A yeast Golgi E-type ATPase with an unusual membrane topology

Abstract

E-type ATPases are involved in many biological processes such as modulation of neural cell activity, prevention of intravascular thrombosis, and protein glycosylation. In this study, we show that a gene of Saccharomyces cerevisiae, identified by similarity to that of animal ectoapyrase CD39, codes for a new member of the E-type ATPase family (Apy1p). Overexpression of Apy1p in yeast cells causes an increase in intracellular membrane-bound nucleoside di- and triphosphate hydrolase activity. The activity is highest with ADP as substrate and is stimulated similarly by Ca2+, Mg2+, and Mn2+. The results also indicate that Apy1p is an integral membrane protein located predominantly in the Golgi compartment. Sequence analysis reveals that Apy1p contains one large NH2-terminal hydrophilic apyrase domain, one COOH-terminal hydrophilic domain, and two hydrophobic stretches in the central region of the polypeptide. Although no signal sequence is found at the NH2-terminal portion of the protein and no NH2-terminal cleavage of the protein is observed, demonstrated by the detection of NH2-terminal tagged Apy1p, the NH2-terminal domain of Apy1p is on the luminal side of the Golgi apparatus, and the COOH-terminal hydrophilic domain binds to the cytoplasmic face of the Golgi membrane. The second hydrophobic stretch of Apy1p is the transmembrane domain. These results indicate that Apy1p is a type III transmembrane protein; however, the size of the Apy1p extracytoplasmic NH2 terminus is much larger than those of other type III transmembrane proteins, suggesting that a novel translocation mechanism is utilized.