Genes that are essential for cellular growth are very difficult to study at the genetic level; such gene deficiencies often cause lethality. To investigate loss-of-function mutations in genes that are essential for growth, we developed an inducible RNAi system, for conditional knock-down expression. We coupled long-hairpin RNAs to a """"""""TET off"""""""" expression system and confirmed that targeted essential mRNAs are depleted upon induced RNAi expression, leading to attenuation of cell growth.? Regulated protein destruction involving SCF (Skp1/Cullin/F-box, E3 ubiquitin ligase) complexes is required for eukaryotic cellular function. The COP9 signalosome (CSN) regulates cullin within SCF in all eukaryotes, but there is extreme sequence divergence of CSN subunits of the yeasts compared to plants and animals. Using the yeast two-hybrid system, we identified the CSN5 subunit as a potential interacting partner of a 7-transmembrane, cell surface receptor for cAMP. We further identified and characterized all 8 CSN subunits in Dictyostelium. Remarkably, despite the ancient origin of Dictyostelium and in contrast to the yeasts, these CSN protein sequences cluster very closely with their plant and animal counterparts. We additionally showed that the Dictyostelium subunits, like those of other systems, are capable of multi-protein interactions within the CSN complex and that CSN5 and CSN2 are essential for cell proliferation in Dictyostelium, a phenotype similar to that of multicellular organisms, but distinct from the yeasts.? The complex cytopathology of mitochondrial diseases is usually attributed to insufficient ATP. AMP-activated protein kinase (AMPK) is a highly sensitive cellular energy sensor that is stimulated by ATP-depleting stresses. By antisense-inhibiting chaperonin 60 expression, we produced mitochondrially diseased strains with gene dose-dependent defects in phototaxis, growth, and multicellular morphogenesis. Mitochondrial disease was phenocopied in a gene dose-dependent manner by overexpressing a constitutively active AMPK alpha subunit (AMPKalphaT). The aberrant phenotypes in mitochondrially diseased strains were suppressed completely by antisense-inhibiting AMPKalpha expression. Phagocytosis and macropinocytosis, although energy consuming, were unaffected by mitochondrial disease and AMPKalpha expression levels. Consistent with the role of AMPK in energy homeostasis, mitochondrial """"""""mass"""""""" and ATP levels were reduced by AMPKalpha antisense inhibition and increased by AMPKalphaT overexpression, but they were near normal in mitochondrially diseased cells. We also found that 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, a pharmacological AMPK activator in mammalian cells, mimics mitochondrial disease in impairing Dictyostelium phototaxis and that AMPKalpha antisense-inhibited cells were resistant to this effect. The results show that diverse cytopathologies in Dictyostelium mitochondrial disease are caused by chronic AMPK signaling not by insufficient ATP.? We have collaborated with Dr. C. Londos, LCDB/NIDDK, to study the function of proteins that associate specifically with the intracellular lipid storage droplets (LSDs) that assemble triacylglycerols and cholesteryl esters for energy metabolism, steroid hormone synthesis, membrane biosynthesis, and cell signaling. We had identified the novel PAT domain as a defining feature for LSD proteins Perilipin (Peri), ADRP, TIP47, et al. and had determined the first structure of a PAT family member, TIP47. Functional studies using native and mutated forms of Peri confirmed its role in PKA-mediated lipolysis of triacylglycerols and showed that Peri is essential for the translocation of hormone-sensitive lipase (HSL) during lipolytic activation. We now show that while the adipose mass of peri-null mice is <30% that of WT, the mutant mice are susceptible to peripheral insulin resistance in liver, but not muscle. We also show that the lipid droplet protein TIP47 is unable to functionally compensate for Peri but can complement the role of ADRP in a cell culture model system. To analyze the various functions of the 5 genes that comprise the PAT family of mammalian proteins, we have initiated a systematic series for production of singly and multiply targeted mutations of these genes in mouse ES cells and their germline transmission in mice.
