Ltc1 is required for sterol internalization and localization to ER-PM contact sites. (A) Neighbor-joining phylogenetic trees of S. pombe and S. cerevisiae OSH and LAM family proteins. The S. pombe genes were named in analogy to the S. cerevisiae names, with Osh = oxysterol-binding protein homologue, Oss1 = oxysterol-binding split-domain protein, and Ltc1/2 = lipid transfer at contact site. Protein domains identified by SMART are indicated (http://smart.embl-heidelberg.de/). (B) mCherry-D4H in OSH family or LAM family deletion mutants treated or not with CK-666 for 1 h. (C) Quantification of mCherry-D4H fluorescence intensity at cell tip versus cell side. Bars show the mean (n ≥ 50 cells). (D) Ltc1-GFP expressed from the native genomic locus localizes to cortical and internal dots (arrowheads) at the ER, labeled with mCherry-AHDL. Right panels show an enlargement of the nucleus. (E) Ltc1-GFP localizes to residual ER-PM contacts (arrowheads) in the scs2Δ scs22Δ mutant. Panel represents middle plane view and four subsequent planes at 0.4 µm distance. Scale bars, 0.5 µm in D, right panel, and 2 µm elsewhere.