Quick-freezing and freeze replica preparation

Yeast cells were quick-frozen either by metal-contact freezing using a Slammer (Valiant Instruments, Ellisville, MO, USA) or by high-pressure freezing using an HPM 010 high-pressure freezing machine (Leica Microsystems, Wetzlar, Germany). For metal-contact freezing, cell pellets were placed on a glass coverslip and slammed to a copper block pre-cooled with liquid nitrogen. For high-pressure freezing, an EM grid (200 mesh) impregnated with yeast cells was sandwiched between a 20-μm–thick copper foil and a flat aluminum disc (Engineering Office M. Wohlwend, Sennwald, Switzerland) and frozen according to the manufacture’s instructions (Cheng et al., 2014).

For freeze-fracture, the specimens were transferred to the cold stage of a Balzers BAF 400 apparatus and fractured at −120 to −100°C under a vacuum of ~1 × 10⁻⁶ mbar. Replicas were made by electron-beam evaporation in a process involving three steps: carbon (C) (2–5 nm in thickness) at an angle of 90° to the specimen surface, platinum-carbon (Pt/C) (1–2 nm) at an angle of 45°, and C (10–20 nm) at an angle of 90° (Fujita et al., 2010). The thickness of the deposition was adjusted by referring to a crystal thickness monitor. Thawed replicas were treated with 2.5% SDS in 0.1 M Tris-HCl (pH 7.4) at 60°C overnight. To remove the cell wall, yeast replicas were treated for 2 hr at 37°C with one of the following solutions: (1) 20 μg/ml Zymolyase 100T (Nacalai Tesque) in PBS containing 0.1% Triton X-100, 1% BSA, and a protease inhibitor cocktail (Nacalai Tesque) or (2) 0.1% Westase (Takara Bio, Kusatsu, Japan) in McIlvain buffer (37 mM citrate, 126 mM disodium hydrogen phosphate, pH 6.0) containing 10 mM EDTA, 30% fetal calf serum, and a protease inhibitor cocktail. Replicas for labeling PtdIns(3)P were generally treated with Zymolyase, whereas those for protein labeling were treated with Westase. After the enzyme treatment, the replicas were further treated in 2.5% SDS and stored in buffered 50% glycerol at −20°C until use.

For freeze-fracture/etching, the specimens were fractured at −102°C under a vacuum of ~1 × 10⁻⁶ mbar and kept at the same temperature for 2 min to induce sublimation of water from the fractured surface. The samples were then exposed to rotary evaporation of Pt/C (4 nm) at an angle of 20°, followed by C (20 nm) at an angle of 80°. The replicas were treated with household bleach to digest biological materials before mounting on EM grids for observation. The method of freeze-fracture/etching is described in detail at Bio-protocol (Tsuji and Fujimoto, 2017).