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 Ca (2+), Mg(2+), and Mn(2+). 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 NH(2)-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 NH(2)-terminal portion of the protein and no NH(2)-terminal cleavage of the protein is observed, demonstrated by the detection of NH(2)-terminal tagged Apy1p, the NH(2)-terminal domain of Apylp 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 Apylp is a type III transmembrane protein; however, the size of the Apy1p extracytoplasmic NH(2) terminus is much larger than those of other type III transmembrane proteins, suggesting that a novel translocation mechanism is utilized.
Highlights
Plasma gondii NTPase [9]
Expression of a putative yeast 71.9-kDa protein (Apy1p)—To determine whether this gene can be expressed in yeast cells, the sequence for a hemagglutinin epitope (HA) tag was added in-frame to the 3Ј-end of the open reading frame, and the resulting cDNA was expressed behind a constitutive promoter in the yeast vector pG1
These results indicate that the NH2terminal domain is in the lumen, that glycosylation of Asn-371 contributes to the formation of the 73-kDa form of Apy1p, and that Asn-532 is not utilized for glycosylation, which is consistent with the cytoplasmic location of the COOH-terminal domain
Summary
Plasma gondii NTPase [9]. All of these proteins contain four highly conserved sequences called apyrase conserved regions (ACR1– 4). After transfer of sugar residues to proteins and lipids by glycosyltransferases, the resulting nucleoside diphosphates are converted to nucleoside monophosphates by nucleotide diphosphatases In this way, nucleoside diphosphates that are inhibitors of glycosyltransferases do not accumulate in the lumen of the Golgi apparatus. A sequence homology search in the GenBank data base revealed another gene from S. cerevisiae (GenBank accession number P40009) with high similarity in the ACR1– 4 motifs to members of the E-ATPase family. This gene is on chromosome V, encodes a hypothetical 71.9-kDa protein, and was proposed recently to be the second E-ATPase found in yeast [14]. It is located in the Golgi and has an unusual membrane topology
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