Abstract
The ciliate Tetrahymena thermophila can either synthesize tetrahymanol or when available, assimilate and modify sterols from its diet. This metabolic shift is mainly driven by transcriptional regulation of genes for tetrahymanol synthesis (TS) and sterol bioconversion (SB). The mechanistic details of sterol uptake, intracellular trafficking and the associated gene expression changes are unknown. By following cholesterol incorporation over time in a conditional phagocytosis-deficient mutant, we found that although phagocytosis is the main sterol intake route, a secondary endocytic pathway exists. Different expression patterns for TS and SB genes were associated with these entry mechanisms. Squalene synthase was down-regulated by a massive cholesterol intake only attainable by phagocytosis-proficient cells, whereas C22-sterol desaturase required ten times less cholesterol and was up-regulated in both wild-type and mutant cells. These patterns are suggestive of at least two different signaling pathways. Sterol trafficking beyond phagosomes and esterification was impaired by the NPC1 inhibitor U18666A. NPC1 is a protein that mediates cholesterol export from late endosomes/lysosomes in mammalian cells. U18666A also produced a delay in the transcriptional response to cholesterol, suggesting that the regulatory signals are triggered between lysosomes and the endoplasmic reticulum. These findings could hint at partial conservation of sterol homeostasis between eukaryote lineages.
Highlights
The ciliate Tetrahymena thermophila can either synthesize tetrahymanol or when available, assimilate and modify sterols from its diet
In order to support the notion that the triggering of signals is confined to the endoplasmic reticulum (ER) or in NPC1-containing compartments, we examined the effect of Brefeldin A, which has been used in Tetrahymena as a Golgi apparatus-disrupting a gent[34]
We demonstrate that cholesterol enters the cells through two types of actin-dependent transport: phagocytosis and pinocytosis
Summary
The ciliate Tetrahymena thermophila can either synthesize tetrahymanol or when available, assimilate and modify sterols from its diet. Tetrahymena acquired the capacity to synthesize tetrahymanol in one step without the need for molecular oxygen by means of the squalene-tetrahymanol cyclase (THC) a ctivity[11,12,13,14] Despite this functional substitution, Tetrahymena possesses enzymes that modify dietary-source sterols, transforming them into the tri-unsaturated molecule 7,22-bisdehydrocholesterol (BDHC)[15,16,17,18,19]. Sterol conversion and its influence on tetrahymanol production has been studied since the 1960s, but only recently has the identification and characterization of the four enzymes involved in BDHC synthesis been achieved Included in these findings are two desaturases that insert double bonds at the C5 and C7 positions of the sterols B ring (DES5 and DES7, respectively)[16,18], a de-ethylase that removes the ethyl group from the aliphatic lateral chain of phytosterols (DES24)[17] and two paralogs of a novel desaturase which introduce double bonds at the C22 position of the lateral chain (DES22A and B)[19]. Images obtained by fluorescence microscopy using a BODIPY-cholesterol probe showed that in comparison with a conditional mutant in phagocytosis (II8G-IA) at the restrictive temperature[22,23], only a phagocytosis proficient strain (CU428) was able to accumulate sterols after a 16-h incubation period
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