Abstract:
Members of the IpaH superfamily constitute a novel class of E3 ubiquitin ligases which are useful for engineering products which modulate trafficking and destruction of target proteins inside a cell and useful targets for identifying new antimicrobial molecules which modulate, especially inhibit, E3 ligases.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     Polypeptides in the IpaH superfamily have been discovered to be a new class of E3 ubiquitin ligases unrelated to known E3 ubiquitin ligases such as RING. U box, and HECT-domain ligases. A ubiquitin ligase attaches the protein ubiquitin to a substrate protein. This post-translation modification affects the intracellular trafficking of the ubiquitinated substrate protein and has been shown to direct a ubiquitinated substrate protein to the proteasome for destruction. The ligase of the invention may be used to construct polypeptides which recognize a specific substrate protein and ubiquitinate it or to identify molecules which block or inhibit E3 ubiquitin ligase activity of the IpaH superfamily, such as E3 ligases expressed by the bacterial pathogens  Salmonella  and  Shigella.    
     2. Description of the Related Art 
     The specific and covalent addition of ubiquitin to proteins, known as ubiquitination, is a eukaryotic-specific modification central to many cellular processes, such as cell cycle progression, transcriptional regulation, and hormone signalling. Ubiquitination involves the conjugation of one or more ubiquitin moieties on to a substrate or target protein. Mono- and multi-ubiquitinations can trigger an alteration of the localization and/or activity of a target protein, while poly-ubiquitination can modulate the properties of the target protein or constitute a signal for its degradation by the 26s proteasome, Angot et al., PLOS Pathogens 3:0001 (January, 2007). 
     The regulated destruction of proteins via the ubiquitin proteasome pathway governs many cellular processes including cell-cycle progression and signal transduction pathways, such as the NF-κB pathway. Invading pathogens are sensed by host cells through surveillance systems that initiate signalling cascades alerting the immune system to the presence of pathogens. These signalling cascades include both MAPK and nuclear factor-κB (NF-κB) programs that induce cytokine production and ultimately result in inflammation (Inohara et al., 2005). 
     The process of ubiquitination requires a ubiquitin-activating enzyme (E1) which uses ATP to activate the ubiquitin protein, a limited number of ubiquitin-conjugating enzymes (E2) which receive the activated ubiquitin and can transfer an activated ubiquitin molecule to an ubuitin ligase or to a substrate protein in the presence of an ubiquitin ligase, and a large number of ubiquitin-ligase enzymes (E3) which recognize and recruit particular substrate proteins and thus control the nature and the specificity of ubiquitination. 
     The C-terminal Gly residue of ubiquitin is charged via a thioesther linkage onto a Cys residue of E1 and transferred to a Cys residue of E2s. E3s recruit ubiquitinated E2s to specific substrates that are ubiquitinated on Lys residues by an amide linkage. RING and U-box E3s promote the transfer of ubiquitin from E2s to targets, whereas HECT-domain E3s transfer ubiquitin onto one of their Cys residues and then to targets (Ardley and Robinson, 2005; Liu, 2004). 
     The ubiquitin moiety of ubiquitinated targets can then be ubiquitinated on Lys residues 48 or 63 to produce polyubiquitinated targets. Ubiquitin chains constructed by Lys-48 linkages target proteins for destruction by the proteasome whereas those constructed by Lys-63 linkages leads to altered protein function, such as the activation of kinases (Liu, 2004), or localization. 
     Bacteria of  Shigella  spp. cause shigellosis in humans by invading the colonic mucosa. Their virulence is dependent upon a 200-kb plasmid encoding a type III secretion (T3S) system (Parsot, 2005). The type III secretion (T3S) apparatus involves the injection of bacterial effector proteins into eukaryotic host cells by many gram-negative bacteria pathogenic for plants or animals, (Galan and Cossart, 2005).  Shigella  effector proteins that promote bacterial entry are produced and stored within the bacterium at 37° C. and transit through the T3S apparatus upon contact with epithelial cells (Menard et al., 1994). A second wave of effectors, whose functions are unknown, are produced only after contact with host cells (Demers et al., 1998). These latter effectors include nine closely related IpaH proteins that are the effectors most abundantly produced by  Shigella  (Demers et al., 1998). Expression of these effectors is dependent upon an AraC family member, MxiE, which activates transcription in response to the activation of the T3S apparatus (Mavris et al., 2002; Penno et al., 2005). 
     Defining the activity of T3S effectors is key to understanding pathogenesis, however, many effectors share little sequence similarity with proteins of known function. T3S effectors are injected into eukaryotic cells and their molecular targets are intracellular. Since yeast have many proteins and processes well conserved in higher eukaryotes, they have been used to model and gain clues as to the roles of effector proteins, such as T3S effectors (Valdivia, 2004). 
     Recently, studies in yeast helped to elucidate the function of the  Shigella  effectors IpgB1 and IpgB2 that act as G protein mimics (Alto et al., 2006). To gain insight to IpaH activity, the inventors utilized  Saccharomyces cerevisiae  as a surrogate model. It was found that expression of the effector molecule IpaH9.8 in yeast disrupts signalling through the pheromone response MAPK pathway by promoting the proteasome-dependent degradation of the MAPKK Ste7. In vitro assays were used to demonstrate that effectors of the IpaH superfamily, including IpaH9.8 from  Shigella  and SspH1 from  Salmonella , constitute a novel class of E3 ubiquitin ligases. 
     BRIEF SUMMARY OF THE INVENTION 
     One aspect of the invention is a chimeric polynucleotide which encodes a polypeptide construct comprising an E3 ligase catalytic domain from a member of the IpaH superfamily and a recognition domain recognizing a particular substrate, or target, protein. 
     The term “recognition domain” refers to an amino acid sequence that recognizes or binds to a substrate or target protein. For example, the N-terminal domains, which contain the leucine rich repeat (LRRs) region of IpaH9.8 from  Shigella flexneri  and SspH1 from  Salmonella enterica , are examples of recognition domains for the yeast protein Ste7 and the mammalian protein PKN1, respectively. 
     The term “construct” includes fusion protein constructs, for example, those expressed by recombinant DNA encoding a C-terminal segment of an Ipah-superfamily E3 ligase and encoding an exogenous an N-terminal recognition domain. Constructs also include chimeric or hybrid proteins produced by means other than gene fusion, e.g., by chemical synthesis or by chemical conjugation of the ligase and recognition domains. 
     An “exogenous” sequence is a sequence not naturally associated with a given sequence. For example, an exogenous (non-Shigella flexerni IpaH9.8) recognition domain may be fused to the C-terminal domain of IpaH9.8 from  Shigella flexerni  to produce a fusion protein containing a recognition domain exogenous to the IpaH9.8 protein of  Shigella flexerni.    
     The chimeric polynucleotide of the invention encodes an active site of the IpaH E3 ligase catalytic domain which includes the sequence Cys-(Xaa) n -Asp (SEQ ID NO: 1), wherein “n” represent at least one up to 20 amino acids or the motif: 
     
