Ste11p/Ste50p Related Yeast Two-Hybrid and one-hybrid Systems PRIOR APPLICATION INFORMATION

This application claims the benefit of US Provisional Patent Application 60/726,177, filed October 14, 2005. BACKGROUND OF THE INVENTION

Many biological processes are regulated by protein-protein interactions; analyses of these interactions account for a major part of functional proteomics. Methods commonly used for analyzing theses interactions include biochemical methods such as co-immunoprecipitation, and yeast two-hybrid systems. However, all the known current methods have limitations, and there is need to develop new methods to extend the ability to detect protein-protein interactions of biological relevance.

The yeast two-hybrid systems, based principally on reconstitution of two non-functional protein modules through bait-prey protein interaction, are typically methods of choice to analyze protein-protein interactions, especially in large scale functional proteomics studies, due to the relatively minor requirement for technical expertise and instrumentation.

In addition to the conventional yeast two-hybrid (YTH) system developed by Fields & Song (1989), and later further developed by others (Durfee et al., 1993; Vojtek et al., 1993; Gyuris et al., 1993), other yeast two-hybrid systems have been developed in attempts to address special needs designed to overcome the limitations of the original system. Of these, the more commonly used ones are the split-ubiquitin based membrane yeast two-hybrid (MbYTH) to analyze membrane protein-protein interactions, and the CytoTrap yeast two-hybrid designed to analyze protein-protein interactions in the cytoplasm. These systems, although highly useful, have intrinsic limitations. Because it is based on reconstitution of a functional transcription factor needed to activate reporter genes in the nucleus to detect protein-protein interactions, conventional YTH limits one's ability to analyze the transcription factors and membrane proteins. Split-ubiquitin based MbYTH on the other hand, is not generally applicable to non-membrane proteins of interest, and the system is sensitive to expression level of bait constructs. Both these two systems are prone to false positives. Based on the ability of membrane targeted hSOS to complement the function of yeast CDC25, CytoTrap offers the possibility

of analyzing protein-protein interactions in the cytoplasm. However, the system has drawbacks such as using temperature-sensitive yeast strain for screening, and limited cloning capacity of the bait due to the large size of hSOS. SUMMARY OF THE INVENTION According to a first aspect of the invention, there is provided an expression vector comprising, in the 5' to 3' direction, a yeast promoter, a nucleotide sequence encoding a first peptide of interest and a nucleotide sequence encoding an Ste11 p peptide lacking a functional SAM domain.

According to a second aspect of the invention, there is provided a method of detecting interaction between a first peptide of interest and a second peptide of interest comprising: providing a yeast cell comprising or having been transformed with: an expression vector comprising, in the 5' to 3' direction, a yeast promoter, a nucleotide sequence encoding a first peptide of interest, and a nucleotide sequence encoding an Ste11 p peptide lacking a functional SAM domain; and an expression vector comprising, in the 5' to 3' direction, a yeast promoter, a nucleotide sequence encoding a second peptide of interest, and a nucleotide sequence encoding an Ste50p peptide lacking a functional SAM domain; and incubating said yeast cell on a high osmotic growth media, wherein growth of said yeast cell on said high osmotic growth media indicates protein-protein interaction between the first peptide of interest and the second peptide of interest.

According to a third aspect of the invention, there is provided a method of detecting interaction between a first peptide of interest and a second peptide of interest comprising: providing a yeast cell comprising or having been transformed with: an expression vector comprising, in the 5' to 3' direction, a yeast promoter, a nucleotide sequence encoding a first peptide of interest, and a nucleotide sequence encoding an Ste11p peptide lacking a functional SAM domain; and

an expression vector comprising, in the 5' to 3' direction a yeast promoter operably linked to a nucleotide sequence encoding a second peptide of interest fused to a plasma membrane localization signal; and incubating said yeast cell on a high osmotic growth media, wherein growth of said yeast cell on said high osmotic growth media indicates protein-protein interaction between the first peptide of interest and the second peptide of interest.

According to a fourth aspect of the invention, there is provided an expression vector comprising a yeast promoter operably linked to a nucleotide sequence encoding a peptide comprising a peptide of interest fused to SteδOp SAM domain.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic presentation of the HOG pathway to show the involvement of Ste11 p/Ste50p in osmoregulation in yeast Sacchromyces cerevisiae.

Figure 2. Schematic presentation of an embodiment of the Ste11 p/Ste50p based yeast two-hybrid system. (A) The interchangeability of the Ste11 p and SteδOp SAM domains. (B) The SAM domains of Ste11 p and SteδOp can be replaced by the synthetic protein-protein interacting modules K-and E-coils. The functionality of various combinations are indicated by their ability to enable yeast cells of ste11δ steδOδ ssk2δ ssk22δ to grow on high osmolarity medium (+). (C) and (D) represent two configurations in which the SAM domains of Ste11 p and SteδOp are replaced by prey (X) and bait (Y) and whose interaction is indicated by the ability of the yeast cell to grow on high osmolarity medium. PM-X indicates plasma membrane-targeted preys.

Figure 3. Schematic presentation of an embodiment for using Ste11 p/Ste50p based systems for studying membrane proteins and their topology. (A) The use of SteδOp SAM domain (1) or Ste1 1δSAMp (2) to identify query-X of plasma membrane localization. (B) The use the systems to study the topology of membrane proteins (X). Only the SteδOp SAM domain configuration is shown, and Ste1 iδSAMp configuration can be used in a similar manner.

Figure 4. The SAM domains of Ste11 p and SteδOp are functionally interchangeable. (A) Schematic representation of an embodiment of the SAM domain swapped chimeras of Ste11p and SteδOp. (B) Yeast cells (ste11δ steδOδ ssk2δ ssk22δ) were co-transformed with either the wild-type STE11 and STE50 or SAM domain swapped chimeras of STE11 and STE50, and tested for the ability to grow on hyperosmotic medium.

