Abstract
1,3-beta-D-Glucan, a major filamentous component of the cell wall in the budding yeast Saccharomyces cerevisiae, is synthesized by 1,3-beta-glucan synthase (GS). Although a yeast gene whose product is required for GS activity in vitro, GNS1, was isolated and characterized, its role in GS function has remained unknown. In the current study we show that Deltagns1 cells accumulate a non-competitive and non-proteinous inhibitor(s) in the membrane fraction. Investigations of inhibitory activity on GS revealed that the inhibitor(s) is mainly present in the sphingolipid fraction. It is shown that Deltagns1 cells contain phytosphingosine (PHS), an intermediate in the sphingolipid biosynthesis, 30-fold more than wild-type cells do. The membrane fraction isolated from Deltasur2 cells contains an increased amount of dihydrosphingosine (DHS) and also exhibits reduced GS activity. Among constituents of the sphingolipid fraction, PHS and DHS show striking inhibition in a non-competitive manner. The intracellular level of DHS is much lower than that of PHS in wild-type cells, suggesting that PHS is the primary inhibitor of GS in vivo. The localization of PHS to the endoplasmic reticulum in wild-type cells coincides with that of the inhibitor(s) in Deltagns1 cells. Taken together, our results indicate that PHS is a potent inhibitor of yeast GS in vivo.
Highlights
In plant and fungi, remodeling of the cell wall is one of the essential processes for cell shape determination
GNS1 interacts genetically with FKS1: a ⌬gns1 ⌬fks1 double mutant grows more slowly and exhibits more reduced glucan synthase (GS) activity in the membrane fraction than single mutants [10]. These results suggest that GNS1 is somehow involved in GS activity, the physiological function of GNS1 remained unsolved since the ⌬gns1 mutant has a normal glucan content [10]
These results indicate that the reduced GS activity in the ⌬gns1 mutant is due to the reduced maximum velocity
Summary
Media and Strains—Media for growth of S. cerevisiae and Escherichia coli are as described previously [13]. GS was solubilized from the membrane fraction by adding 0.2 M NaCl, 20 M GTP␥S, 5 mM dithiothreitol, 0.5% CHAPS, and 0.1% cholesteryl hemisuccinate. This suspension was left on ice for 20 min and centrifuged at 100,000 ϫ g for 30 min. The pellet was washed 3 times with extraction buffer (4 M GTP␥S, 1 mM dithiothreitol, 0.5% CHAPS, and 0.1% cholesteryl hemisuccinate in membrane buffer) containing 5 mM UDP-Glc and was centrifuged at 4,750 ϫ g for 5 min at 4 °C. The tight pellet was homogenized in the extraction buffer and the purified GS fraction was recovered in the supernatant after re-centrifugation at 421,000 ϫ g for 10 min. Mitochondria [25] and Golgi membranes [26] were prepared as described
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