Interactions between the transmembrane domains of CD39: identification of interacting residues by yeast selection

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.

Keywords: CD39; Helical wheel; Hydrophobic moment; Packing moment; Transmembrane domains.

Figure 1

Growth of acid phosphatase-negative yeast strain on uracil-deficient medium supplemented with phosphate or ATP. The cells of the yeast strain carrying pVT101-CD39 (CD39) or pVT101 (Cont.) were streaked on a synthetic minimal DO-U medium plate containing phosphate (A) or 0.3 mM ATP at pH 7.2 as phosphate source (B). A total of 10 μl of a cell culture OD₆₅₀ = 0.1 was plated on both plates. The plates were incubated at 30°C for 3 days.

Figure 2

Growth of acid phosphatase-negative yeast strain in liquid uracil-deficient medium containing ATP. Yeast carrying pVT101-CD39 (■), pVT101-CD39CT (▲), pVT101-CD39NT (●), and pVT101 (□) were grown in DO-U medium, pH 7.2 at 30°C containing 0.3 mM ATP.

Figure 3

Helical wheel projections of TM1 and TM2 of CD39 using the program of Kael Fisher (http://kael.net/helical.htm). TM1, starting at A34, and TM2, starting at L481, are shown going from the extracellular surface to the cytoplasmic surface of the plasma membrane. The residues colored red have helix packing values larger than that of Gln (38). The black arrow indicates the direction of the hydrophobic moment (37); the red arrows show residues with helix packing moments greater than that of Gln suggesting helical packing interfaces (38).

Figure 4

Helical net diagrams (40) of TM1 and TM2. The transmembrane domains are shown with the extracellular surface at the bottom and the cytoplasmic surface at the top of the figure. The residues colored red have helix packing values larger than that of Gln (38). The residues grouped by blue lines in TM1 and TM2 correspond to those in Figure 3 indicated by the red arrows suggesting helical packing interfaces.

Figure 5

Expression of wtCD39, and the G22R and G22R-LTE (L489L, M492T, G496E) mutants in yeast. A total of 15 μg of crude membrane proteins of samples pVT101-CD39G22R (lanes 1–2), pVT101-CD39 G22R-LTE (lines 3–4), pVT101-CD39 (lanes 5–6), and pVT101 (lanes 7–8) were treated (*) or not treated with glycopeptidase F. The samples were subjected to 7.5% SDS-PAGE and examined by immunoblotting with anti-HA11 monoclonal antibody.

Figure 6

Helical wheel heptad projections (45) of TM1 and TM2 showing the arrangement of the amino acids on the surface of the helix. TM1, starting with A34, and TM2, starting with L481, are shown from the extracellular surface to the cytoplasmic surface of the membrane going into the plane of the page. The residues colored red have helix packing values larger than that of Gln (38). The surfaces of TM1 with G22 and S24 are proposed to interact with the surfaces of TM2 with G496/M492 and T495/A491, respectively. (A) TM domain interactions in the open state and (B) TM domain interactions in the closed state. The arrows indicate the direction of rotation of the TM domains through the stated angles in order for the TM domains to move from one stable interaction to the other.