Figure s. Examples showing that the SAM domains of Ste11 p and SteδOp can be replaced by other interaction modules. (A) Schematic diagrams of the STE11 and STE50 constructs with or without the E-coil or the K-coil. The heterotypic interaction between the E- and K-coil is indicated by a diamond symbol. (B) Yeast cells (ste11δ steδOδ ssk2δ ssk22δ) co-transformed with the indicated STE11 and STE50 constructs were tested for their ability to activate the HOG pathway by assaying the ability to grow on hyperosmotic medium.

Figure 6. Fusion of the RA-domain-containing fragment of SteδOp bypasses the requirement for an interaction between the SAM domains of Ste11p and SteδOp. (A) Schematic representation of the constructs encoding fusions of the Ste50p RA-domain-containing fragments and the Ste11 pδSAM fragment. (B) Yeast cells {ste11δ steδOδ ssk2δ ssk22δ) transformed with STE11 and STE50 constructs as indicated were assayed for their ability to activate the HOG pathway by testing their ability to grow on hyperosmotic medium. (C) Western blot analysis of protein extracts of cells as in (B), either treated (+) or non-treated (-) with NaCI at 0.5 M for 5 min. before the extraction of protein samples, was performed with either anti-phospho-p38 antibody to detect phosphorylated Hogip, (p~Hog1 p), or with anti-p38 antibody to detect the total amount of Hogi p. Figure 7. Plasma membrane targeting of the Ste50p SAM domain complements SteδOp function in the HOG pathway. (A) Schematic representation of embodiments of the constructs encoding fusions of the GFP carrying a myristoylation signal and various fragments of Ste50p. (B) Yeast cells {steδOδ ssk2δ ssk22δ), transformed with either the vector control or constructs encoding various fragments of SteδOp with a myristoylation signal, were assayed for the ability to grow on hyperosmotic media. (C) Yeast cells (ste11δ steδOδ ssk2δ ssk22δ) co-transformed with a myristoylated SteδOp SAM domain in combination

with either the wild-type Ste11 p or the Ste11 pδSAM construct were assayed for the ability to grow on hyperosmotic medium. (D) Plasma membrane localization of myristoylated GFP fusion of the SAM domain and the RA domain of Ste50p, as well as the full-length Ste50p in ste50δ ssk2δ ssk22δ cells. (E) Western blot analysis of protein extracts of cells as in (B), for detecting Hogip phosphorylation as described in Fig. 3.

Figure 8. Plasma membrane localization of Ste11p leads to simultaneous activation of multiple signaling pathways. (A) Yeast cells of genotypes as indicated transformed with various STE11 constructs or with the control vector were assayed for their ability to grow on hyperosmotic media. (B) The morphology of yeast cells with relevant genotype (indicated at the bottom of the figure) transformed either with myristoylated GST-Ste11 p (upper panels) or with GST-Ste11 p (lower panels). Yeast strains used were: W303-1A (wt), YEL206 (ste20δ), YCW555 (steδOδ steHδ ssk2δ ssk22δ), and YCW757 (steδOδ ste5δ ste11δ ssk2δ ssk22δ). (C) The Ste11 p plasma membrane localization is sufficient for activation of the HOG pathway, but still needs Steδp for mating pathway activation. Yeast cells of indicated genotype (top) transformed with STE11 constructs as indicated on the right were assayed for their ability to grow on hyperosmotic medium (middle panel), and to form diploids (right panel). Figure 9. Detecting interaction between mammalian MP1 and P14.

MATα yeast cells {steSOδ ssk2δ ssk22δ) transformed with MP-Ste50pδSAM (left column of patches) mated with the isogenic MATa cells with P14-Ste11pδSAM (right column of patches) to produce diploid cells bearing both fusion constructs (middle column of patches). All cells were checked for the presence of the expected plasmid marker and assayed for their ability to grow on hyperosmotic media by replicating the master plate (upper right) to all other selective plates as indicated by the template on the left.

Figure 10. Ste11 pδSAM is able to activate the HOG pathway when fused to transmembrane protein Ste2p. Yeast cells (steSOδ ssk2δ ssk22δ) transformed with Ste2-Ste1 1pδSAM (upper row) and Ste11 pδSAM (lower row) were assayed for their ability to grow on hyperosmotic media.

Figure 11. Amino acid sequence of Ste11 p (SEQ ID No. 1). This

sequence represents the long version of Ste11 p with 738 amino acid residues. The numbering information provided in the SAM domain swapping experiment is based on this sequence, and the swapped fragment is in bold (amino acid residues Met22 to E1 19 inclusive). Figure 12. Amino acid sequence of Ste50p (SEQ ID No. 2). Note: The numbering information provided in the SAM domains swapping experiment is based on this sequence, and the swapped fragment is in bold (amino acid residues Met27 to K149 inclusive).

DESCRIPTION OF THE PREFERRED EMBODIMENTS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

There is provided herein a novel yeast two-hybrid system that is based on the interaction of Ste11p (MEKK) and Ste50p leading to the HOG pathway activation and osmoadptation (Fig. 1). The survival of yeast cells under hyperosmotoc stress, in the absence the two-component osmosensing branch, depends on the function of Ste11p whose activation requires the interaction with SteδOp and subsequent localization to the plasma membrane where to be phosphorylated by Ste20p. Activated Ste11 p phosphorylates and activates Pbs2p (MEK), which in turn phosphorylates and activates the MAP kinase Hogi p. Hogi p activation will eventually leads to the synthesis of intracellular glycerol or HOG (high osmotic glycerol) activation to combat the extracellular hyperosmotic stress. Ste11 p and SteδOp interact through their respective SAM domains, and this interaction is required for the HOG pathway activation.

Substitution of bait and prey proteins for the respective SAM domains offers a unique potential to analyze bait-prey interactions using the activation of the HOG pathway as a reporter, that is, the yeast cells are able to grow on high osmotic

medium if the bait interacts with the prey. This is mode one of the Ste11 p/Ste50p related yeast two-hybrid (SRYTH) (Fig. 2C).

