Abstract
A full-length E(ecto)-ATPase (Plesner, L. (1995) Int. Rev. Cytol. 158, 141-214) cDNA was cloned from a human brain cDNA library; it encodes a 610-amino acid protein that contains two putative transmembrane domains. Heterologous expression of this protein in COS-7 cells caused a significant increase in intracellular membrane-bound nucleoside phosphatase activity. The activity was highest with UDP as substrate and was stimulated by divalent cations in the following order: Ca2+ >> Mg2+ > Mn2+. The results of immunofluorescence staining indicate that this protein is located in the Golgi apparatus. UDP hydrolysis was increased in the presence of Triton X-100 or alamethicin, an ionophore that facilitates movement of UDP across the membrane, suggesting that the active site of this UDPase is on the luminal side of the Golgi apparatus. This is the first identification of a mammalian Golgi luminal UDPase gene. Computer-aided sequence analysis of the EATPase superfamily indicates that the human UDPase is highly similar to two hypothetical proteins of the nematode Caenorhabditis elegans and to an unidentified 71.9-kDa yeast protein and is less related to the previously identified yeast Golgi GDPase.
Highlights
A highly specific Saccharomyces cerevisiae Golgi GDPase has been described and purified to homogeneity [7]
Expression of the protein in COS-7 cells resulted in an increase in Ca2ϩ-dependent nucleoside phosphatase activity
Since rat liver UDPase [1] and yeast GDPase [8] are involved in protein and lipid glycosylation in the Golgi apparatus, it is likely that the human UDPase might have the same function
Summary
(Received for publication, January 28, 1998, and in revised form, February 25, 1998). UDP hydrolysis was increased in the presence of Triton X-100 or alamethicin, an ionophore that facilitates movement of UDP across the membrane, suggesting that the active site of this UDPase is on the luminal side of the Golgi apparatus. After transfer of sugar residues to proteins and lipids by galactosyltransferases, the resulting UDP is hydrolyzed to UMP by UDPase [4] In this way, UDP, which is highly inhibitory to galactosyltransferases [5], does not accumulate in the lumen of the Golgi apparatus. A highly specific Saccharomyces cerevisiae Golgi GDPase has been described and purified to homogeneity [7] This enzyme appears to be involved in protein and lipid mannosylation, as the GDP is generated from the mannose donor, GDP-mannose, by mannosyltransferase. We show that this new E-ATPase is a Golgi luminal UDPase
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