CONSEJO ACADÉMICO

All solutions required for cultivation of yeast were autoclaved or sterile filtered before usage. All yeast methods were adapted from “Yeast Protocols” (Evans, 1996), if not stated otherwise.

3.2.2.1 Culture and storage

Saccharomyces cerevisiae yeast strains were cultivated on YPD or SC medium containing agar plates or as liquid culture with vigorous agitation at the required temperature in YPD or SC medium. Media were supplemented with 0,003 % adenine, since all used yeast strains harbor an auxotroph ade2-101 mutation within the adenine gene. For short-term storage, yeast was streaked on agar plates, grown until colony formation and stored at 4 °C. For long-term storage, glycerol stocks were performed by mixing a liquid culture, grown to saturation, in 1:1 ration with 50 % glycerol. Cell suspension is shock frozen in liquid nitrogen and stored at -80 °C.

3.2.2.2 Determination of cell density

The density of cells in a culture was determined spectrophotometrically by measuring its optical density (OD) at 600 nm. For reliable measurements, cultures were diluted such that the OD600 was below 1. In this range, each 0.1 OD600 unit corresponds to ~ 3x106 cells/ml.

Consequently, an OD600 of 1 is equal to 3x107 cells/ml.

3.2.2.3 Preparation and transformation of competent yeast cells

For generation of competent yeast cells, 5 ml appropriated media was inoculated with a single yeast colony and grown to stationary phase at 30 °C. This preculture was used to inoculated 50 ml main culture to a cell density of OD600 = 0.15 and incubated as described

above. Cells were harvested at OD600 = 0.5 – 0.6 by centrifugation at 1000 x g for 3 min at

room temperature, resuspended in 50 ml ddH2O and centrifuged again. Cells were

resuspended in 12.5 ml lithium/sorbitol-buffer and pelleted once more. After an additional centrifugation step in order to remove the supernatant completely, pellets were finally resuspended in 300 µl lithium/sorbitol-buffer and mixed with 30 µl yeast carrier DNA (10 mg/ml). Next, cell suspension was aliquoted and stored at -80 °C. For transformation, 50 µl cells were incubated with 1 µg plasmid DNA and 300 µl lithium/PEG-buffer for 20 min at

Material and Methods 46 RT. 35 µl DMSO were added to the cells suspension followed by heat shock incubation at 42 °C for 15 min. Cells were pelleted at 4000 x g for 5 min, resuspended in appropriated medium and plated on selective plates followed by incubation at 30 °C until colonies developed.

Lithium/sorbitol buffer 100 mM LiOAc, 10 mM Tris-HCl pH 8.0, 1 mM EDTA

1 M sorbitol

Lithium/PEG buffer 100 mM LiOAc, 10 mM Tris-HCl pH 8.0, 1 mM EDTA

40% PEG3350

3.2.2.4 Purification of DNA from yeast cells

2 ml yeast cultures were grown overnight to early stationary phase at 30 °C. Cells were pelleted by centrifugation at 2000 x g, washed once with ddH2O and resuspended in 200

µl buffer A followed by incubation for 1 h at 30 °C with gently agitation. Subsequently, 200 µl buffer B were added and incubated for 20 min at 65 °C. Cells were placed on ice prior to addition of 200 µl KAc and 15 min incubation. Cells were centrifuged at 20.000 x g and supernatant transferred to fresh Eppendorf tube. DNA was precipitated from supernatant by addition of 600 µl isopropanol. After short incubation at RT, DNA was pelleted by centrifugation at 20.000 x g for 10 min, washed once with 70 % ethanol and incubated at RT for 10 min. DNA was pelleted again by centrifugation at 20.000 x g for 10 min, air-dried and resuspended in 30 µl TE buffer.

Buffer A 100 mM Tris-HCl pH 7.5, 10 mM EDTA pH 7.5, 10 µl/ml 2-

mercaptoethanol, 0.2 mg/ml Zymolase-20T

Buffer B 0.2 M NaOH, 1 % SDS

3.2.2.5 Protein expression in yeast cells

For expression of proteins in yeast, galactose, copper and doxycycline regulated promoters, GAL1, CUP1 and tetO respectively, were used. Since glucose inhibits the GAL1

promoter, glucose was only used as carbon source for yeast in media when expression was driven by CUP1 promoter. For galactose inducible expression, raffinose was used as carbon source instead of glucose. A stationary phase preculture was used to inoculated a main culture to an optical density of OD600 = 0.15 and grown at 30 °C while shaking (230 rpm). Expression

of the protein of interest was induced with 2 % Galactose or 100 µM CuSO4, for GAL1 or CUP1 promoter respectively, once the culture reached OD600 = 0.4 – 0.5 and cells were

Material and Methods 47 expression was induced by withdrawal of doxycycline from the media. Cells were washed 3 times in media and were allowed to grow for desired period.