One of the functions of Ste50p on Ste11 p regulation is to bring Ste11p to the plasma membrane, and direct plasma membrane localization of Ste11 p bypasses the requirement of Ste50p (see below in example A section). As an alternative, the prey protein can be fused to a plasma membrane localization signal, such as a myristoylation signal, to direct the prey protein to the plasma membrane. The ability of yeast cells co-transformed with the prey proteins and the bait protein fused Ste11 p lacking its SAM domain to grow on high osmotic medium will depend on the interaction between the prey and the bait protein (Fig. 2D). In this case, the plasma membrane targeted prey is mimicking the function of Ste50p if the prey interacts with the bait-fused Ste11p. This is mode two of the SRYTH. The myristoylation signal has been tested with SteδOp SAM domain, which can partially complement the function of the wildtype SteδOp, as discussed below. In addition to its ability to detect protein-protein interactions, with minor modifications the system disclosed herein allows identification of proteins that localize to the plasma membrane, and studies of the topology of such classes of proteins. A plasma membrane localized SteδOp SAM domain complements the function of wild-type SteδOp in osmoadaptation, and this property can be exploited to identify proteins having plasma membrane localization by fusing the query protein to either the N- or C-terminus of SAM domain of SteδOp. The strain's subsequent ability to grow on high osmotic medium suggests that the query protein contains plasma membrane localization potential (Fig.3A-1). (see Table III for examples) Furthermore, by using the combination of two constructs in which the query protein of plasma membrane localization is fused to either the N- or C- terminus of the SAM domain of SteδOp, the topology of the query protein is analyzed by scoring for the growth on high osmotic medium (Fig. 3B). As an alternative, the property that plasma membrane localization of Ste11 p is able to activate the HOG pathway in the absence of Ste50p can be exploited to study the plasma membrane localization and topology of prey proteins by fusing them to Ste11δSAMp (Fig. 3A-2). (see Fig. 10) In addition, both the properties of the SAM domains of Ste50p and Ste11δSAMp described above can be exploited to monitor in yeast the translocation of proteins of interest to the plasma membrane, which

may be used in applications including those high-throughput screens (HTS) that are designed to identify compounds that modulate such translocation processes.

The plasmid constructs used in the system are easy to use to create libraries at customized needs. The inserts can be either cloned into multiple cloning sites (MCS) of choice by standard cloning procedures, or by in vivo recombination (IVR) in yeast at Smal site of the MCS, except the C-terminal fusion to Ste50p SAM in which cloning at Xhol site is required (see Materials and Methods for details).

Accordingly, in a first aspect of the invention, there is provided an expression vector comprising, in the 5' to 3' direction, a yeast promoter, a nucleotide sequence encoding a first peptide of interest, and a nucleotide sequence encoding an Ste11 p peptide lacking a functional SAM domain. In some embodiments, the expression vector may include a yeast transcription termination sequence. As will be apparent to one of skill in the art, the promoter is operably linked to the nucleotide sequences and is arranged for expression of these sequences so that a first peptide of interest: Ste11pδSAM fusion peptide is produced.

As will be appreciated by one of skill in the art, any suitable yeast promoter may be used, including the native STE1 1 promoter. Similarly, any suitable yeast transcription termination sequence may be used, including the native STE11 transcription termination sequence. It is to be understood that as used herein, 'yeast promoter' and 'yeast transcription termination sequence' refers to any promoter or transcription termination sequence functional to a suitable extent in yeast for use within the expression system as described herein, not just to native yeast promoters and transcription terminators.

Furthermore, the Ste11p peptide lacking a functional SAM domain may comprise a fragment of Ste11p lacking all or part of the SAM domain such that the SAM domain is non-functional, that is, is incapable of forming a heterodimer with another SAM domain or may comprise Ste11 p having one or more mutations which render the SAM domain non-functional for forming such a heterodimer for example with the Ste50p SAM domain. As shown in Figure 11 , the SAM domain of Ste11 p comprises amino acids 22 to 119 of SEQ ID No. 1. It is noted that targeted mutagenesis within this region to identify suitable mutants as well as progressive

or nested deletions to determine minimal and maximal deletions which render SAM interaction non-functional are well within the skill of one knowledgeable in the art. In preferred embodiments, the Ste11p lacking a functional SAM domain may comprise an amino acid sequence as set forth in amino acids 131-738 of SEQ ID No. 1 or as set forth in amino acids 120-738 of SEQ ID No. 1.

The first peptide of interest may, as discussed above, be 'bait' or 'prey', as shown in Figure 2C. For example, the peptide may be a fragment or domain of a larger peptide or a full-length peptide for which binding partners are being sought or may be a library of peptides, for example, a cDNA library or a random peptide library. It is noted that potential 'bait' and 'prey' peptides and sources are well known in the art, as discussed above.

As discussed above, the above-described Ste11 vector partner may be used in combination with an expression vector comprising, in the 5' to 3' direction, a yeast promoter, a nucleotide sequence encoding a second peptide of interest, and a nucleotide sequence encoding an Ste50p peptide lacking a functional SAM domain. In some embodiments, the expression vector may further include a yeast transcription termination sequence. As will be apparent to one of skill in the art, the promoter is operably linked to the nucleotide sequences and is arranged for expression of these sequences so that a second peptide of interest: SteδOpδSAM fusion peptide is produced.

As will be appreciated by one of skill in the art, any suitable yeast promoter may be used, including the native STE50 promoter. Similarly, any suitable yeast transcription termination sequence may be used, including the native STE50 transcription termination sequence. Furthermore, the Ste50p peptide lacking a functional SAM domain may comprise a fragment of Ste50p lacking all or part of the SAM domain such that the SAM domain is non-functional, that is, is incapable of forming a heterodimer with another SAM domain or may comprise SteδOp having one or more mutations which render the SAM domain non-functional for forming such a heterodimer, for example, with the Ste11 p SAM domain. As shown in Figure 12, the SAM domain of Ste11 p comprises amino acids 27 to 149 of SEQ ID No. 2. It is noted that targeted mutagenesis within this region to identify suitable mutants as well as progressive or nested deletions to determine minimal and maximal deletions which

render SAM interaction non-functional are well within the skill of one knowledgeable in the art and can be identified by many means, including failure to grow on hyperosmotic media, as discussed herein. In preferred embodiments, the Ste50p lacking a functional SAM domain may comprise an amino acid sequence as set forth in amino acids 115-346 of SEQ ID No. 2 or as set forth in amino acids 149-346 of SEQ ID No. 2.