       
         
               
               
             
               
               
             
           
               
                 (SEQ ID NO: 2) 
                   
               
             
          
           
               
                 Trp-Xaa(59,72)-Cys-Xaa-Asp-Xaa(29,31)-Leu-Xaa(8)- 
                   
               
               
                   
               
               
                 Arg-Xaa(7)-Ala-Xaa(13,22)-Leu-Xaa(9)-Leu-Xaa-Leu. 
               
             
          
         
       
     
     The catalytic domain may also obtained from a gene encoding IpaH9.8 from  Shigella flexneri  (SEQ ID NO: 3) or the gene encoding SspH1 of  Salmonella enterica  (SEQ ID NO: 7) or any other gene encoding a member of the IpaH superfamily. The term “catalytic domain” refers to an amino acid sequence that will ligate ubiquitin to a substrate protein. While its catalytic activity in this respect is similar to that of E3 ubiquitin ligases containing an HECT domain, it will have little or no sequence similarity with HECT E3 ligases. The C-terminal domains of IpaH9.8 from  Shigella flexneri  and SspH1 from  Salmonella enterica  are examples of catalytic domains. 
     The IpaH C-terminal domain shares 25-40% identity with two groups of bacterial proteins that contain a T3S system. Examples of IpaH superfamily members include IpaH9.8 from  Shigella flexneri  and SspH1 from  Salmonella enterica . Comparisons of other members of this family are provided by  FIG. 4  which depicts the alignment of the C-terminal domains of different IpaH superfamily members. IpaH9.8 is a virulence factor expressed by  Shigella  which facilitates colonization of host epithelial cells by its affect on host inflammatory responses. Deletion of the gene encoding IpaH9.8 reduced the ability of  Shigella  to colonize host cells by a factor of thirty, Okuda et al.,  Biochem Biophys Res Commun.  333(2):531. SspH1 is a  Salmonella enterica  serovar Typhimurium Type III secretion system effector that localizes to the mammalian nucleus and down-modulates production of proinflammatory cytokines by inhibiting nuclear factor (NF)-κB-dependent gene expression, Haraga et al.,  Cell Microbiol.  8(5):837. 
     “IpaH superfamily” refers to a class of bacterial polypeptides containing a conserved C-terminal domain which is characterized by a shared nine amino acid motif: 
     
