Abstract
Under nitrogen starvation, Ustilago maydis forms lipid droplets (LDs). Although the dynamics of these organelles are known in the literature, the identity of the lipases implicated in their degradation is unknown. We determined lipase activity and identified the intracellular lipases expressed during growth under nitrogen starvation and YPD media by zymograms. The results showed that cytosolic extracts exhibited higher lipase activity when cells were grown in YPD. Under nitrogen starvation, lipase activity was not detected after 24 h of culture, resulting in lipid accumulation in LDs. This suggests that these lipases could be implicated in LD degradation. In the zymogram, two bands, one of 25 and the other of 37 kDa, presented lipase activity. The YPD extracts showed lipase activity in olive and almond oils, which contain triacylglycerols with mono and polyunsaturated fatty acids. This is the first report about U. maydis cytosolic lipases involved in LD degradation.
Highlights
We reported that lipid droplets (LDs) are dynamic organelles that can be mobilized when a nitrogen source is present, and we suggested that under these conditions the lipase activity should be enhanced (Romero-Aguilar et al, 2017)
U. maydis was grown for 72 hours in YPD and minimal medium without a nitrogen source (MM-N) (Figure 1)
We previously reported the accumulation of lipids as LDs in U. maydis under nitrogen starvation (MM-N) (Zavala-Moreno et al, 2014)
Summary
U. maydis FB2 (a2b2) cells were kept in 25% glycerol at −70 °C, streaked on YPD plates (1.0% glucose, 0.25% peptone, 0.5% yeast extract and 2.0% agarose), and incubated at 28 °C for 24 h. The strain was pre-inoculated in 100 ml YPD medium (1.0% glucose, 0.25% peptone, and 0.5% yeast extract) and shaken at 175 rpm at 28 °C for 24 h. The cells were harvested by centrifugation at 1000 g for 10 min, washed twice with H2O, and inoculated in a new medium with an initial optical density at 600 nm (OD600) of 0.05 units. 18 aliquots of cell cultures were taken every 2 h in the first 24 h, and afterwards every 3 h, measuring the OD600 in a Genesys 20 spectrophotometer (Z376035, Sigma-Aldrich, St. Louis, USA)
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