The second peptide of interest may, as discussed above, be 'bait' or 'prey', as shown in Figure 2C. For example, the peptide may be a fragment or domain of a larger peptide or a full-length peptide for which binding partners are being sought or may be a library of peptides, for example, a cDNA library or a random peptide library. It is noted that potential 'bait' and 'prey' peptides and sources are well known in the art, as discussed above.

In a preferred embodiment, there is provided a method of detecting interaction between a first peptide of interest and a second peptide of interest comprising: providing a yeast cell comprising or having been transformed with: an expression vector comprising, in the 5' to 3' direction, a yeast promoter, a nucleotide sequence encoding a first peptide of interest, and a nucleotide sequence encoding an Ste11 p peptide lacking a functional SAM domain; and an expression vector comprising, in the 5' to 3' direction, a yeast promoter, a nucleotide sequence encoding a second peptide of interest, and a nucleotide sequence encoding an SteδOp peptide lacking a functional SAM domain; and incubating said yeast cell on a high osmotic growth media, wherein growth of said yeast cell on said high osmotic growth media indicates protein-protein interaction between the first peptide of interest and the second peptide of interest.

Preferably, in these embodiments, the yeast cell is ste11δ steδOδ. In other embodiments, the yeast strain may be steδOA, as discussed below. As will be appreciated by one of skill in the art, this refers to not only a deletion of STE50 or STE11 but also to disruption or other forms of inactivation known in the art.

Examples of suitable high osmotic or hyperosmotic growth media will be readily known to one of skill in the art.

In another embodiment of the invention, the first peptide of interest is the bait protein and the Ste11p expression vector is used in combination with an expression vector comprising in a 5' to 3' direction a yeast promoter operably linked to a nucleotide sequence encoding a second peptide of interest fused to a plasma membrane localization signal, for example, a myristoylation signal.

In a preferred embodiment, there is provided a method of detecting interaction between a first peptide of interest and a second peptide of interest comprising: providing a yeast cell comprising or having been transformed with: an expression vector comprising, in the 5' to 3' direction, a yeast promoter, a nucleotide sequence encoding a first peptide of interest, and a nucleotide sequence encoding an Ste11 p peptide lacking a functional SAM domain; and an expression vector comprising, in the 5' to 3' direction a yeast promoter operably linked to a nucleotide sequence encoding a second peptide of interest fused to a plasma membrane localization signal; and incubating said yeast cell on a high osmotic growth media, wherein growth of said yeast cell on said high osmotic growth media indicates protein-protein interaction between the first peptide of interest and the second peptide of interest. In another aspect of the invention, there is provided an expression vector comprising a yeast promoter operably linked to a nucleotide sequence encoding a peptide comprising a peptide of interest fused to SteδOp SAM domain. The peptide of interest may be fused to either the C-terminus or N-terminus of the Ste50p SAM domain, as discussed above. The Ste50p SAM domain may comprise amino acids 27 to 149 of SEQ ID No. 2, as discussed above.

Other suitable expression systems can be constructed as described herein.

There is disclosed herein one embodiment, a novel system and method for detecting protein-protein interactions based on the fact that such interactions activate the HOG pathway. There is no forced nuclear localization, as is in the case of conventional YTH, required to detect the interaction. The disclosed system and method is able to detect interactions in cytoplasm and/or on (plasma) membrane. It is believed to be applicable to substantially all proteins, and thus will in some instances be superior to the split-ubiquitin MbYTH, which is limited to a

subset of membrane proteins. Compared to CytoTrap YTH, the relative small size of the tag parts of Ste11p and Ste50p used in the disclosed system makes it easier to manipulate in terms of cloning large-size bait or prey proteins. As the size of the hSOS gene is fairly big, it is difficult to accommodate larger bait proteins. The disclosed system also uses normal yeast strains for easier screening for growth as compared to CytoTrap which uses special temperature sensitive mutant yeast strains. This system is believed to be less prone to false positive due to the principal difference for detecting protein-protein interaction and the activation of reporter genes etc. In addition, this system offers the versatility of identifying plasma membrane proteins and their membrane topology. Examples Part A Results

The SAM domains of Ste11p and SteδOp are functionally exchangeable, but a heterotypic configuration is required for function It has been shown that a protein-protein interaction through the respective

SAM domains of Ste1 1 p and SteδOp is essential for the function of Ste11 p in the HOG pathway, and that both the Ste50 SAM domain and the C-terminal RA domain are required for SteδOp function.

The SAM domains between SteδOp and Ste11 p were exchanged to make chimeras in which the SAM domain (aa 22-119) of Ste11 p replaced that of SteδOp (aa1-114), creating SAM _1r Ste50pδSAM (11/50); and in which the SAM domain (aa 27-149) of SteδOp replaced that of Ste11p (aa 25-131), creating SAM ₅₀ - Ste11 pδSAM (50/1 1) (Fig. 1A, and see Materials and Methods for details). The ability of these chimeras to function in the HOG and the pheromone response pathways was measured. YCW555 (ste11δ steδOδ ssk2δ ssk22δ) cells are sensitive to high osmotic stress and cannot grow on hyperosmotic medium. However, this strain was able to grow on hyperosmotic medium when the yeast cells harbored both the chimeras, indicating that the interaction between the two chimeras leads to the activation of the SAM ₅ o-Ste11pδSAM kinase, and thus the activation of the HOG pathway (Fig.4B). In contrast, cells transformed either with plasmids combination of SAM ₅₀ -Ste11 pδSAMchimera and wild-type SteδOp, or with wild-type Ste11p and SAM _{i r} Ste50pδSAM chimera were unable to grow on

hyperosmotic media indicating their inability to activate the HOG pathway. In both cases, only one type of SAM domain is present in each pair of the molecules, the Ste50p SAM domain in the former, and the Ste1 1 p SAM in the latter.

