Identification and characterization of a glucan synthesis regulatory gene designated ACU1

An attempt was made to clone the glucan synthetase gene of Saccharomyces cerevisiae by a procedure termed segmental aneuploidy. This procedure involves the endowment of cellular resistance to a particular inhibitor by the over-expression of a cloned gene encoding the protein target of the particular inhibitor. An S. cerevisiae library in the vector YEp13 was used to transform S. cerevisiae DBY747. Transformants that were resistant to Aculeacin A, a glucan synthetase inhibitor, were obtained. These transformants theoretically contained the gene for glucan synthetase or a regulator of glucan synthetase.

One of the aculeacin A resistant S. cerevisiae transformants contained a plasmid designated pGS4A which carried a 4.5kb insert. Using a mixed-membrane glucan synthesis assay it was found that the plasmid pGS4A increased glucan synthesis activity by two-fold in an S. cerevisiae DBY747 transformant.

Various fragments of the insert in pGS4A were subcloned and it was determined that a 1.2kb fragment was responsible for increasing glucan synthesis activity. The 1.2kb fragment was sequenced and the sequence was used in a search of the GENBANK database. The putative amino acid sequence of the only open reading frame (designated the ACU1 gene), which is composed of 236 amino acids, found was used in a search of the PIR and Swissprot databases. No significant matches were found in either database search. The ACU1 gene was found to be located on chromosome X by both pulsed-field gel electrophoresis and using a portion of the chromosome sequenced previously by others. A gene disruption experiment was performed and again a two-fold increase in glucan synthesis was observed suggesting the gene codes for a negative regulator of glucan synthesis. Northern analysis indicated a predicted transcript size of - 800 bases and that the ACU1 gene was poorly transcribed. Potential transcription induction of the ACU1 gene by pheromone treatment or growth on glycerol was predicted by putative upstream activating sequences of ACU1 but Northern analysis showed induction did not occur. The complete mechanism by which glucan synthesis occurs is unknown at this time, but a model is presented which may explain how the protein encoded by the ACU1 gene regulates glucan synthesis.