Performing a hunt by interaction mating

互联网2013-09-05

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Abstract

When more than one bait will be used to screen a single library, significant time and resources can be saved by performing the interactor hunt by interaction mating. In this protocol one strain is transformed with library DNA and the transformants are collected and frozen in aliquots. For each interactor hunt, an aliquot of this frozen "pretransformed library strain" is thawed and mixed with an aliquot of a bait strain transformed with the bait expression plasmid. Overnight incubation of the mixture on a YPD plate results in mating - i.e., cells of one strain fuse with cells of the other strain to form diploids. The diploids are then exposed to galactose to induce expression of the library encoded proteins, and interactors are selected in the same manner as in the standard hunt protocols. The advantage to this approach is that it requires only one high-efficiency library transformation for multiple hunts with different baits. It is also useful for bait proteins that are somewhat toxic to yeast; yeast expressing toxic baits can be difficult to transform with the library DNA.

Background and Introduction

An alternate way of conducting an interactor hunt is to mate a strain that expresses the bait protein with a strain pretransformed with the library DNA, and screen the resulting diploid cells for interactors (Bendixen et al., 1994; Finley and Brent, 1994). This "interaction mating" approach can be used for any interactor hunt, and is particularly useful in three special cases. The first case is when more than one bait will be used to screen a single library. Interaction mating allows several interactor hunts with different baits to be conducted using a single high-efficiency yeast transformation with library DNA. This can be a considerable savings of time a resources, since the library transformation is one of the most challenging tasks in an interactor hunt . Moreover, some yeast strains pretransformed with libraries are becoming commercially available, which may eliminate altogether the need to conduct a high-efficiency library transformation for some researchers. The second case is when a constitutively expressed bait interferes with yeast viability. For such baits, performing a hunt by interaction mating avoids the difficulty often encountered when trying to achieve high-efficiency transformation of a strain expressing a toxic bait. Moreover, the actual selection for interactors will be conducted in diploid yeast, which are more vigorous than haploid yeast and generally can better tolerate expression of toxic proteins. The third case is when a bait cannot be used in a traditional interactor hunt using haploid yeast strains because it activates transcription of even the least sensitive reporters. In diploids the reporters are less sensitive to transcription activation than they are in haploids. Thus, the interaction mating hunt provides an additional method to reduce background from transactivating baits.

In the protocol described below, the library DNA is used to transform a strain with a LEU2 reporter. This pretransformed library strain is then frozen in many aliquots, which can each be thawed and used for individual interactor hunts. The bait is expressed in a strain of mating type opposite to that of the pretransformed library strain, and also bearing the lacZ reporter. A hunt is conducted by mixing the pretransformed library strain with the bait strain and allowing diploids to form on YPD medium overnight. The diploids are then incubated in galactose to induce expression of the library encoded proteins, and screened for interactors in the same manor as in a traditional haploid interactor hunt.

NOTE: Strain combinations other than those described below can also be used in an interaction mating hunt. The key to choosing the strains is to ensure that the bait and prey strains are of opposite mating types and that both have auxotrophies to allow selection for the appropriate plasmids and reporter genes. Also, once the bait plasmid and lacZ reporter plasmid have been introduced into the bait strain, and the library plasmids have been introduced into the library strain, the resulting bait strain and library strain must each have auxotrophies that can be complemented by the other, so that diploids can be selected.

The interaction mating protocol presented here is based on the LexA version of the two-hybrid system (Fields and Song, 1989) developed in Roger Brent''s lab (e.g., Gyuris et al., 1993). Go here to find links to background information and other protocols for the Brent lab system. Briefly, the essential features of the Brent lab system are:

The reporter is a LEU2 gene with upstream LexA binding sites or operators (LexAop-LEU2) integrated into the yeast genome in strains like EGY48 (Estojak et al., 1995) or a derivative of EGY48, RFY231 (Kolonin and Finley, 1998). Different strains with different numbers of LexA, and hence different levels of sensitivity, are available (Estojak et al., 1995).
The interaction assay is usually conducted in the presence of an additional lacZ reporter. The LexAop-lacZ reporter usually resides on a multicopy (2µm) plasmid bearing a URA3 marker, such as pSH18-34. Again, various lacZ reporters with different numbers of upstream LexA operators exist. Strains with the entire lacZ plasmid integrated into the ura3 locus also are available; these lacZ reporters are the least sensitive as they are in single copy.
The LexA fusion or bait is expressed from a multicopy (2µm) bait plasmid bearing a HIS3 marker, such as pEG202 (Estojak et al., 1995). The LexA bait is constitutively expressed from the yeast ADH1 promoter. Version of the bait plasmid also exist with a nuclear localization signal fused to LexA, or with the ADH promoter replaced by the GAL1 promoter for conditional expression of bait.
The library or "prey" plasmid (i.e., expressing the activation domain (AD) -fused protein that may interact with the bait) is a multicopy (2µm) plasmid bearing the TRP1 marker, such as pJG4-5 (Gyuris et al., 1993). The AD-fused protein is expressed from the yeast GAL1 promoter - on in galactose but off in glucose.

Protocol (20 steps)

Materials

Yeast Strains:

Bait strains: Either RFY206 (Finley and Brent, 1994), YPH499 (Sikorski and Hieter, 1989; ATCC#6625), or an equivalent MATa strain with auxotrophic markers ura3, trp1, his3, and leu2.

Library strains: Either RFY231 (Kolonin and Finley, 1998), EGY48 (Estojak et al., 1995), or other MATalpha strain with LexAop-LEU2 reporter and auxotrophic markers ura3, trp1, his3, and leu2.

Media and Solutions

See previous protocols.

Construct the bait strain

The bait strain will be a MATa yeast strain (mating type opposite of RFY231) containing a lacZ reporter plasmid like pSH18-34 and the bait-expressing plasmid, pBait.

1. Construct the bait plasmid (pBait) to express the LexA fused bait protein, for example using the HIS3 2µm plasmid pEG202 (Estojak et al., 1995).

2. Co-transform the MATa yeast strain (e.g., either RFY206 or YPH499) with pBait and a lacZ reporter, such as the very sensitive pSH18-34, using the lithium acetate method. Select transformants on Glu/CM-Ura,-His plates. Incubate plates at 30^o C for 3-4 days until colonies form. Combine 3 colonies for all future tests and for the mating hunt.

The bait strain (RFY206/pSH18-34/pBait or YPH499/pSH18-34/pBait) can be tested by immunoblotting to ensure that the bait protein is expressed.. Synthesis and nuclear localization of the bait protein can also be tested by the repression assay, as described in previous protocols.

3. (optional) Assay lacZ gene activation in the bait strain, for example, by plating to Glu/CM-Ura,-His X-Gal plates.

If the bait activates the lacZ reporter, a less sensitive lacZ reporter plasmid, or an integrated version of the lacZ reporter should be tried. A bait that strongly activates the lacZ reporters usually cannot be used in a hunt based on selection of interactors with the LEU2 reporter because the LEU2 reporters are more sensitive than even the most sensitive lacZ reporters. However, both reporters are less sensitive to activation by a bait in diploid cells, as compared to haploid cells. Thus, a more important test of the transactivation potential of a bait is to test the leucine requirement of diploid cells expressing it, as described in steps 6 - 19 of this protocol.

Prepare the pretransformed library strain (RFY231 + library plasmids)

4. Perform a large-scale transformation of EGY48 with library DNA using the lithium acetate method , starting with RFY231 bearing no other plasmids. To prepare for transformation, grow RFY231 in YPD liquid medium. Select library transformants on Glu/CM-Trp plates at 30^o C for 3 days.

5 . Collect primary transformants by scraping plates, washing yeast, and resuspending in 1 pellet vol glycerol solution (65% (v/v) glycerol, 0.1M MGSO₄ , 25mM Tris, pH8.0). Freeze 0.2-1.0-ml aliquots at ?70 to ?80^o C. The cells will be stable for at least 1 year.

Re-freezing a thawed aliquot will result in loss of viability. Thus, many frozen aliquots should be made so that each thawed aliquot can be discarded after use.

Prepare the pretransformed control strain (RFY231 + pJG4-5)

6. Transform RFY231 grown in YPD liquid medium with the empty library vector, pJG4-5, using the lithium acetate method. Select transformants on Glu/CM-Trp plates at 30^o C for 3 days.