       
         
               
               
             
               
               
             
           
               
                 (SEQ ID NO: 2) 
                   
               
             
          
           
               
                 Trp-Xaa(59,72)-Cys-Xaa-Asp-Xaa(29,31)-Leu-Xaa(8)- 
                   
               
               
                   
               
               
                 Arg-Xaa(7)-Ala-Xaa(13,22)-Leu-Xaa(9)-Leu-Xaa-Leu. 
               
             
          
         
       
     
     This motif is shared by IpaH9.8 from  Shigella  and SspH1 from  Salmonella . For example, the motif emboldened in the sequence depicted below is from IpaH9.8 of  Shigella flexneri  (SEQ ID NO: 4); 
     
       
         
               
               
               
             
           
               
                 1 
                 mlpinnnfsl pqnsfyntis gtyadyfsaw dkwekqalpg eerdeavsrl keclinnsde 
                   
               
               
                   
               
               
                 61 
                 lrldrlnlss lpdnlpaqit llnvsynqlt nlpelpvtlk klysasnkls elpvlppale 
               
               
                   
               
               
                 121 
                 slqvqhnele nlpalpdsll tmnisyneiv slpslpqalk nlratrnflt elpafsegnn 
               
               
                   
               
               
                 181 
                 pvvreyffdr nqishipesi lnlrnecsih isdnplssha lqalqrltss pdyhgpriyf 
               
               
                   
               
               
                 241 
                 smsdgqqntl hrpladavta wfpenkqsdv sqi   w   hafehe ehantfsafl drlsdtvsar 
               
               
                   
               
               
                 301 
                 ntsgfreqva awleklsasa elrqqsfava adates   c   ed   r    valtwnnlrk tllvhqaseg 
               
               
                   
               
               
                 361 
                 lfdndtga l l slgremf   r   le iledi   a   rdkv rtlhfvdeie vy   l   afqtmla ek   l   q   l   stavk 
               
               
                   
               
               
                 421 
                 emrfygvsgv tandlrtaea mvrsreenef tdwfslwgpw havlkrtead rwalaeeqky 
               
               
                   
               
               
                 481 
                 emleneypqr vadrlkasgl sgdadaerea gaqvmreteq qiyrqltdev lalrlpengs 
               
               
                   
               
               
                 541 
                 qlhhs 
               
             
          
         
       
