| S. cerevisiae - EUROFAN II - Node N5 |
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Coordinator: H Riezman (Basel, CH)
Participants: M Aebi (Zürich, CH), R Haguenauer-Tsapis (Paris, FR), S Keränen (Helsinki, FI), C Stirling (Manchester, GB), H Riezman (Basel, CH), H Ronne (Uppsala, SE).
N5 Secretion and protein trafficking
Markus Aebi (CH), Rosine Haguenauer-Tsapis (F), Sirkka Keränen (SF), German Larriba (E), Hans Ronne (S), Colin Stirling (GB), Howard Riezman (CH, coordinator)
The secretory/endocytic pathway is in fact a complex network of interconnected pathways between which proteins are transported by vesicular flow and perhaps other mechanisms. A large number of essential and nonessential genes have been shown to be required for these transport steps, but the pathway has not been saturated as judged by the continual discovery of new genes involved in these processes.
During transport of secretory proteins to the cell surface, these proteins undergo a series of modifications (folding, glycosylation, proteolytic processing, GPI anchoring) that allow their progress through the pathway to be followed easily by examination of their molecular weight or their modifications. The technique of Western blotting with specific antibodies is the pillar of our strategy and will be used to detect progress through the secretory pathway in all available viable disruptants. If conditional alleles of genes are developed they will also be tested as many genes involved in secretion are essential. The technique of Western blotting (1) will also detect mutants which are defective in various aspects of glycosylation. For mutants affecting protein folding, we will look for induction of the unfolded protein response (UPR).
Transport through the endocytic pathway will be followed by assaying the accumulation of the small fluorescent dye, Lucifer Yellow CH in the vacuole (2). A defect in the endocytic pathway can be confirmed by testing for synthetic lethality with mutants in the vacuolar ATPase and the site of the endocytic block can be localized by following the endocytic marker, a-factor (3).
Some functions of the secretory pathway are not accompanied by a change in size detected by Western blotting. Among these are protein folding in the endoplasmic reticulum and transport from the Golgi to the cell surface. Recent evidence suggests that there are at least two and maybe more pathways from the Golgi to the cell surface. In order to identify genes affecting these functions and pathways we will use synthetic lethality approaches that are currently being developed similar to those used for endocytosis.
Another step that is not associated with a difference in molecular weight is retrograde transport through the Golgi and from the Golgi to the endoplasmic reticulum. We will set up a test for this by disrupting the genes with the long homology cassettes in a strain that is sterile if the pathway is fully functional, but acquires the ability to mate when there is a defect in retrieval from the Golgi to the endoplasmic reticulum (4). The appropriate strain will be disrupted with the long homology cassettes and the disruptants will be tested for their ability to mate by replica plating.
We will also examine the ability of the disruptants to phosphorylate the carbohydrate chains of cell surface glycoproteins. Phosphorylation of these chains renders the cell surface highly negatively charged and causes the cells to bind tightly to positively charged beads or to be stained more heavily by Alcian Blue. A simple Alcian Blue staining protocol and bead binding assay will be used to evaluate this phosphorylation step.
Several very small membrane proteins have been shown to be involved in various aspects of the secretory pathway; for example translocation into the ER (5), and full oligosaccharide tranferase activity (6). These ORFs are too small to have been considered by EUROFAN, but we expect that many will be important in the secretory pathway. To identify such small ORFs, we will extract small hydrophobic membrane proteins from membranes, separate them by HPLC and analyze individual peaks by mass spectroscopy. This sequence data will be used to search the yeast genome for the corresponding locus and standard techniques will be used to analyze their function.
