Regulation of Inositol Transport in Saccharomyces cerevisiae Involves Inositol-induced Changes in Permease Stability and Endocytic Degradation in the Vacuole

Git1p mediates the transport of the phospholipid metabolite, glycerophosphoinositol, into Saccharomyces cerevisiae. We report that phosphate limitation and inositol limitation affect GIT1 expression and Git1p transport activity via distinct mechanisms that involve multiple transcription factors. GIT1 transcript levels and Git1p activity are greater in cells starved for phosphate, with or without inositol limitation, than in cells only limited for inositol. Furthermore, the kinetics of GIT1 transcript accumulation and Git1p activity upon transfer of cells to phosphate starvation media are different from those obtained upon transfer of cells to inositolfree media. Pho2p and Pho4p are required for GIT1 expression and for Git1p transport activity under all growth conditions tested. In contrast, Ino2p and Ino4p are required for full GIT1 expression when inositol is limiting, with or without phosphate limitation, but not when only phosphate is limiting. Greatly reduced transport activity was detected in ino2⌬ and ino4⌬ cells under all growth conditions. A 300-base pair region of the GIT1 promoter containing potential Pho4p binding sites was shown to be required for full GIT1 expression. Git1p appears to act as a H ؉ -symporter, and neither inositol nor phosphate effectively compete with glycerophosphoinositol for transport by Git1p. Glycerophosphoinositol was shown previously to support the growth of an inositol auxotroph. Remarkably, we now report that glycerophosphoinositol can act as the sole source of phosphate for the cell, providing functional relevance for the regulation of Git1p transport activity by phosphate.

Section: Deletion Of Potential Pho4p Binding Sites Results In Loss Ofmentioning

confidence: 99%

Glycerophosphoinositol, a Novel Phosphate Source Whose Transport Is Regulated by Multiple Factors in Saccharomyces cerevisiae

Almaguer

Cheng

Nolder

et al. 2004

“…Alternatively, expression of the gene encoding PS synthase may be derepressed at low cellular CDP-DAG synthase levels, as is INO1 (inositol 1-phosphate synthase) expression in some mutants of phospholipid metabolism. Constitutive expression of INO1 causes inositol excretion, the opi phenotype (44), which has been found in a series of mutants involved in synthesis of the aminophospholipids and inositol uptake (36,(45)(46)(47)(48). Low cellular CDP-DAG synthase in cdg1 and CDS1* mutants may affect the biosynthesis of the aminophospholipids due to the limited amount of CDP-DAG available for de novo synthesis and consequently may result in derepression of INO1 gene expression.…”

Section: Discussionmentioning

confidence: 99%

Reduction of CDP-diacylglycerol Synthase Activity Results in the Excretion of Inositol by Saccharomyces cerevisiae

Shen

Dowhan

1996

3 Tyr substitution) within the CDS1 gene (encodes CDP-DAG synthase) of this mutant. Expression of CDP-DAG synthase activity from a plasmid-borne copy of the CDS1 gene in the cdg1 mutant was not down-regulated, and this expression also corrected the inositol excretion phenotype. Introduction of the above mutated gene (CDS1*) controlled by its endogenous promoter on a single copy plasmid into a cds1-null background reconstituted a transformant with the cdg1 phenotype, including reduced CDP-DAG synthase activity, elevated phosphatidylserine synthase activity, and inositol excretion into the growth medium. Expression of CDS1* in a single copy in the cdg1 mutant raised CDP-DAG synthase activity from 15 to 30% of derepressed wild-type yeast levels but still did not correct the inositol excretion phenotype. CDP-DAG synthase activity was not regulated in response to precursors of phospholipid biosynthesis in the cdg1 mutant either with or without a trans copy of the CDS1* gene. An open reading frame was identified 5 to the CDS1 locus, YBR0314, which also resulted in inositol excretion when present in trans in multiple copies.

“…A large body of work has pointed to intracellular inositol availability as the primary mode of regulation of PI synthase (54 -56). Inositol is taken up from the media by the inositol permease encoded for by the ITR1 gene (57). Inositol uptake may be compromised in temperature-shifted erg26-1 cells due to the accumulation of zymosterol intermediates.…”

Section: Fig 8 Erg26p and Erg27p Localize To The Er In Yeast Cellsmentioning

confidence: 99%

The Effect of the erg26-1 Mutation on the Regulation of Lipid Metabolism in Saccharomyces cerevisiae

Baudry¹,

Swain²,

Rahier

et al. 2001

A temperature-sensitive Saccharomyces cerevisiae mutant harboring a lesion in the ERG26 gene has been isolated. ERG26 encodes 4␣-carboxysterol-C3 dehydrogenase, one of three enzymatic activities required for the conversion of 4,4-dimethylzymosterol to zymosterol. Gas chromatography/mass spectrometry analyses of sterols in this mutant, designated erg26-1, revealed the aberrant accumulation of a 4-methyl-4-carboxy zymosterol intermediate, as well as a novel 4-carboxysterol. Neutral lipid radiolabeling studies showed that erg26-1 cells also harbored defects in the rate of biosynthesis and steady-state levels of mono-, di-, and triglycerides. Phospholipid radiolabeling studies showed defects in the rate of biosynthesis of both phosphatidic acid and phosphatidylinositol. Biochemical studies revealed that microsomes isolated from erg26-1 cells contained greatly reduced 4␣-carboxysterol-C3 dehydrogenase activity when compared with microsomes from wild type cells. Previous studies have shown that loss of function mutations in either of the fatty acid elongase genes SUR4/ELO3 or FEN1/GNS1/ELO2 can "bypass" the essentiality of certain ERG genes (Ladeveze, V., Marcireau, C., Delourme, D., and Karst, F. (1993) Lipids 28, 907-912; Silve, S., Leplatois, P., Josse, A., Dupuy, P. H., Lanau, C., Kaghad, M., Dhers, C., Picard, C., Rahier, A., Taton, M., Le Fur, G., Caput, D., Ferrara, P., and Loison, G. (1996) Mol. Cell. Biol. 16, 2719 -2727). Studies presented here have shown that this sphingolipiddependent "bypass" mechanism did not suppress the essential requirement for zymosterol biosynthesis. However, studies aimed at understanding the underlying physiology behind the temperature-sensitive growth defect of erg26-1 cells showed that the addition of several antifungal compounds to the growth media of erg26-1 cells could suppress the temperature-sensitive growth defect. Fluorescence microscopic analysis showed that GFP-Erg26p and GFP-Erg27p fusion proteins were localized to the endoplasmic reticulum. Two-hybrid analysis indicated that Erg25p, Erg26p, and Erg27p, which are required for the biosynthesis of zymosterol, form a complex within the cell.