photoreceptor cell excitation (10,21).
Drosophila phototransduction is one of the best model systems for the study of G protein-coupled PLC signaling (10,22,23). Not only is the system amenable to molecular genetic analysis but also it can report activity with exquisite sensitivity and specificity: photoreceptor cells are sensitive to single photons, and the signaling pathway can be turned on and off with millisecond kinetics (phototransduction in Drosophila is the fastest known G protein cascade, taking just a few tens of milliseconds to go from light activation of rhodopsin to the generation of a receptor potential). As described above, active PLC catalyzes the hydrolysis of the minor membrane phospholipid PIP2 into the second messengers inositol IP3 and DAG. IP3 mobilizes calcium from internal stores, which affects and modulates many cellular processes, and DAG activates members of the PKC family of proteins. Given the central role of PIP2 in signaling, its levels may be expected to be tightly regulated in the cell.
Fig. 2 shows an expanded view of the PIP2 cycle. CDP-DAG synthase (CDS) is an enzyme required to convert phosphatidic acid into CDP-DAG, the acceptor for the inositol head group. Using enhancer trap technology, we identified an eye-specific form of CDP-DAG and isolated mutations in this gene (eyecds) (21). To determine whether eye-cds mutants have a defect in their signaling properties, wild-type and mutant animals were assayed for their ability to maintain a continuously activated state of the photoreceptor cells because such a state would require the continuous availability of the second messenger PIP2. Our results demonstrated that light activation depletes a pool of PIP2 necessary for excitation that cannot be replenished in eye-cds mutants (Fig. 3a–d). This phenotype is due exclusively to a defect in eye-cds, because introduction of the wild-type eye-CDS cDNA into mutant hosts fully restores wild-type physiology (Fig. 3e–f). Furthermore, inclusion of PIP2 in the patch pipette is sufficient to restore signaling in the depleted eye-cds mutants (Fig. 3g–h). These results suggest, contrary to expectations, that the pool of PIP2 required for signaling is quite small and likely synthesized “on demand”. On the basis of these findings, we reasoned that it should be possible to modulate the output of this cascade by experimentally manipulating the levels of eye-CDS. Indeed, we made