The RNA interference (RNAi) pathway is initiated by processing long double-stranded RNA into small interfering RNA (siRNA). The siRNA-generating enzyme was purified from Drosophila S2cells and consists of two stoichiometric subunits: Dicer-2(DCR-2) and a previously unknown protein that we named R2D2. R2D2 is homologous to the Caenorhabditis elegans RNAi protein RDE-4. Association with R2D2 does not affect the enzymatic activity of DCR-2. Rather, the DCR-2/R2D2 complex, but not DCR-2 alone, binds to siRNA and enhances sequence-specific messenger RNA degradation mediated by the RNA-initiated silencing complex (RISC). These results indicate that R2D2 bridges the initiation and effector steps of the Drosophila RNAi pathway by facilitating siRNA passage from Dicer to RISC.

Odorant binding proteins (OBPs) are extracellular proteins localized to the chemosensory systems of most terrestrial species. OBPs are expressed by nonneuronal cells and secreted into the fluid bathing olfactory neuron dendrites. Several members have been shown to interact directly with odorants, but the significance of this is not clear. We show that the Drosophila OBP lush is completely devoid of evoked activity to the pheromone 11-cis vaccenyl acetate (VA), revealing that this binding protein is absolutely required for activation of pheromone-sensitive chemosensory neurons. lush mutants are also defective for pheromone-evoked behavior. Importantly, we identify a genetic interaction between lush and spontaneous activity in VA-sensitive neurons in the absence of pheromone. The defects in spontaneous activity and VA sensitivity are reversed by germline transformation with a lush transgene or by introducing recombinant LUSH protein into mutant sensilla. These studies directly link pheromone-induced behavior with OBP-dependent activation of a subset of olfactory neurons.

Detection of volatile odorants by olfactory neurons is thought to result from direct activation of seven-transmembrane odorant receptors by odor molecules. Here, we show that detection of the Drosophila pheromone, 11-cis vaccenyl acetate (cVA), is instead mediated by pheromone-induced conformational shifts in the extracellular pheromone-binding protein, LUSH. We show that LUSH undergoes a pheromone-specific conformational change that triggers the firing of pheromone-sensitive neurons. Amino acid substitutions in LUSH that are predicted to reduce or enhance the conformational shift alter sensitivity to cVA as predicted in vivo. One substitution, LUSH(D118A), produces a dominant-active LUSH protein that stimulates T1 neurons through the neuronal receptor components Or67d and SNMP in the complete absence of pheromone. Structural analysis of LUSH(D118A) reveals that it closely resembles cVA-bound LUSH. Therefore, the pheromone-binding protein is an inactive, extracellular ligand converted by pheromone molecules into an activator of pheromone-sensitive neurons and reveals a distinct paradigm for detection of odorants.

The only known volatile pheromone in Drosophila, 11-cis-vaccenyl acetate (cVA), mediates a variety of behaviors including aggregation, mate recognition, and sexual behavior. cVA is detected by a small set of olfactory neurons located in T1 trichoid sensilla on the antennae of males and females. Two components known to be required for cVA reception are the odorant receptor Or67d and the extracellular pheromone-binding protein LUSH. Using a genetic screen for cVA-insensitive mutants, we have identified a third component required for cVA reception: sensory neuron membrane protein (SNMP). SNMP is a homolog of CD36, a scavenger receptor important for lipoprotein binding and uptake of cholesterol and lipids in vertebrates. In humans, loss of CD36 is linked to a wide range of disorders including insulin resistance, dyslipidemia, and atherosclerosis, but how CD36 functions in lipid transport and signal transduction is poorly understood. We show that SNMP is required in pheromone-sensitive neurons for cVA sensitivity but is not required for sensitivity to general odorants. Using antiserum to SNMP infused directly into the sensillum lymph, we show that SNMP function is required on the dendrites of cVA-sensitive neurons; this finding is consistent with a direct role in cVA signal transduction. Therefore, pheromone perception in Drosophila should serve as an excellent model to elucidate the role of CD36 members in transmembrane signaling.CD36 ͉ olfaction ͉ olfactory ͉ sexual behavior ͉ signal transduction C VA (11-cis-vaccenyl acetate) mediates social behaviors in Drosophila, and its reception requires the odorant receptor Or67d and the extracellular pheromone-binding protein LUSH (1-4). Misexpression of Or67d receptors in trichoid neurons that are normally insensitive to pheromone confers cVA sensitivity but only if LUSH is present (3). However, Or67d and LUSH are not sufficient to confer cVA sensitivity to basiconic neurons (T.S.H. and D.P.S., unpublished work). This finding reveals that there are additional factors required for cVA sensitivity present in trichoid sensilla that are lacking in basiconic sensilla. Using a genetic screen, we set out to identify additional components important for cVA sensitivity. We screened Ϸ3,000 mutagenized third-chromosome lines selected for homozygous viability (5). We screened each mutant line for T1 electrophysiological responses to cVA using single sensillum electrophysiological recordings (2, 3, 6). We identified five complementation groups that were cVA-insensitive yet retained spontaneous activity in the pheromone-sensing neurons (the vains phenotype) ( Fig. 1 and Table 1). The presence of spontaneous activity indicates that the neurons are present, are viable, and can sustain action potentials, thereby eliminating nonspecific mutants affecting development or general neuronal function. Of the five complementation groups recovered, two, Or67d and Or83b, affect genes previously implicated in cVA or general odorant detection, two remain unmapped, and the fifth encodes SNMP, a new cVA...