Experiments with yeast

2.13.1 Complementation (drop test)

For complementation analysis transformed yeast cells were grown in 10 ml selective SD media (with glucose) until an OD600 of 0.5. The cultures were split into two aliquots and pelleted via centrifugation for 10 min at 800 g. The first pellet was diluted in selec-tive SD media (with glucose) in a way to obtain 0.5 OD600 after 12 h of incubation. The second pellet was diluted in the same manner using selective inductive media (without glucose, supplemented with 1% galactose and 1% fructose [w/v]). After incubation five successive 1:5 dilutions starting with an OD600 of 0.3 were set up for each culture.

Drops of 3 µl were spotted on a set of plates (selective SD plates [with glucose], selective inductive SD plates [without glucose, supplemented with 1% galactose and 1% fructose (w/v)], normal and inductive YPD plates [glucose or galactose/ fructose] as control). One set of plates was incubated at 30 °C for 2-3 days, to check for comple-mentation of cold-sensitive phenotypes an additional plate set was incubated at 20 °C for 5-7 d.

2.13.2 Lipid uptake assey

100 ml yeast cell culture within the exponential phase (OD₆₀₀ 0.3-0.7) grown on selec-tive SD media (with glucose) was pelleted via centrifugation for 10 min at 800 g and dissolved in the same amount selective inductive SD media (without glucose, supple-mented with 2% galactose [w/v]). Cells were incubated for 12 h under gene expression inducing conditions driven by the GAL1-10-promoter on the transformed plasmid (Johnston

and Davis, 1984). Cells transformed with an empty vector were treated the same way and used as a negative control. OD600 was determined and the culture split in two aliquots. Probes of the first aliquot were diluted in a way to achieve the same OD. 40 ml were pelleted and used for protein analysis (chapter 2.13.3). Probes of the second ali-quot were pelleted and diluted with selective SD media (without glucose, supplemented with 4% galactose [w/v]) in a way to achieve an OD₆₀₀ of 10.

2.13.2.1 Lipid labeling of cells

Uptake experiments with fluorescently labeled PL were performed as described by Pomorski et al. (2003) with the optimizations from Poulsen et al. (2008b). Cells were incubated 5 min at 30 °C prior to labeling. 1.5 µl 10 mM NBD-labeled phospholipids (my-ristoyl-[NBD-hexanoyl]-PC, -PE and -PS were tested, Avanti Polar Lipids, Alabaster, AL, USA) dissolved in DMSO were added and incubated for 30 min at 30 °C. A negative control was prepared in the same way with DMSO only. Cells were thoroughly mixed every 5 min. The reaction was stopped by addition of 1 ml ice-cold SSA/BSA (2mM NaN3, 2% sorbitol, 3% BSA [w/v] in selective SD media) and cells were immediately chilled on ice. Cells were pelleted (5 min at 700 g and 4 °C), redissolved in 1 ml SSA/ BSA und transferred into new glass tubes. In order to remove non-internalized PL, cells were washed two times with SSA/BSA and the pellet finally dissolved in 250 µl PBS (compo-sition given in Table 26) and kept on ice until flow cytometry measurement.

2.13.2.2 Measurement by flow cytometry

Flow cytometry was used to evaluate internalization of NBD-labeled PL of yeast cells.

50 µl cell suspension was mixed with 1 ml PBS in FACS vials. Immediately before meas-urement 1 µl propidium iodide (5 mg/ml [w/v]) was added under mixing to the cells.

Flow cytometry was performed on a Becton Dickinson flow cytometer (San Jose, CA, USA) equipped with a 488 nm argon laser. Detection of propidium iodide (PI) stained cells was recorded at 570 nm, NBD fluorescence at 545 nm. Forward scatter indicated cell size, sideward scatters the fluorescence intensity of PI and NBD. 20000 cells, ga-ting PI stained dead cells, were counted and the accumulation of NBD-labeled lipids was calculated as percentage compared to a negative control (cells transformed with an empty plasmid).

2.13.3 Protein analysis

2.13.3.1 Preparation of total cell lysate and membrane concentration

Cellular membranes were prepared similar to the method from Villalba et al. (1992).

Pelleted yeast cells were resuspended in 250 µl ice-cold lysis buffer (composition given in Table 26) and transferred to a new 10 ml glass tube. 0.5 g glass beads (600 µm ø) were added to the cell suspension and cells disrupted by vigorously mixing for 5 min.

Table 26: Lysis buffer for yeast cells.

