FIGURE 1.
The ORMDLs suppress in vivo activity of wild-type and mutant SPT. A, HEK293 cells were transfected with empty vector (lane 1) or hLCB1, hLCB2a, and HA-ssSPTa in the absence (lane 2) or presence of HA-ORMDL1 (lane 3), HA-ORMDL2 (lane 4), HA-ORMDL3 (lane 5), or ORMDL1-GFP (lane 6). Protein expression was verified by immunoblotting. B, total levels of sphingosine (So), dihydrosphinganine (Sa), and 1-deoxySa were determined in cells transfected with pcDNA, or hLCB1, hLCB2a, ssSPTa, and the indicated ORMDLs as described under “Experimental Procedures.” Results reported are the average of three independent experiments ± S.D. C and D, same as A and B with the exception that the hLCB1C133W mutant subunit, which results in the efficient synthesis of 1-deoxySa, was used. E, ORMDL expression was quantified as described under “Experimental Procedures.” Results are presented as the mean ± S.D. from three independent experiments.