Figure 6. ORM-dependent relocalization of SPT is induced by LCBs.

A. Yeast cells expressing Lcb2 containing a multimerizing version of GFP inserted between residues 82 and 83 (Lcb2^GFP@82) were grown in YPD medium with and without LCBs (PHS, DHS, or 3-ketosphinganine (3-KDS) and the localization of Lcb2 was observed using fluorescence microscopy. The non-physiological LCB stearylamine (STA) served as a negative control. B. Lcb1-RFP was co-expressed with Lcb2^GFP@82, and the localization of the proteins was compared by fluorescence microscopy. C. RFP-Orm2 and Lcb2^GFP@82 were co-expressed and their localization visualized by fluorescence microscopy. D. PHS-induced relocalization of SPT in wild-type (-PHS, no bars, n>500 cells; +PHS, 51 ± 4% with bars, n=247 cells, 5 experiments) and orm1Δorm2Δ mutant cells (-PHS, no bars, n=93 cells; +PHS, no bars, n=187 cells, 3 experiments) expressing Lcb2^GFP@82 was compared as in Panel A. E. PHS-induced relocalization of SPT in cells expressing Lcb2^GFP@82 and the non-phosphorylatable Orm2-3A (-PHS, 49 ± 9% with bars, n=116 cells; +PHS, 52 ± 16% with bars, n=67 cells, 3 experiments) or the phosphomimetic Orm2-3D mutant (-PHS, no bars, n=80 cells; +PHS, no bars, n=118 cells, 3 experiments) was determined as in Panel A. F. PHS-induced relocalization of SPT in wild-type and sac1Δ cells (-PHS, no bars, n>100 cells; +PHS, 23 ± 10% with bars, n=99 cells, 4 experiments) expressing Lcb2^GFP@82 was determined as in Panel A. Scale bar, 5 microns.