PBS containing 1% PMFS-stock and 0.1% protease inhibitor mix [v/v]

Name Concentration Substance

PBS 136.89 mM NaCl

2.68 mM KCl 10.14 mM Na2HPO4

1.67 mM KH2PO4; pH 7.4

PMFS-stock 100 mM PMSF in isopropanol

protease inhibitor mix 1 mg/ml apoprotein 1 mg/ml leupeptin 1 mg/ml pepstatin 5 mg/ml antipain

157 mg/ml banzamidine in DMSO

During cell disruption, probes were shortly chilled on ice app. every 30 sec. After cell disruption the supernatant was transferred to a new reaction vial, the glass beads were washed with 250 µl ice-cold lysis buffer and the buffer combined with the previous su-pernatant. Cell debris and intact cells were separated via centrifugation for 5 min at 500 g and 4 °C and total cell lysate stored at -20 °C.

Membranes were concentrated from total cell lysate via ultra-centrifugation for 1 h at 100000 g and 4 °C. 25 µl of the supernatant (total lysate) was kept as a control and the membrane pellet was resuspended in 25 µl ice-cold lysis buffer.

2.13.3.2 SDS-PAGE

Electrophoretically separation of proteins was performed after Laemmli (1970) via discontinuous SDS-PAGE. Polyacrylamide gels of 0.75 mm thickness consisting of a

10% resolving gel (375 mM Tris-HCl; pH 8.8) combined with a 4.5% stacking gel (125 mM Tris-HCl; pH 6.8) were generated and run using the Mini-PROTEAN 3 cell system (BioRad, Munich, Germany) according to the manufacturer’s instructions with a running buffer consisting of 25 mM Tris, 192 mM glycine and 0.1% SDS (w/v). Prior to electrophoresis 25 µl sample buffer (10% SDS, 0.0025% bromo-phenol blue [w/v], 12.5%

glycerol [v/v], 100 mM DTT in 100 mM Tris; pH 6.8) was added to each probe (25 µl), followed by incubation for 15 min at 37 °C and centrifugation for 10 min at 5000 g. 15 µl of the supernatant was loaded together with 8 µl of a size marker (Prestained SDS-PAGE standard, BioRad, Munich, Germany) on an PA gel. Electrophoretical separation was performed at 180 V for 50 min.

2.13.3.3 Protein transfer

Prior to their detection proteins were transferred from the PA matrix to a membrane based on Towbin et al. (1979). Protein transfer occurred in a wet blot chamber (Trans Blot Cell, BioRad, Munich, Germany) onto a PVDF membrane (Hybond-P, Amersham Pharmacia Biotech, Freiburg, Germany) according to the manufacturer’s instructions using 4 °C chilled transfer buffer (20% methanol [v/v], 10 mM Tris, 77 mM glycine, 0.1% SDS [w/v]) and applying an amperage of 3.5 mA/cm² membrane for 50 min.

2.13.3.4 Immunological protein detection

After protein transfer the membrane was incubated in TBS (150 mM NaCl, 10 mM Tris;

pH 8.0) for 10 min followed by a 1% blocking solution (Western Blocking Reagent, Roche Applied Science, Mannheim, Germany) for 1 h at RT. The immunological detection of tagged proteins was performed as follows: First, the membrane was incubated with pri-mary antibody (monoclonal mouse anti-HA 1:1000 and monoclonal mouse anti-RGSH6

1:5000 [Santa Cruz Biotechnology, Santa Cruz, CA, USA]) in 0.5% blocking solution ([v/v] in TBS) over night at 4 °C. Next, the membrane was washed twice with TBST (TBS supplemented with 0.1% Tween 20 [v/v]) for 10 min, followed by two wash steps with 0.5% blocking solution for 10 min. Then the membrane was incubated with secon-dary antibody (peroxidase-linked goat anti-mouse 1:5000 [Santa Cruz Biotechnology, Santa Cruz, CA, USA]) in 0.5% blocking solution for 1 h at RT. Subsequently, the membrane was washed four times 15 min with TBST. All washing and incubation steps occurred under shaking.

Visualization of detected proteins was done via chemiluminescence with ECL Plus Reagenz (Amersham Pharmacia Biotech, Freiburg, Germany) according to the

manu-facturer’s instructions. Prepared reagent was carefully dropped on the wet membrane and incubated for 5 min at RT. Depending on the signal strength the membrane was exposed to a film (X-OMAT AR, Kodak, Stuttgart, Germany) for 10 sec - 5 min and sub-sequently developed.