These results indicate that the SAM-SAM domain interaction between Ste11 p and Ste50p must be heterotypic to permit Ste50p regulation of the Ste11p kinase, and that the relative configuration of the SAM domain to the rest of the molecule does not seem to be functionally important.

The interaction of SAM domains can be substituted by artificially designed protein - protein interacting modules

It was determined that the major function of the interaction of the SAM domains of Ste11p and SteδOp was to bring the two proteins together. To test if other protein-protein interaction could replace that between the SAM domains of SteδOp and Ste11 p, plasmid constructs were made in which the SAM domains of Ste11 p and SteδOp were replaced by artificially designed coiled-coiled protein- protein interaction modules, namely the E-coil (E) and K-coil (K). These coiled- coils have been shown to exhibit a specific E/K heterotypic interaction (Tripet et al. 1996; Tripet et al. 2002). An E-coil domain was N-terminally fused into a SAM domain deleted Ste1 1 p to create E-Ste11δSAMp, and the SAM domain of SteδOp was replaced with a K-coil or an E-coil to make K-Ste50δSAMp and E- SteδOδSAMp. These constructs were assayed for their ability to activate the HOG pathway in response to high osmotic stress in ste11δ steδOδ ssk2δ ssk22δ cells. As positive control, co-transformation of the yeast strain with both wild-type Ste11p and SteδOp restores the ability of the strain to grow on hyperosmotic medium. As shown in Fig. 5, only those yeast cells co-transformed with the combination of heterotypic chimeras, K-Ste50pδSAM and E-Ste11pδSAM, were able to grow on high osmotic medium, albeit not as efficiently as the combination containing the wild-type proteins. In contrast, the cells with a combination of homotypic chimeras, E-Ste50pδSAM and E- Ste11pδSAM, like the cells co-transformed with Ste11 pδSAM and Ste50pδSAM as negative control, were not able to grow on the same medium. These results indicated that the functionality of the heterotypic

interaction between the SAM domains of Ste11 p and Ste50p could be replaced by the interaction between the E- and K-coils.

Fusing a nonfunctional SteδOpδSAM with Ste11pδSAM created a functional chimera protein that complements the function of the Ste11 p-Ste50p complex in the HOG pathway

It has been shown that both the SAM domain and the C-terminal RA domain of SteδOp are required for SteδOp function, and these domains can function relatively independently since the removal of the inter-region between the domains does not seem to significantly affect the function of the protein (Wu et al., 1999). The two domains, when provided in trans on separate plasmids, did not complement the function of SteδOp. Based on the results of the SAM domain swapping and the replacement with coiled-coil interaction modules, an investigation was made to determine if the C-terminal RA domain of SteδOp, provided in cis relative to the kinase domain of Ste11p, could modulate a Ste11p without its SAM domain. The SteδOp C-terminal fragments of either amino acid residues 1 16-346 or 219-346 were cloned into Ste11 pδSAM. This process created constructs containing the first 24 amino acid residues of Ste11 p, followed by the C- terminal region of SteδOp fused to the rest of Ste11 p containing amino acid residues 132-738, but lacking the region containing the SAM domain. These chimeric proteins were expressed from low copy plasmids under the control of the STE11 promoter (Fig. 6A). These chimeras were examined to determine if they were able to complement the function of both SteδOp and Ste11 p in the HOG pathway in steiiδ steδOδ ssk2δ ssk22δ cells. Yeast cells with either Steδ0p(116- 346), Ste11δSAM alone, or both together in trans, were unable to grow on high osmotic media. However, cells with either of the fused proteins were resistant to hyperosmotic stress and able to grow on high osmotic medium, indicating that the chimeric constructs were able to complement the wild-type Ste1 1 p-Ste50p function to activate the HOG pathway (Fig. 6B).

The level of expression of the chimeras was examined to rule out the possibility that the phenotypes observed were due to changed levels of protein expression. In the HOG pathway even overexpression of Ste1 1 pδSAM has no effect on pathway activity, but overexpression does have a significant effect on the

pheromone response. However, Western blot analysis indicated the steady-state protein expression levels of the chimeras of SteδOp and Ste11p showed no significant difference from that of the control Ste11 pδSAM. The possibility the chimeras created constitutive activity of the HOG pathway was considered by examining the activating phosphorylation level of Hogi p by Western blot analyses using specific anti-phospho-p38 antibodies. As shown in Fig. 6C, the chimera of Ste50p(219-346)-Ste11pδSAM was able to induce the phosphorylation of Hogip under high osmotic stress condition, whereas Ste1 1 pδSAM was unable to do so under the same conditions. Membrane targeting of the Ste50p SAM domain bypasses the requirement of the C-terminal domain of SteδOp

Previous studies have shown that in addition to the SAM domain at its N- terminus, the C-terminal region of SteδOp is necessary for the function of SteδOp. The sequence alignment indicates that the C-terminal region of Ste50p has similarity to Ras association (RA) domain. Members of the Ras superfamily are small GTPases that are typically localized to membranes through a lipid modification at their C-terminal tails. To test if membrane localization could mimic part of the SteδOp function in the HOG pathway, the myristoylation signal of Gpai p was added to the N-terminus of a GFP tagged SAM domain of SteδOp (aa1-110), creating myrGFP-SteδOpSAM. As controls, the myristoylation signal was also added to GFP only, generating myrGFP, as well as to the full-length and the C- terminal region (aa 1 15-346) of SteδOp to generate myrGFP-SteδOp and myrGFP- SteδOpδSAM, respectively (Fig. 7A). The ability of these constructs to complement the function of SteδOp in the HOG pathway was then examined. Yeast cells of YCW36δ {steδOδ ssk2δ skk22δ) were transformed with the plasmid constructs mentioned above and scored for the ability to grow on hyperosmotic medium. Cells with myrGFP (vector) alone could not grow on high osmotic media. However, the transformants with myrGFP-Ste50pSAM were able to grow on the same media, showing that the SAM domain of SteδOp with a membrane association signal is capable of complementing the function of wild-type SteδOp to activate the HOG pathway (Fig. 7B). In contrast, transformants with myrGFP-SteδOpδSAM failed to grow on the same medium, indicating that membrane targeting the C-terminal RA-

