Data associated with Figs. 2 and 3 . Fld1 and Nem1 localize to punctae in cells devoid of neutral lipids or LDs. (A) Localization of Fld1 or Nem1 to ER foci is independent of neutral lipid synthesis or the presence of LDs. BODIPY staining of 4ΔKO cells expressing genomic Fld1-mCherry or Nem1-mCherry. Scale bar, 5 µm. (B and C) Time-lapse images of Fld1-GFP and Nem1-GFP. 4ΔKO cells expressing genomic Fld1-GFP (B) or Nem1-GFP (C) were grown to early stationary phase, and time-lapse images were recorded. The boxed region in B and C is shown in higher magnification. Snapshots of images at the indicated time points are shown. White arrowheads indicate Fld1 or Nem1 punctae that stay immobile in the ER membrane. Yellow arrowheads denote rapidly moving Fld1 punctae. Scale bars, 5 µm. (D) Fld1 colocalizes with Nem1. WT cells expressing Nem1-GFP and coexpressing Fld1-mCherry were imaged. White arrowheads denote colocalization between Fld1 and Nem1. Scale bar, 5 µm. (E) Quantification of colocalization between Nem1-GFP and Fld1-mCherry. Data are means ± SD, n > 50 cells.