Abstract
Rat CD39, a membrane-bound ectonucleoside triphosphate diphosphohydrolase that hydrolyzes extracellular nucleoside tri- and diphosphates, is anchored to the membrane by two transmembrane domains at the two ends of the molecule. The transmembrane domains are important for enzymatic activity, as mutants lacking one or both of these domains have a fraction of the enzymatic activity of the wild-type CD39. We investigated the interactions between the transmembrane domains by using a strain of yeast that requires surface expression of CD39 for growth. Random mutagenesis of selected amino acid residues in the N-terminal transmembrane domain revealed that the presence of charged amino acids at these positions prevents expression of functional protein. Rescue of the growth of these mutants by complementary mutations on selected residues of the C-terminal transmembrane domain indicates that there is contact between particular faces of the transmembrane domains.
Highlights
Ectoapyrase activities are present in virtually all eukaryotic cells, starting with protozoa [1]
The approach was based on the view that the surfaces of TM1 and TM2 defined by the hydrophobic moment would be likely to be exposed to lipid [37], while the helix surfaces characterized by helix packing moments would be involved in interactions between the transmembrane helical domains [39]
We conclude that G22 and S24 are likely to be on interfacial faces of TM1 and that the α-helices in question are likely to be closer to one another at the extracellular ends and farther apart at the cytoplasmic ends to account for the ability of large nonpolar amino acids to occupy these positions in a functional protein
Summary
Ectoapyrase activities are present in virtually all eukaryotic cells, starting with protozoa [1]. The active site of the E-NTPDases is extracytoplasmic, either on the cell surface or in the lumen of intracellular membrane compartments, and it catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside tri- and diphosphates. There are eight members of the E-NTPDase family, six with two transmembrane domains [3]. CD39 is a monomer in the first group of detergents and a dimer in digitonin solutions; the soluble form of CD39 lacking both transmembrane domains is a monomer. These results suggested that interactions between the transmembrane domains are required to form the active form of CD39 and that the dimer may be the active form of the enzyme.
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