7. Pick and combine three transformant colonies and use to inoculate 30 ml of Glu/CM-Trp medium. Incubate 15-24 hours at 30^o C to OD₆₀₀ >3.

8. Centrifuge 5 min at 1000 to 1500 X g at room temperature. Resuspend in 10 ml sterile water to wash cells.

9. Centrifuge 5 min at 1000 to 1500 X g at room temperature. Resuspend in 1 pellet vol glycerol solution and freeze 100 µl aliquots at ?70 to ?80^o C.

Determine plating efficiency of pretransformed library and pretransformed control strains.

10. After freezing (at least 1 hour) thaw an aliquot of each pretransformed strain (from step 5 and step 9) at room temperature. Make several serial dilutions in sterile water, including aliquots diluted 10⁵ - 10⁶ -, and 10⁷ -fold. Plate 100 µl of each dilution on 100-mm Glu/CM-Trp plates and incubate at 30^o C for 2-3 days.

11. Count the colonies and determine the number of colony-forming units (cfu) per aliquot of transformed yeast.

The plating efficiency for a typical library transformation and for the control strain will be ~10⁸ cfu/100 µl.

Mate the bait strain with the pretransformed library strain and the pretransformed control strain

In steps 12 through 20, an interactor hunt is conducted concurrently with testing for LEU2 reporter activation by the bait itself. For most baits this approach will be the quickest way to isolate interactors. However, for some baits, such as those that have a high transactivation potential, or those that affect yeast mating or growth, steps 12 through 20 will serve as a pilot experiment to determine the optimal parameters for a subsequent hunt.

12 . Grow a 30 ml culture of the bait strain in Glu/CM-Ura,-His liquid dropout medium to mid- to late-log phase (OD₆₀₀ =1.0-2.0, or 2-4 x 10⁷ cells/ml)

A convenient way to grow the bait strain is to inoculate a 5 ml culture with ~ three colonies from a plate and grow it overnight at 30^o C with shaking. In the morning measure the OD₆₀₀ , dilute into a 30 ml culture to a final OD₆₀₀ =0.2, and grow at 30^o C with shaking. The culture should reach mid- to late-log phase before the end of the day.

13 . Centrifuge the culture 5 minutes at 1,000 - 1,500 X g at room temperature to harvest cells. Resuspend the cell pellet in sterile water to make a final volume of 1 ml. This should correspond to ~1 x 10⁹ cells/ml.

14. Set up two matings. In one sterile eppendorf tube mix 200 µl of the bait strain with 200 µl of a thawed aliquot of the pretransformed control strain from step 9. In a second eppendorf tube mix 200 µl of the bait strain with approximately 10⁸ cfu (~0.1 - 1 ml) of the pretransformed library strain from step 5.

The library mating should be set up so that it contains a ~2-fold excess of bait strain cfu over pretransformed library strain cfu. Because the bait strain was harvested in log phase, most of the cells will be viable (i.e., cells/ml = ~cfu/ml), and the number of cfu can be sufficiently estimated from optical density (1 OD₆₀₀ =~2 X 10⁷ cells/ml). Under these conditions, ~10% of the cfu in the pretransformed library strain will mate with the bait strain. Thus, a complete screen of 10⁷ library transformants will require a single mating with at least 10⁸ cfu of the pretransformed library strain and at least 2 X 10⁸ cfu of the bait strain.

To screen more library transformants, set up additional matings. The number of pretransformed library transformants to screen depends on the size of the library and the number of primary transformants obtained in step 5. If the size of the library is larger than the number of transformants obtained in step 5, the goal will be to screen all of the yeast transformants. In this case, complete screening of the library will require additional transformations of RFY231 and additional interactor hunts. If the size of the library is smaller than the number of transformants obtained in step 5, the goal will be to screen at least a number of transformants equivalent to the size of the library.

15. Centrifuge each cell mixture for 5 minutes at 1,000 - 1,500 X g , pour off media and resuspend cells in 200 µl YPD. Plate each suspension onto a 100-mm YPD plate. Incubate 12-15 hours at 30^o C.

