A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.
In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.
Instituto de Bioquímica Médica, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.
Bruno L
BL
Bozaquel-Morais
Juliana B
JB
Madeira
Clarissa M
CM
Maya-Monteiro
Claudio A
CA
Masuda
Mónica
M
Montero-Lomeli
D015153:Blotting, Western; D005456:Fluorescent Dyes / Q000737:chemistry*; D050356:Lipid Metabolism; D008856:Microscopy, Fluorescence; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*
D005456:Fluorescent Dyes; D010749:Phosphoprotein Phosphatases