Growth-dependent oligomerization of SAC1 regulates its ER–Golgi distribution. (A) Sequence motifs in SAC1 orthologues. Leucine residues within the putative LZ motif and lysine residues within the C-terminal ER retrieval motif are highlighted in magenta. (B and C) COS7 cells transiently expressing flag-SAC1 mutants and/or GFP-SAC1 were cultured either in the presence (10%) or absence (0.5%) of serum for 24 h and lysed, and flag-SAC1 was immunoprecipitated with M2 agarose beads. The bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-GFP, β-COP, or SEC23 antibodies. Black lines indicate that intervening lanes have been spliced out. (D and E) COS7 cells transiently expressing GFP-SAC1-K2A (D, green) or GFP-SAC1-LZ (E, green) were cultured either in the presence (10%) or absence (0.5%) of serum for 24 h and then subjected to immunofluorescence microscopy using either polyclonal anti-TGN46 (D, red) or anti-Sec61β (E, red). Bars, 15 μm. (F) COS7 cells were infected with adenoviruses to express flag-tagged versions of SAC1, SAC1-LZ, and phosphatase-dead SAC1-C/S. Cells were then lysed and SAC1 was collected on M2 agarose beads. After the elution with 200 μg/ml of M2 peptide, 2 μg of each purified SAC1 protein was assayed for phosphatase activity using dioctanoyl-PI(4)P as a substrate. Data are from three independent phosphatase measurements. Data represent means ± SD from three independent experiments.