Inositol and Phosphate Regulate GIT1 Transcription and Glycerophosphoinositol Incorporation in Saccharomyces cerevisiae

Abstract

Glycerophosphoinositol is produced through deacylation of the essential phospholipid phosphatidylinositol. In Saccharomyces cerevisiae, the glycerophosphoinositol produced is excreted from the cell but is recycled for phosphatidylinositol synthesis when inositol is limiting. To be recycled, glycerophosphoinositol enters the cell through the permease encoded by GIT1. The transport of exogenous glycerophosphoinositol through Git1p is sufficiently robust to support the growth of an inositol auxotroph (ino1⌬). We now report that S. cerevisiae also uses exogenous phosphatidylinositol as an inositol source. Evidence suggests that phosphatidylinositol is deacylated to glycerophosphoinositol extracellularly before being transported across the plasma membrane by Git1p. A genetic screen identified Pho86p, which is required for targeting of the major phosphate transporter (Pho84p) to the plasma membrane, as affecting the utilization of phosphatidylinositol and glycerophosphoinositol. Deletion of PHO86 in an ino1⌬ strain resulted in faster growth when either phosphatidylinositol or glycerophosphoinositol was supplied as the sole inositol source. The incorporation of radiolabeled glycerophosphoinositol into an ino1⌬ pho86⌬ mutant was higher than that into wild-type, ino1⌬, and pho86⌬ strains. All strains accumulated the most GIT1 transcript when incubated in media limited for inositol and phosphate in combination. However, the ino1⌬ pho86⌬ mutant accumulated approximately threefold more GIT1 transcript than did the other strains when incubated in inositol-free media containing either high or low concentrations of P i . Deletion of PHO4 abolished GIT1 transcription in a wild-type strain. These results indicate that the transport of glycerophosphoinositol by Git1p is regulated by factors affecting both inositol and phosphate availabilities and suggest a regulatory connection between phosphate metabolism and phospholipid metabolism.

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

“…Through the action of phospholipase B, S. cerevisiae hydrolyzes external phosphatidylinositol to release GroPIns (13). Thus, the combination of external phospholipases and Git1p provide yeast with the capability of obtaining crucial nutrients (inositol and phosphate) from environments in which PI or its deacylation product, GroPIns, are available.…”

“…2) in cells grown in IϪ, high P i medium, a condition which clearly supports Git1p-mediated transport (12,13), prompted us to perform a more in-depth analysis of GIT1 expression and its relationship to transport activity. GIT1 expression was monitored via a reporter construct in which the GIT1 promoter was fused to the bacterial chloramphenicol acetyltransferase (CAT) gene (cat) (13). GIT1 promoter activity as well as Git1p transport activity were monitored as a function of time following the transfer of cells from fully repressing medium (Iϩ, high P i ) to various starvation media (Fig.…”

“…For the inositol auxotrophs, ino2⌬ and ino4⌬, 5IϪ media were substituted for IϪ media. At the indicated times, aliquots of the cultures were harvested, and CAT activity was determined using the FAST CAT green (deoxy) chloramphenicol acetyltransferase assay kit as described (13).…”

“…For the inositol auxotrophs, ino2⌬ and ino4⌬, 5IϪ media were substituted for IϪ media. RNA was extracted, and Northern blots were performed as described (13).…”

“…The predicted GIT1 protein product bears similarity to other S. cerevisiae transport proteins, including the inositol transporters Itr1p and Itr2p, and the inorganic phosphate transporter, Pho84p. Recent studies (13) have shown that in addition to inositol limitation, phosphate limitation induces increased uptake of GroPIns. In the present report, we characterize further the nature of the regulation of GroPIns transport via inositol and phosphate limitation.…”