Figure 3.

Overexpression of budding yeast Ndc1 or its N-terminal domain induces ER membrane expansion with pore-like structures. (A) Electron micrographs of WT yeast cells overexpressing ProtA-ScNdc1 showing the membrane proliferation. Cytoplasmic tubular membrane assemblies are marked with black arrows. Bars, 500 nm. ER-expansions with local membrane discontinuities (II), marked with red arrows, are magnified. Bars, 250 nm. (B) Fluorescence microscopy of cells overexpressing eGFP-Ndc1 for 0.5 or 5 h using DsRed-HDEL as an ER marker. Chromatin is stained with Hoechst 33258. Bars, 5 µm. (C) Electron micrograph of a WT yeast cell overexpressing ProtA-tagged 3-hydroxy-3-methylglutaryl-coenzymeA reductase 1 (ProtA-ScHmg1). The observed karmellae are shown at higher magnification (bars, 250 and 50 nm for the magnified panel). (D) Electron micrographs of yeast cells overexpressing the N-terminal part of ScNdc1 (ProtA-ScNdc1 (1-260)). Black arrows mark NE/ER proliferations, red arrows cytoplasmic membrane assemblies. Tubular membrane assemblies, similar to the ones observed upon overexpression of full-length Ndc1, are shown in I, karmellae-like multi-layered membrane whorls are shown in II. Bars, 250 nm. (E) Growth tests with yeast cells overexpressing ProtA-tagged full-length Ndc1 and Ndc1 truncations from both C. thermophilum and S. cerevisiae, ProtA-ScHmg1 or ProtA alone using LEU2 as selection marker. 10-fold serial dilutions of corresponding overnight cultures were spotted onto either glucose- (expression suppressing) or galactose- (expression inducing) containing plates lacking leucine (SDC-Leu or SGC-Leu) and incubated at 30 or 37°C for up to 3 d. For expression control, whole cell lysates were prepared from overnight cultures and analyzed by Western blotting. Source data are available for this figure: SourceData F3.