Determination Of The Topology Of The Hydrophobic Segment Of Mammalian Diacylglycerol Kinase Epsilon In A Cell Membrane And Its Relationship To Predictions From Modeling

The epsilon isoform of diacylglycerol kinase (DGKepsilon) is unique among
mammalian DGKs in having a segment of hydrophobic amino acids comprising
approximately residues 20 to 41. Several algorithms predict this segment to be a
transmembrane (TM) helix. Using PepLook, we have performed an in silico analysis
of the conformational preference of the segment in a hydrophobic environment
comprising residues 18 to 42 of DGKepsilon. We find that there are two distinct
groups of stable conformations, one corresponding to a straight helix that would
traverse the membrane and the second corresponding to a bent helix that would
enter and leave the same side of the membrane. Furthermore, the calculations
predict that substituting the Pro32 residue in the hydrophobic segment with an
Ala will cause the hydrophobic segment to favor a TM orientation. We have
expressed the P32A mutant of DGKepsilon, with a FLAG tag (an N-terminal 3xFLAG
epitope tag) at the amino terminus, in COS-7 cells. We find that this mutation
causes a large reduction in both k(cat) and K(m) while maintaining k(cat)/K(m)
constant. Specificity of the P32A mutant for substrates with polyunsaturated acyl
chains is retained. The P32A mutant also has higher affinity for membranes since
it is more difficult to extract from the membrane with high salt concentration or
high pH compared with the wild-type DGKepsilon. We also evaluated the topology of
the proteins with confocal immunofluorescence microscopy using NIH 3T3 cells. We
find that the FLAG tag at the amino terminus of the wild-type enzyme is not
reactive with antibodies unless the cell membrane is permeabilized with
detergent. We also demonstrate that at least a fraction of the wild-type
DGKepsilon is present in the plasma membrane and that comparable amounts of the
wild-type and P32A mutant proteins are in the plasma membrane fraction. This
indicates that in these cells the hydrophobic segment of the wild-type DGKepsilon
is not TM but takes up a bent conformation. In contrast, the FLAG tag at the
amino terminus of the P32A mutant is exposed to antibody both before and after
membrane permeabilization. This modeling approach thus provides an explanation,
not provided by simple predictive algorithms, for the observed topology of this
protein in cell membranes. The work also demonstrates that the wild-type
DGKepsilon is a monotopic protein.