domain of SteδOp, unlike membrane targeting the SAM domain, does not complement the SteδOp function. A similar phenotype was observed when myrGFP was replaced with myrGST. As shown in figure 7C, a myrSte50SAM was unable to active the HOG pathway in stei iδ ste50δ ssk2δ ssk22δ cells when the SAM domain of Ste11p was deleted, demonstrating that the ability of myrSteδOpSAM to complement the function of SteδOp depended on its ability to interact with the SAM domain of Ste11p. The localization of the SteδOp constructs bearing the myristoylation signal sequence was examined by fluorescent microscopy. As shown in Fig. 7D, the GFP signal was enriched at the cell periphery, and therefore presumably the plasma membrane. To demonstrate that the SteδOpSAM bearing a myristoylation signal can activate the HOG pathway, we examined the phosphorylation of the activation loop of Hogi p. In the absence of signaling from the Sln1 p-Ssk1 p-Ssk2p/Ssk22p module, phosphorylation of the activation loop of Hogi p in response to high osmotic stress is dependent on the function of the Sho1 p-Ste11 p-Ste50p pathway. The phosphorylation of Hogi p in ste50δ ssk2δ ssk22δ cells, when treated with high osmotic stress, is dependent on the presence of a functional of SteδOp. As shown in Fig. 7E by Western blot analysis, Hogi p was phosphorylated upon hyperosmotic treatment in cells transformed with a myristoylated SAM domain of SteδOp, but not with a myristoylated SteδOpδSAM. These results together indicate that plasma membrane targeting of SteδOpSAM complements the SteδOp function for the regulation of Ste11p in the HOG pathway, and thus it appears that the C-terminal RA-like domain of SteδOp may play a role in localizing the Ste11p-Ste50p complex to the plasma membrane. Membrane targeting Ste11p bypasses the requirement for Ste50p

It was proposed that direct plasma membrane targeting of Ste11p could bypass the requirement of SteδOp in the HOG pathway. Constructs generating myristoylated GST-Ste1 1 p (myrGST-Ste11 p) were made, and myrGST, non- myristoylated GST-Ste11 p and Ste11 p were used as controls. These constructs were expressed under control of the G AU promoter. The ability of these constructs to activate the HOG pathway in ste11δ ste50δssk2δ ssk22δ cells that depend on the activity of the Ste50p-Ste11p complex was tested. Cells of ste11δ

ste50δssk2δ ssk22δ overexpressing either Ste11 p or GST-Ste11 p could not grow on high osmotic media. However, the cells expressing myrGST-Ste11 p could grow slowly on galactose-based hyperosmotic media, but were able to generate spontaneous fast-growing papillae after prolonged periods of incubation (Fig. 8A, left two panels). When examined for their cellular morphology, we found that these slow-growing cells had aberrant shapes (Fig. 8B). The cultures consisted of enlarged cells with multiple projections, which resembled the morphology of cells responding to high concentrations of mating pheromone; these cells were largely arrested for proliferation, and the spontaneous formation of colonies on high osmotic media apparently arose from the escape of this cell cycle arrest.

To abrogate the potential mating response pathway activation, STE5 was deleted in the host strain. Cells of the resulting strain transformed with myrGST- Ste11 p, but not with the other plasmid constructs mentioned above, were able to grow healthily on high osmotic media (Fig. 8A, the right two panels), indicating that the myrGST-Ste11 p was able to activate the HOG pathway in the absence of Ste50p. As expected, the aberrant morphology associated with the expression of myrGST-Stel 1 p also disappeared in the absence of Ste5p, confirming that the aberrant morphology was due mainly to the improper activation of the pheromone response pathway. Indeed, although the myrGST-Ste11 p bypassed the requirement for SteδOp in the HOG pathway, this construct still requires the scaffold protein Steδp for mating (Fig. 8C). The aberrant morphology caused by the overexpression of myrGST-Ste11 p also required the presence of Ste20p (Fig. 8B), suggesting that while Ste11 activation involves plasma membrane targeting as a necessary step, it still requires the function of Ste20p. It appears to be clear that SteδOp is an adaptor with two modules, the SAM and the RA-like domains, which act together as a necessary step for the activation of Ste11 p. The SAM domain serves as interaction surface for binding Ste11 p, and the RA-like domain may direct membrane localization potentially through a protein capable of plasma membrane localization. Materials and Methods

Materials -Restriction endonucleases and DNA-modifying enzymes were obtained from New England Biolabs (Beverly, MA, U.S.A) and Amersham Biosciences (Quebec, Canada). High fidelity Expand thermostable DNA

polymerase, anti-His monoclonal antibody, and tablet protease inhibitors were purchased from Roche Molecular Biochemicals (Quebec, Canada). Acid-washed glass beads (450-600 μm), synthetic α-mating factor, protease inhibitors, and bovine serum albumin were purchased from Sigma (Ontario, Canada). Geneticin was purchased from Life Technologies (Ontario, Canada), and nourseothricin (clonNAT) from Werner BioAgents (Jena-Cospeda, Germany). The antibody against GST was described previously (Wu et al. 1999). Anti-Myc(9E10) monoclonal antibody, anti-Ste1 1 p, and horseradish peroxidase conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal antibodies, anti-p38 and anti-phospho-p38, were from Cell Signaling Technology (Beverly, MA). Nitrocellulose membranes were purchased from Bio-Rad (Hercules, CA). The enhanced chemiluminescence (ECL) assay system was purchased from Amersham Biosciences.

Yeast strains and manipulations - Yeast media, culture conditions and manipulations of yeast strains were as described (Rose et al. 1990). Yeast transformations with circular or linearized plasmid DNA were carried out after treatment of yeast cells with lithium acetate (Rose et al. 1990). The yeast strains used in this study are listed in Table I.