3.2.2.6 Growth assays

Growth curves and plate assays were performed to compare viability of various yeast strains. For plate assays, a serial dilution was performed with the appropriated medium starting from an exponential phase culture adjusted to a cell density of OD600 = 1. 5 µl of each

dilution was dropped on selective plate and incubated at the required temperature until colonies developed. Sensitivity to the microtubule-destabilizing drug benomyl was assayed by spotting cells on YPD agar plates containing 0 and 40mg/ml benomyl. For growth curves, cell density was recorded in liquid media supplemented with 50 mM 3-AT at 30 °C by reading OD600 at various time points.

3.2.2.7 Luciferase assay

Luciferase activity was measured by mixing approximately 5 µl exponential phase cells with 50 µl of beetle luciferin (Promega). The samples were immediately transferred into a luminometer (EG&G Berthold) and measured for 5 – 30 seconds. Each measurement was repeated at least three times and the results were averaged. Cell density was determinated by measuring OD600 in order to calculate relative luciferase activity.

3.2.2.8 β-Galactosidase assay

β-galactosidase converts the colorless ONPG substrate into galactose and the chromophore ο-nitrophenol, yielding a bright yellow solution. 100 µl lysate were incubated with 700 µl Z buffer and 160 µl ONPG at 30 °C. 400 µl Stop solution were added when all samples showed a weak yellow coloring and absorbance were measured at 420 nm. β- galactosidase activity was expressed in nmoles of β-galactose formed per minutes per mg of lysates at 30 °C (Nielsen et al., 1983).

Z Buffer 100 mM NaH2PO4 pH 7.0, 10 mM KCl, 1 mM MgSO4, 50 mM β-

mercaptoethanol ONPG 4 mg/ml in Z Buffer Stop solution 1 M Na2CO3

Material and Methods 48

3.2.2.9 Preparation of yeast cell lysates

Glass bead lysis was performed for preparation of lysates under native conditions. Yeast cultures were harvested by centrifugation at 2000 x g. Pellets were washed once with ddH2O and mixed with 0.5 – 1 ml ice-cold lysis buffer and one volume of acid-washed glass

beads (diameter 0.45 – 0.5 mm). Lysis buffers TG&I or TP were only used for gel filtration and immunoprecipitation or NiNTA pull down of TRiC, respectively. Suspensions were repeatedly shaken vigorously for 1 min and chilled on ice in between. Cell debris and beads were pelleted at 1000 x g at 4 °C and supernatants were transferred to new tubes.

Lysis buffer 25 mM Tris-HCl pH 7.5, 50 mM KCl, 10 mM MgCl2, 1 mM EDTA,

5% glycerol, 1% Triton-X-100, 1x protease-inhibitor-mix Lysis buffer TG&I 1 x PBS, 1 mM EDTA, 1x protease-inhibitor-mix

Lysis buffer TP 1 x PBS, 10 mM MgCl2, 5 % glycerol, 0.1% Triton-X-100, 1x

protease-inhibitor-mix

3.2.2.10 Immunofluorescence

For immunofluorescence microscopy, 5 ml exponential phase culture were fixed by addition of 0.5 M K3PO4 (pH 6.5) and 3.7% formaldehyde for one hour at RT. Cells were

harvested by centrifugation at 1000 x g, washed five times with sorbitol buffer and resuspended in 1 ml sorbitol buffer supplemented with 2.5 mg Zymolase T20 and 19 µM 2- mercaptoethanol. Suspension was incubated at 30 °C under agitation until ~ 90 % of cells were spheroblasts. After thoroughly washing with sorbitol buffer, spheroblasts were resuspended in 500 µl sorbitol buffer. 20 µl of suspension were transferred onto a multiwell microscopy slide coated with 0,3 % poly-L-lysine and incubated for 10 min. Unbound spheroblasts were washed away with PBS supplemented with 1 % BSA (PBS-B) before air- drying slides. For permeabilization, slides were incubated 5 min in ice-cold 100 % ethanol following 30 sec in ice-cold 100 % acetone. After air-drying, cells were incubated with PBS- B for one hour at RT to prevent unspecific antibodies binding. Spheroblasts were incubated with appropriate primary antibody (20 – 50 mg/ml in PBS-B) for one hour. After thoroughly washing with PBS-B, cells were incubated with corresponding fluorescent-labeled secondary antibody (5 – 10 µg/ml in PBS-B) as before. Slides were washed as before, overlaid with mounting medium and covered with coverslip sealed by nail polisher.

Sorbitol buffer 0.1 M KPO4, pH 6.5, 1.2 M sorbitol

Material and Methods 49