Golgi Localization and Functional Expression of Human Uridine Diphosphatase*
- From the Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
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 nematodeCaenorhabditis elegans and to an unidentified 71.9-kDa yeast protein and is less related to the previously identified yeast Golgi GDPase.
Footnotes
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↵* This work was supported in part by Grant HL08893 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF016032.
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↵‡ Supported in part by a Maria Moors Cabot fellowship.
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↵§ To whom correspondence should be addressed: Dept. of Molecular and Cellular Biology, Harvard University, 7 Divinity Ave., Cambridge, MA 02138. Tel.: 617-495-2301; Fax: 617-495-8308; E-mail:guidotti{at}fas.harvard.edu.
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↵1 The abbreviations used are: BFA, brefeldin A; 5′-RACE, rapid amplification of 5′-cDNA ends; PCR, polymerase chain reaction; ACR, apyrase conserved region; RER, rough endoplasmic reticulum; EATPase, ecto-ATPase.
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- Received January 28, 1998.
- Revision received February 25, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
