Figure 3.

The ER anchored CD of HMGR was sufficient to cause membrane reorganization. (A) Diagrams of Ole1pⁱ variants examined. TDH3pr-driven triple-myc–tagged Ole1p with the H161A, H166A-inactivating mutations was fused to various soluble proteins as indicated. Nomenclature is as follows: Ole1pⁱ, Ole1p with H161A and H166A mutations; Ole1pⁱ-GFP, inactive Ole1p fused to GFP; Ole1pⁱ-2hN-GFP, inactive Ole1p fused to the hN-domain of Hmg2p and GFP; Ole1pⁱ-Hmg2p CD, inactive Ole1p fused to the CD of Hmg2p; and Ole1pⁱ-Hmg1p CD, inactive Ole1p fused to the CD of Hmg1p. White- and gray-striped area corresponds to Hmg2p linker domain; hatched area corresponds to Hmg1p linker domain. Quantitation of cells harboring one or two copies of the expression plasmids are listed on the right. Expression plasmids were integrated at one or two loci. Cells were prepared for IF and examined for structures. A minimum of 200 cells was counted in three independent experiments; averages and standard deviations are shown. Nucleus is visualized with DAPI. (B) Representative IF of strains with two expression constructs is shown. Arrows indicate ER membrane structures. (C) Electron micrographs of cells with two copies of TDH3pr-driven Ole1pⁱ-Hmg2p CD and Ole1pⁱ-Hmg1p CD. Log phase cells were grown in YPD. Double arrowheads indicate structures.