Finally, the analysis done in the first part of this proposal should lead to the identification of disruptants that affect various aspects of the secretory pathway. These disruptants will be analyzed further in year 2 by the groups which are expert in this particular part of the secretory/endocytic pathway.
| Group | Expertises |
| MA | oligosaccharide synthesis and glycosylation, protein folding |
| RHT | secretion to the cell surface, endocytosis |
| GL | secretion to the cell surface, phosphorylation of carbohydrate chains |
| SK + HRonne | Golgi to cell surface |
| HR | endocytosis, ER retention, vacuole biogenesis |
| CS | insertion into the ER, protein folding in the ER |
Alcian Blue Test
General: Alcian blue is a cationic stain that binds those cells carrying a negative charge on the cell surface (Fries and Ottolenghii, 1970). When the assay is performed at acid pH, only the sugar-linked phosphates are responsible for the negative charge of the cell wall, so that the method allows detection of cells with an altered amount of phosphate groups. For this purpose, the stain is prepared as a 0.1% solution in 0.02 N HCl. The solution should be centrifuged to eliminate insoluble precipitates.
Method: Cells are grown for 48 hr on YPD plates. A small amount of cells is taken using a tooth pick and transferred to a disposable glass test tube avoiding the transfer of agar which would give false results. Cells are washed with 0.5 ml of 0.9% NaCl and then supplemented with 0.2 ml of the alcian blue solution. The mixture is vortexed until cells and stain are well mixed, maintained at room temperature for 5 to 10 min and centrifuged at 2000 rpm. The supernatant is discarded (the upper part of the tube is wiped with tissue paper). The pellet is washed with 2 ml of 0.02 N HCl, and the last supernatant above the undisturbed pellet is retained.
The test can be done with a large number of samples, by using microtiter
plates. For this purpose each well was treated as follows:
Cells: place at the bottom of the well with a toothpick
Wash with 0.2 ml of 0.9% NaCl
Add 0.1 ml of alcian blue solution (0.1% in 0.02 N HCl). Wait 10-15
min at room temperature.
Centrifuge and wash twice with 0.2 ml of 0.02 N HCl.
Results with Standard Strains:
| Strain | Phosphate content | Color | |
| mm6 | 10% | White | |
| mnn1 mnn9 | medium | light blue | |
| FY1679 (wt) | 100% | blue | |
| mnn1 | 100% (more accessible) | deep blue |
Staining with Alcian blue, coloration rated from 1 to 6 (white to deep blue)
The assay was essentially performed as described by Dulic et al. (1991 Methods Enzymol. 194:697-710).
Cultures of yeast cells are grown to early logarithmic phase (0.5-2x106 cells/ml respectively to an OD600 of 0.1-0.4) in YPUAD (1% yeast extract, 2% peptone, 20mg/l uracil, 40 mg/l adenine and 2% glucose).
Cells are harvested by 1 minute low speed centrifugation in a benchtop microfuge. The yeast cells are resuspended in 90m l fresh YPUAD medium. Lucifer Yellow (40mg/ml in water) is then added to a final concentration of 4mg/ml. The cells are incubated like this at room temperature for 1 hour. Following this they are washed 3 times with 600m l ice-cold buffer (50mM NaPO4 pH 7.0, 10mM NaN3, 10mM NaF).
The final pellet is resuspended in a small volume (about 10m l of washing buffer). The cells can be kept on ice like this for up to 2 hours.
2-3m l of this suspension are mixed with an equal volume of low melting point agarose (1,6% kept at 42oC) and mounted on a microscope slide.
Lucifer Yellow is visualized by fluoresence microscopy using FITC optics. Exposure times of approximately 20s are necessary for optimal detection of vacuolar Lucifer yellow staining in wild-type cells.
Results in the file are presented as follows:
1: strongly reduced or no accumulation of Lucifer Yellow
0: Lucifer Yellow accumulation similar to wild-type
Work done by Andreas Wiederkehr in the laboratory of Howard Riezman:
wiederkehra@ubaclu.unibas.ch
Preparation of total protein extracts for western immunoblots
The following protocol can be applied to cells grown in YPD, minimal YNB medium, low Pi rich or minimal media etc.
- Centrifugation of a volume of culture corresponding to 3 A600nm. Leave 0.5 ml of medium.
- Add 50 µl of NaOH 1.85 M (with or without added 2% 2-mercaptoethanol), mix and leave for 10 min on ice.
- Add 50 µl of TCA 50%, mix and leave 10 min (or more...) on ice.
- Centrifugation for 5 min (12000xg), and take away the whole supernatant.
- Resuspend the pellet in 70 µl of
2 volumes sample buffer (1x)
1 volume Tris Base 1 M
2% 2-mercaptoethanol
and heat for 10 min at 95°C (for soluble proteins, also OK for the membrane-bound Gas1p) or 10 min at 37°C for multispanning membrane-bound proteins (such as plasma membrane [H+] ATPase, uracil permease, general amino acid permease, ...).
even if the pellet is not entirely dissolved, you can apply directly the resulting sample on gels (volumes of 2-10µl depending upon the protein of interest).
The samples can be frozen at -20°C for months (even years...).
2-fold concentrated sample buffer:
100 mM Tris HCl pH 6.8
4 mM EDTA
4% SDS
20% glycerol
0.002% bromophenol blue
This rapid protocol is derived from that published by Riezman et al, 1983 EMBO. J., 14, 1329-1339, and slightly modified as described in Volland et al, 1994, J. Biol. Chem. 269, 9833-9841. The TCA precipitation step was found to be critical to avoid proteolytic degradation of a number of proteins. The protocol can be modified in numerous ways. It is possible for instance to use it with higher volumes of growth medium, with adjustments of the volumes of NaOH and TCA...
Deletion mutants in the FY1679 MATalpha background (Eurofan strains FY10001B-FY10796B) were grown on YPD plates, suspended in sterile water at a density of approximately 3 x 106 cells/ml, and then sequentially diluted in water using either 10-fold or 25-fold dilution steps. Approximately 3 µl of the each dilution was spotted onto synthetic complete plates containing either brefeldin A (500 µg/ml), monensin (25 µg/ml) or C2-ceramide (500 µg/ml). The plates were incubated at 25°C and 30°C for up to five days and monitored for growth.
Howard Riezman, Andreas Wiederkehr
University of Basel
Biocenter
Klingelbergstrasse 70
CH-4056 Basel, CH
Tel. +41 61 267 21 60; FAX +41 61 267 21 49
e-mail: riezman@ubaclu.unibas.ch
e-mail: wiederkehra@ubaclu.unibas.ch
Markus Aebi
ETH Zentrum
Mikrobiologisches Institut
Schmelzstrasse 7
CH-8092 Zürich, CH
Tel. +41 1 632 64 13; FAX +41 1 632 11 48
e-mail: aebi@micro.biol.ethz.ch
Colin J. Stirling
School of Biological Sciences
2.205 Stopford Building
University of Manchester
Oxford Road
Manchester M13 9PT
UK
Tel. +44-(0)-161-275-5104 (Office) -5644 (Lab); FAX +44-(0)-161-275-5082
e-mail: stirling@fs2.scg.man.ac.uk
German Larriba
University of Extremadura
Depto. de Microbiologica. F. de Ciencias
Avda. de Elvas s/n
ES-6071 Badajoz, ES
ES
Tel. +34 24 289428; FAX +34 24 289428
e-mail:glarriba@ba.unex.es
Hans Ronne
Uppsala University
Dept. of Medical Biochemistry & Microbiology
Uppsala Biomedical Center, Box 582
751 23 Uppsala, SE
SE
Tel. +46 18 714211; FAX +46 18 509848
e-mail: Ronne@bmc.uu.se
Rosine Haguenauer-Tsapis, Sandrine Avaro
Institut Jacques Monod
CNRS-Universite Paris VII
2 place Jussieu
Tour 43-44 5eme etage
75251 PARIS Cedex 05
France
Tel. +33 1 44 27 63 86; +33 1 44 27 47 24 (Avaro); FAX +33 1 44 27 59 94
e-mail: haguenauer@ijm.jussieu.fr
e-mail: avaro@ijm.jussieu.fr
Sirkka Keranen, Jukka Juselius
VTT, Biotechnology and Food Research
P.O. Box 1500
FIN-02044 VTT
Finland
Tel. +358 9 456 5138; FAX +358 9 455 2103
e-mail: Sirkka.Keranen@vtt.fi
e-mail: Jukka.Juselius@vtt.fi
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