Phosphorylation is the major mechanism regulating isocitrate lyase activity in Paracoccidioides brasiliensis yeast cells

Abstract

The glyoxylate cycle plays an essential role for anaplerosis of oxaloacetate during growth of microorganisms on carbon sources such as acetate or fatty acids and has been shown to contribute to virulence of several pathogens. Here, we investigated the transcriptional and post‐translational regulation of the glyoxylate cycle key enzyme isocitrate lyase (PbICL) in the human pathogenic fungus Paracoccidioides brasiliensis. Although sequence analyses on fungal isocitrate lyases revealed a high phylogenetic conservation, their regulation seems to differ significantly. Closely related Aspergillus species regulate the glyoxylate cycle at the transcriptional level, whereas Pbicl was constitutively expressed in yeast cells. However, only low PbICL activity was detected when cells were grown in the presence of glucose. Two‐dimensional gel analyses with subsequent antibody hybridization revealed constitutive production of PbICL, but low PbICL activity on glucose coincided with extensive protein phosphorylation. Since an in vitro dephosphorylation of PbICL from glucose grown cells strongly increased ICL activity and resembled the phosphorylation pattern of highly active acetate grown cells, post‐translational modification seems the main mechanism regulating PbICL activity in yeast cells. In agreement, a transfer of yeast cells from glucose to acetate medium increased PbICL activity without requirement of de novo protein synthesis. Thus, inactivation of PbICL by phosphorylation is reversible, denoting a new strategy for the rapid adaptation to changing environmental conditions.

Transcriptional and Proteomic Responses to Carbon Starvation in Paracoccidioides

“…Membranes were blocked for 1 h at room temperature in a solution containing 10% (w/v) skim milk powder and 0.1% Tween 20 in Tris-buffered saline (TBS-T). The primary polyclonal antibody anti-isocitrate lyase [43] was diluted in blocking solution and incubated with the membrane for 1 h at room temperature. Membranes were washed in Tris-buffered saline and then incubated with alkaline phosphatase conjugated secondary antibodies for another hour at room temperature.…”

“…The proteins were fractionated by one-dimensional gel electrophoresis. The proteins were blotted onto a nitrocellulose membrane and the ∼60 kDa protein species was detected by using the rabbit polyclonal antibody anti- Pb Icl [43]. ( B ) Densitometric analysis of immunoblotting bands was performed using the software AphaEaseFC.…”

3′ Untranslated Region-Dependent Degradation of the aceA mRNA, Encoding the Glyoxylate Cycle Enzyme Isocitrate Lyase, by RNase E/G in Corynebacterium glutamicum

“…AceK phosphorylates ICD; this lowers ICD turnover and prevents a high flux of isocitrate through the TCA cycle (10,16). Since dephosphorylation of ICL leads to loss of catalytic activity (41), phosphorylation is assumed to increase the flux of isocitrate through the glyoxylate cycle in E. coli (11).…”

Proteomic profile response of Paracoccidioides lutzii to the antifungal argentilactone

“…P. lutzii (ATCC-MYA-826) has been extensively studied in different laboratories (Pereira et al, 2010; Cruz et al, 2011; Oliveira et al, 2013; Teixeira et al, 2013). The fungus was cultivated in Fava-Netto's medium (1.0% w/v peptone, 0.5% w/v yeast extract, 0.3% w/v proteose peptone, 0.5% w/v beef extract, 0.5% w/v NaCl, 4% w/v glucose, and 1.4% w/v agar, pH 7.2) (Fava-Netto and Raphael, 1961) at 36°C for growth of the yeast phase.…”