16. Add ~ 1ml of Gal/Raff/CM-Ura,-His,-Trp to the lawns of mated yeast on each plate. Mix the cells into the media using a sterile applicator stick or glass rod.

17. Transfer each slurry of mated cells to a 500 ml flask containing 100 ml of Gal/Raff/CM-Ura,-His,-Trp dropout medium. Incubate with shaking 6 hrs at room temperature to induce the GAL1 promoter which drives expression of the cDNA library.

18. Centrifuge the cell suspensions 5 minutes at 1,000 - 1,500 X g at room temperature to harvest the cells. Wash by resuspending in 30 ml of sterile water and pelleting again. Resuspend each pellet in 5 ml sterile water. Measure OD₆₀₀ and, if necessary, dilute to a final concentration of ~10⁸ cells/ml.

This is a mixture of haploid cells that have not mated and diploid cells. Under a microscope the two cell types can be distinguished by size (diploids are ~1.7 times bigger than haploids) and shape (diploids are slightly oblong and haploids are spherical). Because diploids grow faster than haploids, this mixture will contain about 10 ? 50% diploid cells. The actual number of diploids will be determined by plating dilutions on -Ura, -His, -Trp medium, which will not support the growth of the parental haploids.

19 . For each mating make a series of 1/10 dilutions, at least 200 µl each, in sterile water to cover a 10⁶ -fold concentration range. Plate 100 µl from each tube (undiluted, 10^-1 , 10^-2 , 10^-3 , 10^-4 , 10^-5 , and 10^-6 dilution) on 100-mm Gal/Raff/CM-Ura,-His,-Trp,-Leu plates. Plate 100 µl from tubes 10^-4 , 10^-5 , and 10^-6 on 100-mm Gal/Raff/CM-Ura,-His,-Trp plates. Incubate plates at 30^o C. Count the colonies on each plate after 2-5 days.

Selecting interactors

20. For the mating with the pretransformed library, make an additional 3 ml of a 10^-1 dilution. Plate 100 µl of the 10^-1 dilution on twenty 100-mm Gal/Raff/CM-Ura,-His,-Trp,-Leu plates, and 100 µl of the undiluted cells on twenty 100-mm Gal/Raff/CM-Ura,-His,-Trp,-Leu plates. Incubate at 30^o C. Pick Leu⁺ colonies after 2-5 days and characterize them as described in previous protocols (See Part II D of the Finley, 1997 )

The number of Leu⁺ colonies to pick to ensure that all of the pretransformed library has been screened depends on the transactivation potential of the bait protein itself. The transactivation potential is expressed as the number of Leu⁺ colonies that grow per cfu (Leu+/cfu) of the bait strain mated with the control strain, as determined in step 19. It can be calculated as the ratio of the number of colonies that grow on Gal/Raff/CM-Ura,-His,-Trp,-Leu to the number of colonies that grow on Gal/Raff/CM-Ura,-His,-Trp for a given dilution of the mating between the bait strain and the control strain. A bait with essentially no transactivation potential will produce less than 10^-6 Leu⁺ /cfu. For a bait to be useful in an interactor hunt it should not transactivate more than 10^-4 Leu⁺ /cfu.

To screen all of the pretransformed library, it will be necessary to pick a sufficient number of Leu⁺ colonies in addition to background colonies produced by the transactivation potential of the bait itself. Thus, the minimum number of Leu⁺ colonies that should be picked at step 20 is given by:

(transactivation potential, Leu⁺ /cfu) x (# library transformants screened).

For example, if 10⁷ library transformants were obtained in step 2 (and at least 10⁸ cfu of these transformants were mated with the bait strain in step 14, since only ~10% will form diploids), and the transactivation potential of the bait is 10^-4 Leu⁺ /cfu, then at least 1000 Leu⁺ colonies must be picked and characterized. In other words, if the rarest interactor is present in the pretransformed library at a frequency of 10^-7 , to find it one needs to screen through at least 10⁷ diploids from a mating of the library strain. However, at least 1000 of these 10⁷ diploids would be expected to be Leu⁺ due to the bait background if the transactivation potential of the bait is 10^-4 . The true positives will be distinguished from the bait background in the next step by the galactose dependence of their Leu⁺ and lacZ⁺ phenotypes.