Construction of Plasmids - For SAM domain swapping among Ste11 p and Ste50p, plasmid pCW555 was constructed by cloning a PCR amplified STE50 fragment corresponding to amino acid residues 27-149 into the C/al and EcoRI sites of pCW204 (Wu et al. 1999) resulting in a chimera Ste11 p bearing the Ste50p SAM domain. Plasmid pCW556 was constructed by cloning into the SamHI site of pCW362 a STE11 PCR fragment corresponding to amino acid residues 22-119 creating a SteδOp chimera bearing the SAM domain of Ste11p. The plasmid pCW362 was constructed by exchanging the STE50 fragment encoding aa115- 346 as a BamHI-EcoRI fragment from pCW207 with that of pCW267 (Wu et al. 2003). Plasmids pCW592 and pCW594, which are STE11 δSAM constructs containing the sequences of STE50 encoding the aa115-346 and aa219-346 of the C-terminus of Ste50p in the place of its SAM domain, respectively, were constructed by cloning the CIaI and EcoRI digested STE50 PCR products into the CIaI and EcoRI digested pCW204. To replace the SAM domain of SteδOp with the coiled-coil interaction modules, the E-coil and K-coil (kindly provided by Dr. M.

O'Connor, NRC, Montreal, Canada) were cloned into the EcoRI site of pGREG586 (Jansen, 2005) to create pCW386 and pGJ1273 respectively; and the resulting plasmids were digested with Sail, and recombined in vivo with a PCR amplified STE50 fragment encoding aa115-346 to generate plasmids pCW608 and pCW609 respectively. To generate either the K-coil or the E-coil tagged STE11 or STE11 δSAM, the K-coil and E-coil (as EcoRI fragments from pCW386 and pGJ1273) were cloned into the EcoRI site of pCW199 and pCW204 (Wu et al. 1999) to generate the K-coil tagged STE11 and STE11 δSAM constructs, pCW547 and pCW549; and the E-coil tagged STE11 and STE11 λSAM constructs, pCW548 and pCW550.

To construct myristoylated Ste50p and its various fragments, the corresponding ORF and fragments were cloned into vector plasmid pGREG596 (Jansen et al., 2005) which expresses GFP N-terminally tagged with the myristoylation signal sequence of the N-terminal 10aa of Gpal p, the α-subunit of the heterotrimeric G-protein of S. cerevisiae; the resulting constructs express myrGFP-SteδOp, myrGFP-Ste50pSAM (aa1-110), and myrGFP-Ste50pδSAM (aa115-346) under the control of a galactose promoter GAL1. Similarly, the construct expressing myristoylated GST-Ste11p, myrGST-Stel 1 p, was created by cloning STE11 into plasmid pGREG556 (Jansen et al., 2005). Yeast growth and other assays- Assays for the ability of cells to grow on hyperosmotic media (selective media containing either 1.25M sorbitol or 0.75M NaCI as indicated) to test the function of the HOG pathway, and yeast extract preparation and Western blot analyses were performed as described previously (Wu et al. 1999). Photomicroscopy - Cells were grown as indicated, and fixed with formaldehyde at a final concentration of 3.7% with 150 mM NaCI. Cells were viewed with a microscope equipped with Nomarski optics, and microscopic photographs were acquired with a100 x objective using a Micro Max camera (Princeton Instruments Inc.) with Northern Eclipse imaging software (Empix Imaging Inc.). The fluorescent microscopy of the GFP signals was performed as described (Wu et al. 1997). The microscopic photographs were processed using Adobe Photoshop for Macintosh.

Examples Part B

Exemplary genes illustrating the disclosed system

The procedures described generally in Part A are to be employed using (a) Ste4p/Gpa1 p; (b) Ste4/Ste18; (c) Swi6/Mbp1 ; (d) Mp1/P14 (mammalian); as the bait/prey proteins in place of the naturally occurring SAM domains in Ste11p and Ste50p to test the mode one of the system. In each case HOG pathway activation is measured to indicate protein/protein interaction between the fused bait/prey proteins. The following gene products are used as initial examples of ways of exploiting the disclosed system to study plasma membrane localization, topology and translocation: Ste2p, Gpal p, Ste18p, Opy2p, Trki p and CFTR (mammalian). These genes are to be cloned into suitable vectors for assaying their ability to activate the HOG pathway. Below are some examples of successfully cloned genes.

Results

The detection of protein-protein interaction between mammalian MP1 and

P14

Mammalian MP1 and P14 were cloned into pGJ1605-l/RA3 and pGJ1602- HIS3 respectively to generate MP1-Ste50pδSAM and P14-Ste1 1pδSAM. The resulting plasmid constructs were used to transform steSOδ ssk2δ ssk22δ yeast strains of both mating type, and selected diploid cells from the mating of the transformants of both mating types bore both plasmid constructs. Cells bearing either plasmid alone or both were then subjected to the assay of growth on hyperosmotic selective media. As shown in Fig. 9, only cells bearing both plasmid constructs could grow on hyperosmotic media, whereas cells with either plasmid alone could not. These results show that MP1-Ste50pδSAM and P14- Ste11 pδSAM were able to activate the HOG pathway, indicating that MP1 and P14 interact, and this interaction is able to replace that of the SAM domains of Ste11p and SteδOp to activate a functional HOG pathway. Similarly, we show that Ste4p and Ste18p interact with each other, and so do Swiδp and Mbpi p. These results are summarized in Table II. Ste11 pδSAM is able to activate the HOG pathway when fused to transmembrane proteins

To exploit the applicability of using Ste11pδSAM as a probe to detect/classify membrane proteins and study the topology of plasma membrane proteins, the seven-transmembrane (7-TM) G-protein-coupled receptor Ste2p was fused to Ste11 pδSAM. Yeast cells (steSOδ ssk2δ ssk22δ) transformed with the plasmid construct were then assayed for their ability to activate the HOG pathway by testing growth on hyperosmotic media. As shown in Fig. 10, steSOδ ssk2δ ssk22δ cells bearing the plasmid encoding Ste2-Ste11pδSAM were able to grow on hyperosmotic media, whereas cells with the control vector plasmid were not able to do so. This indicates that Ste11pδSAM can be used to identify/classify membrane proteins. Since the C-terminus of Ste2p is located in the cytosol, this also provides for use of Ste11 pδSAM as a probe to study the topology of plasma membrane proteins. Other gene products shown to be positive, in terms of being able to grow on hyperosmotic media, include Opy2p (single TM) and Trki p (potential 12-TM), and are summarized in Table II. SteδOpSAM is able to activate the HOG pathway when fused to transmembrane proteins