     
     “IpaH family” is a group of proteins expressed by  Shigella  which have substantial homology at the C-terminal; and leucine-rich repeats on the N-terminal end. 
     Variants of an IpaH superfamily catalytic domain or of a recognition domain may be produced and screened by methods well-known in the art, and also by the methods described by  Current Protocols in Molecular Biology  (1987-2007), vols. 1-4, which is hereby incorporated by reference. A mutant or variant of the polynucleotides encoding a recognition domain or a catalytic domain will have 70%, 80%, 90%, 95%, or 99% homology or similarity to the corresponding sequence. Similarly a mutant or variant of the polypeptides forming a catalytic domain or a recognition domain will have 70%, 80%, 90%, 95%, or 99% homology or similarity to the corresponding amino acid sequence. Such mutants or variants may also encode, or be functionally active fragments of, these polypeptide sequences. A variant or mutant of a catalytic domain will exhibit the E3 ligase activity and those of a recognition domain will have the ability to recognize or bind to a substrate or target protein. 
     Similarity or homology may be determined by an algorithm, such as those described by  Current Protocols in Molecular Biology , vol. 4, chapter 19 (1987-2007) or by using software or computer programs such as the BestFit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711). BestFit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of identity or similarity between two sequences. Gap performs global alignments: all of one sequence with all of another similar sequence using the method of Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970). When using a sequence alignment program such as BestFit, to determine the degree of sequence homology, similarity or identity, the default setting may be used, or an appropriate scoring matrix may be selected to optimize identity, similarity or homology scores. Similarly, when using a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores. 
     Polynucleotide variants encoding the E3 ligase catalytic domain of those of  Shigella flexneri  IpaH9.8 (SEQ ID NO: 3) or the E3 ligase domain of SspH1 of  Salmonella  (SEQ ID NO: 7) may be characterized by their ability to hybridize under stringent conditions with the complements of SEQ ID NOS: 3 and 7. Alternatively, such variants may be simply isolated from a  Shigella  or  Salmonella  strain. Hybridization conditions may comprise hybridization at 5×SSC at a temperature of about 50 to 68° C. Washing may be performed using 2×SSC, optionally followed by washing using 0.5×SSC. For even higher stringency, the hybridization temperature may be raised to 68° C. or washing may be performed in a salt solution of 0.1×SSC, or both. Other conventional hybridization procedures and conditions may also be used as described by  Current Protocols in Molecular Biology , (1987-2007), see e.g. Chapter 2. The details of the subject matter described above are incorporated by reference to the corresponding documents cited above. 
     Proteins belonging to the Ipah superfamily may be identified by database mining of fully or partially sequenced bacterial genomes as proteins exhibiting over 25% sequence identity with the C-terminal domain of IpaH9.8 or any other member of the Ipah superfamily already identified (and shown in  FIG. 4 ) or as protein containing the motif defined as: 
     Methods for producing chimeric genes encoding fusion proteins or for producing engineered polypeptides such as peptide aptamer derivatives are well known in the art. Fusion proteins may be produced by conventional fusion protein methods, including those incorporated by reference to Chapter 16 of  Current Protocols in Molecular Biology , vol. 3 (1987-2007). Expression is not limited to eukaryotic cells such as yeast, plant, insect or mammalian cells, but include expression in prokaryotes, such as enterobacteria, e.g.,  E. coli, Shigella , or  Salmonella . Polypeptide constructs containing a catalytic and recognition domain may also be produced by chemical synthesis or by conjugation of separate peptides or polypeptide moieties. Methods for making aptamer constructs are described by Colas et al., PNAS 97(25): 13720, which is incorporated by reference. Methods for purifying engineered proteins, such as fusion proteins, are well-known and are incorporated by reference to Chapter 10 of  Current Protocols in Molecular Biology , vol. 2 (1987-2007). Such polypeptide constructs will preferably express the E3 ligase catalytic domain at the C-terminal and a recognition polypeptide domain, which binds to a particular substrate protein, at the N-terminal end. 
     The isolated chimeric polynucleotides may be placed into vectors, such as plasmid or viral vectors. Such vectors may be transfected into cells to express the corresponding polypeptide constructs or fusion proteins. Vectors suitable for introducing and expressing genes and polynucleotides in eukaryotic host cells are well known in the art. Vectors, host cells and expression methods for recombinant expression of proteins are well-known in the art and generally involve inserting DNA sequences encoding a peptide or polypeptide into a vector, transforming a host cell with the vector and expressing the recombinant protein in the transformed host cells. Recombinant expression of proteins is well known in the art, as are suitable vectors and host cells for expressing proteins, such as polypeptide constructs containing an E3 ligase catalytic domain and a recognition domain. Such methods, vectors and host cells are incorporated by reference to Chapter 1, 2, 3 and 9 of  Current Protocols in Molecular Biology , vol. 1 (1987-2007) and Chapter 16 “Protein Expression” in vol. 3. 
