Functional fat body proteomics and gene targeting reveal in vivo functions of Drosophila melanogaster α-Esterase-7.

Carboxylesterases constitute a large enzyme family in insects, which is involved in diverse functions such as xenobiotic detoxi fi cation, lipid metabolism and reproduction. Phylogenetically, many insect carbox- ylesterases are represented by multienzyme clades, which are encoded by evolutionarily ancient gene clusters such as the a -Esterase cluster. Much in contrast to the vital importance attributed to carbox- ylesterases in general, the in vivo function of individual a -Esterase genes is largely unknown. This study employs a functional proteomics approach to identify esterolytic enzymes of the vinegar fl y Drosophila melanogaster fat body. One of the fat body carboxylesterases, a -Esterase-7 , was selected for mutational analysis by gene targeting to generate a deletion mutant fl y. Phenotypic characterization of a -Esterase-7 null mutants and transgenic fl ies, which overexpress a chimeric a -Esterase-7:EGFP gene, reveals important functions of a -Esterase-7 in insecticide tolerance, lipid metabolism and lifespan control. The presented fi rst deletion mutant of any a -Esterase in the model insect D. melanogaster generated by gene targeting not only provides experimental evidence for the endogenous functions of this gene family. It also offers an entry point for in vivo structure-function analyses of a -Esterase- 7, which is of central importance for naturally occurring insecticide resistance in wild populations of various dipteran insect species

Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas.

We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu(+) accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply