We first modified a set of 24 pRS yeast shuttle vectors (pRS303, pRS304, pRS305, pRS306, pRS413, pRS414, pRS415, pRS416, pRS423, pRS424, pRS425 and pRS426, each with a CYC1 terminator and a GPD or GAL1 promoter) to make them compatible for use with the Gateway® system. To generate this core set of pAG (Advanced Gateway) vectors, we inserted the chloramphenicol/ccdB resistance Gateway® cassette A into the single SmaI restriction site of each pRS plasmid. Proper orientation of the Gateway® cassette was confirmed by DNA sequencing. The resulting plasmids were then used to generate a set of derived vectors that allows for the expression of proteins with C-terminal tags (Please see Supplementary Table 1 for nomenclature and a complete list of pAG plasmids). The coding sequences for the various tags were amplified using the primers and templates listed in and . PCR products were cloned between the HindIII and XhoI restriction sites of pAG426GPD and pAG426GAL (EGFP, ECFP, EYFP, Cerulean, DsRed and 3HA) or between the ClaI and XhoI sites of pAG423GPD and pAG423GAL (TAP). The correct amplification and integration of tag DNA sequences was confirmed by sequencing. To complete the set of pAG plasmids the entire expression cassette was removed from the primary pAG423GPD/GAL-ccdB-tag and pAG426GPD/GAL-ccdB-tag constructs by digestion with SacI and KpnI and subcloned into the remaining pRS vector backgrounds using SacI and KpnI for linearization of the target plasmids.
Primers used for the amplification of tags (restriction sites are underlined).
Primer combinations and templates used for the amplification of tags.
For the cloning of vectors for expression of N-terminal fusions, PCR-generated fragments (See and for details) were inserted into the XmaI and XhoI sites of pRS426GPD and pRS426GAL. The resulting plasmids were cut with XhoI and incubated with mung bean nuclease to remove single-stranded overhangs. Next, the C1 Gateway® cassette was ligated to the blunted plasmids. The resulting plasmids were subsequently analyzed by sequencing to confirm the correct integration of tag sequences and Gateway® cassette. As a final step a SpeI-MluI fragment comprising the Gateway® cassette and the tag sequence was cut out and cloned between the SpeI and MluI sites of the core set of pAG plasmids.
Standard BP and LR reaction protocol
We have developed modified versions of the standard Gateway® LR and BP reactions. The smaller volumes require less enzyme, reducing the cost per reaction.
BP or LR Reaction
- 150 ng expression clone (BP) or entry clone (LR): 1 microliter
- 150 ng entry vector (BP) or destination vector (LR): 1 microliter
- 1X TE (pH 8): 2 microliters
- BP (or LR) Clonase II mix (Invitrogen): 1 microliter
Incubate at room temperature for 1 hour. Transform 2–3 microliters of the reaction into DH5α, TOP10, or other ccdB-sensitive cells. Select for transformants on LB agar plates containing 100 μg/ml ampicillin.
Experiments with α-synuclein
Entry clones containing full-length human α-synuclein with or without a stop codon were used in Gateway LR reactions to generate 426GAL-α–syn-EGFP, 426GAL-α–syn-DsRed, 426GAL-α–syn-HA, 426GAL-α–syn-TAP, and 426GAL-α–syn. These plasmids were transformed into the BY4741 yeast strain. To induce expression of the α–syn fusion proteins, yeast cells were pre-grown overnight in synthetic media containing raffinose to mid log-phase. Cells were switched to synthetic media containing galactose and grown for 6 hours. Cells were either processed for fluorescence microscopy or protein lysates prepared for immunoblotting. Antibodies used for immunoblotting were as follows: GFP (Roche, 1:1,000); HA (Roche, 1:1,000); TAP (Open Biosystems, 1:1,000). Horseradish peroxidase coupled secondary antibodies were used at 1:10,000. For growth assays, serial dilutions of transformants were grown on solid synthetic media containing either glucose (control, α-syn “off”) or galactose (α-syn “on”).