To exploit the applicability of using SteδOpSAM as a probe to detect/classify membrane proteins and study the topology of plasma membrane proteins, the seven-transmembrane (7-TM) G-protein coupled receptor Ste2p was fused to Ste50pSAM either to the N-terminal or the C-terminal of the Ste50 SAM domain. Yeast cells (steSOδ ssk2δ ssk22δ) transformed with the plasmid construct were then assayed for their ability to activate the HOG pathway by testing growth on hyperosmotic media. As predicted, when Ste2p was fused to the N-terminus of the SteδOSAM domain, the chimera is able to complement the Ste50p function; whereas when fused to the C-terminus of the SAM domain it did not complement. Similarly, heterotrimeric G-protein γ-subunit Ste18p, which is C-terminally lipid modified, was also tested. In contrast to the case of Ste2p, it was the fusion of Ste18p to the C-terminus, not to the N-terminus of SteδOSAM that complement the Ste50p, suggesting that Ste50SAM-Ste18p chimera could activate the HOG pathway to allow the growth of steSOδ ssk2δ ssk22δ cells on hyperosmotic media. These data are summarized in Table III.

Materials and Methods

Vector plasmids construction and cloning of candidate genes

The fragment of STE11 encoding Ste11p lacking its SAM domain (aa 131- 738) was cloned into pGREG506 (Jansen et al., 2005) at the Sail site to create pGJ1602 (pGREG506-Ste1 1δSAM), which was then inserted with a HIS3 stuffer marker to give rise to pGJ1602-HIS3. Similarly, the fragments of STE50 encoding SteδOp without its SAM domain (aa 115-346) and the SAM domain only (aa 1-110) were cloned respectively into pGREG503 (Jansen et al., 2005) to give rise to PGJ1605 (pGREG503-Ste50δSAM) and pGJ1603 (pGREG503-Ste50SAM). A URA3 stuffer marker was then cloned into these two constructs at the Smal site to generate pGJ1605-L/R/\3 and pGJ1603-L/Rλ3NT; and pGJ1603-L/R/\3CT was created by cloning the URA3 gene as Sall-Xhol fragment into pGJ1603 at the Xhol site.

All candidate genes chosen are cloned into the vector plasmids, (except pGJ1603-URA3CT), at the Smal site through in vivo recombination (IVR) with PCR products. All the primers used for the PCRs contain gene specific sequences and common sequences used for IVR in a layout as follows: 5'- ATTCTAGAGCGGCCGCACTAGTGGATCCCCCGGG-gene specific sequence (starting with ATG)-3' for the forward orientation (SEQ ID No. 3), and 5'- TCGATAAGCTTGATATCGAATTCCTGCAGCCCGGG-gene specific sequence (delete the stop codon)-3' for the reverse orientation (SEQ ID No. 4). For candidate genes to be cloned into pGJ1603-URA3CT at the Xhol site through IVR with PCR products amplified with primers: 5'-

AAGTTGGAGTGGAAGGACGACAAGCTGGACCTCGAG-gene specific sequence (starting with ATG)-3" (SEQ ID No. 5), and 5'- GTGAATGTAAGCGTGACATAACTAATTACATGACTCGAG- gene specific sequence (with stop codon)-3' (SEQ ID No. 6).

While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

References

Inclusion of a reference is neither an admission nor a suggestion that it is relevant to the patentability of anything disclosed herein.

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TABLE 1. Yeast strains employed

Strain Relevant genotype Source

W303-1 A MA Ta ade2 ura3 his3 Ieu2 trp 1 can 1 R. Rothstein W303- 1 B MATα ade2 ura3 his3 Ieu2 trp 1 can 1 R. Rothstein YCW340 MA Ta ura3 Ieu2 his3 ssk2δ::LEU2 ssk22δ: :LEU2 Wu et al, 1999 ste11δ::Kan ^R YCW365 MA Ta ura3 Ieu2 his3 ssk2δ::LEU2 ssk22δ: :LEU2 Wu et al, 1999 ste50δ::TRP1 YCW555 MA Ta ura3 Ieu2 his3 ssk2δ::LEU2 ssk22δ: :LEU2 Wu et al, 1999 ste11δ::Kan ^R ste50δ::TRP1 YCW757 MA Ta ura3 Ieu2 his3 ssk2δ::LEU2 ssk22δ: :LEU2 This study ste1 iδ::Kan ^R ste50δ::TRP1 ste5δ::hisG YCW1477 MA Ta um3 Ieu2 his3 ssk2δ::LEU2 ssk22δ: :LEU2 This study ste50δ::TRP1 YCW1476 MA Ta ura3 Ieu2 his3 ssk2δ::LEU2 ssk22δ: :LEU2 This study ste50δ::TRP1

Table II.

Detecting protein-protein interaction and membrane localization using the SAM- domain related yeast two-hybrid and membrane identification system based on Ste50p/Ste1 1 p activation

X- Ste11 DSAM Y- Ste50DSAM HOG Activation Comments y_ γ_ (Growth on high

^{~ ~} osmotic media; ~ interaction

Ste4p Ste18p + Yeast proteins of cell signaling Ste18p Ste4p + Yeast proteins of cell signaling P14 MP1 + Mammalian proteins

Swiδp Mbpi p + Yeast transcription factors

Swiδp Ste18p - Negative control

Ste2p + 7-TM GPCR, yeast

Opy2p + Single TM, yeast

Trki p + K+ transporter, 10-TM, yeast

Table III.

Detecting membrane localization using the SAM-domain related yeast two-hybrid and membrane identification system based on Ste50p/Ste11 p activation

HOG Activation Comments

(Growth on high osmotic media)

X= X-Ste50SAM Ste50SAM-X

Yeast proteins of cell

Ste2p + signaling Ste18p Yeast proteins of cell signaling