Appearance of Smaller Lipid Bodies and Protein Kinase Activation in the Lipid Body Fraction Are Induced by an Increase in the Nitrogen Source in the Mortierella Fungus

Abstract

We studied the regulation of lipid body biogenesis in the oleaginous fungus Mortierella ramanniana var. angulispora by investigating culture conditions to modulate lipid body size, which we found was affected by the carbon-to-nitrogen ratio (C/N ratio) in the culture medium. Increasing the nitrogen source or decreasing the C/N ratio from 38 to 9 induced the appearance of lipid bodies with diameters less than 2-3 micro m, which are usually found at a C/N ratio of 38 in this fungus. To determine factors regulating lipid body size, we compared lipid body fractions from fungal cells cultured at different C/N ratios. We found some differences in polypeptide profiles between lipid body fractions from fungal cells cultured at different C/N ratios for 2 days when the lipid bodies were enlarged at a C/N ratio of 38. We then compared the phosphorylation of lipid body proteins, since protein phosphorylation plays a pivotal role in various aspects of signal transduction. In vitro phosphorylation in the lipid body fraction indicated that protein kinase activity toward endogenous and exogenous substrates such as histone IIIS, VIIS, and myelin basic protein increased in the lipid body fraction at a C/N ratio of 9. Further analysis by in-gel protein kinase assay indicated the presence of at least three activated protein kinases with molecular masses of 75, 72, and 42 kDa, which were also autophosphorylated. These results indicate the presence of nutrient-regulated protein kinases and increased phosphorylation in lipid bodies, which correlate with the appearance of smaller lipid bodies in this fungus. Further studies to characterize these protein kinases at the molecular level should provide new insights into the link between nutrient sensing and lipid storage.

A Novel Protein Kinase Localized to Lipid Droplets Is Required for Droplet Biogenesis in Trypanosomes

“…Although we anticipated that our screen might yield genes encoding proteins localized to the plasma membrane, this kinase was instead localized to an intracellular organelle. Previous work has shown some partial localization of mitogen-activated protein kinases to lipid droplets of leukocytes (53), as well as anonymous kinase activity associated with lipid droplets (33). However, these physical associations have not been tied to droplet function.…”

Identification of Genes Affecting Lipid Content Using Transposon Mutagenesis inSaccharomyces cerevisiae

“…We investigated the effect of nitrogen limitation on the lipid content of these disruptants (Table 4), since nitrogen limitation is known to cause lipid accumulation in oleaginous fungi and yeasts. 15,30) The results indicate that cultivation in the nitrogen-limited medium increased the lipid content in all disruptants as well as the wild type. The lipid content of the Ápre9 disruptant was particularly increased by nitrogen limitation.…”

“…After drying, plates were developed with hexane/diethylether/acetic acid (80:40:1) to the top. Free sterol spots were charred by sulfuric acid as described previously, 15) and quantified by densitometry with ergosterol as a standard.…”

“…angulispora, and characterized lipid transport into lipid bodies 13,14) and modulation of protein kinases in lipid bodies in response to the carbon-to-nitrogen ratio. 15) In an attempt to identify the genes affecting lipid accumulation, we took advantage of genetic engineering tools in S. cerevisiae. We obtained mutants with higher lipid content by random transposon insertion, followed by identification of inserted genes.…”

Plant lipid bodies and cell-cell signaling: A new role for an old organelle?

“…67 Chilling upregulates LB-associated GH17s and displaces important during lipolysis and germination in seeds as well as in bud break and apical growth in perennial species. To summarize, LBs in plants, like those in animals 85 and fungi 86 might be multifunctional organelles, participating in a host of cellular processes including membrane trafficking, lipid-based signaling, sterol-homeostasis, nutrient sensing, and transport of enzymes, regulatory proteins and signaling molecules. 6,19,42,53 In the near future it will be of great interest to establish to what degree the LB-and PD-proteome 80,87,88 will converge.…”