     Another aspect of the invention is the use of a fusion protein which comprises an E3 ubiquitin ligase catalytic domain and a recognition domain to modulate protein trafficking in a eukaryotic cell. The fusion protein may be expressed by transfecting or transforming a cell with a vector expressing it, or by transport of the fusion protein directly into a cell, as occurs, for example, with bacterial effector proteins such as SspH1. Modulation and control of trafficking can be used to study intracellular phenomena or to up or down-regulate particular biochemical pathways, such as pathways involved in production of recombinant molecules or other desired products, such as particular chemical compounds by a cell line. 
     The construct or fusion protein of the invention may be used therapeutically to reduce the intracellular levels or reduce the biological activity of intracellular proteins associated with diseases or responsible for various pathological states. By binding to, and ubiquitinating, these intracellular proteins, their amounts and/or biological activities are reduced, and thus the severity of disease or pathology is reduced. By selecting a construct having a recognition domain for an intracellular protein associated with cancer, an autoimmune disease, a genetic disorder or with a pathogen, such as a virus, bacteria, or parasite, intracellular levels of these virulence-associated proteins may be reduced. For example, HMGA proteins are expressed at a high level during embryogenesis, while their expression becomes low to undetectable in adult tissues. High HMGA expression in adult life is associated only with pathological conditions such as carcinomas; antisense HMGA cDNA interferes with tumor growth in vivo; Pierantoni et al., J. Clin. Invest. Doi 10.1172/JCI29852. The constructs of the invention provide a new way to reduce the levels of these proteins without the use of antisense nucleic acids. The constructs of the invention may be introduced into cells through means known in the art, including by their expression within a cell or by introduction of the protein constructs into the cytoplasm. Methods for introduction of proteins into a cell are well-known in the art and are incorporated by reference to  Current Protocols in Molecular Biology  (1987-2007). 
     Host cell lines or transgenic animals expressing the fusion protein of the invention may also be constructed using methods known in the art and are incorporated by reference to  Current Protocols in Molecular Biology  (1987-2007), vols 1-4, especially vol. 4, chapter 23. Transgenic animals expressing a polypeptide construct containing an E3 ligase catalytic domain linked to a recognition domain may be employed to measure or investigate the effects of reducing intracellular expression of the target molecule recognized by the recognition domain. For instance, a transgenic animal expressing a construct containing a recognition domain for an HMGA protein may be used to evaluate the effects of lowering HMGA levels on carcinoma and other pathologies associated with HMGA expression. Transgenic animals expressing or containing the constructs of the invention may be used in place of animals having knock-out mutations. Beneficially, the expression of a construct of the invention may be placed under control of an inducible promoter that permits the expression of the construct to be turned on and off. This permits a comparison of the effects of lowering the amount of, or reducing the activity of, a particular target protein in the same cell line without the need to construct and compare a knockout mutant. 
     The invention is also directed to the polynucleotides such as exogenous inserts which are contained in strains JRE36, JRE52, JRE 63 and JRE66, these strains have deposited at CNCM on Feb. 27, 2007 under the numbers I-3717, I-3718, I-3719 and I-3720. 
     The invention is also directed to methods of screening molecules, such as putative antimicrobial compounds, for their ability to inhibit E3 ligase activity of IpaH superfamily members, such as those expressed by  Shigella  and  Salmonella . This method involves (a) contacting an IpaH superfamily E3 ubiquitin ligase with a test molecule in the presence of a substrate protein, ubiquitin, a ubiquitin activating enzyme (E1) and a ubiquitin conjugating enzyme (E2) for a time and under conditions suitable for ubiquitination of the substrate protein recognized by the E3 ubiquitin ligase. The ligase may be a fusion protein comprising a catalytic domain of an IpaH superfamily E3 ubiquitin ligase and a recognition domain for a particular substrate protein. The ability of a test molecule to modulate, e.g., inhibit, the E3 ligase is determined by comparing the amount of ubiquitinated substrate protein in the presence of and absence of the test molecule. Methods and kits for determining ubiquitin protein ligase (E3) activity are known in the art and are incorporated by reference to US2006/008901 A1, WO02/16633 A2 or WO 2004/038036 A2. 
     Large-scale two-hybrid screens and proteonomics analysis are used to unravel and understand the network of interactions between cellular proteins. However, the functional significance of these interactions is often not understood. As an alternative to the use of siRNA to decrease the translation and stability of mRNA encoding a target protein X, the amount of target protein X can be decreased by using a protein construct of the invention which has a recognition domain for protein X and which reduces it&#39;s the amount or activity of protein X through ubiquitinating it. For example, the known binding partner Y of target protein X may be used to form a construct with an IpaH superfamily E3 ubiquitin ligase domain and the polynucleotide encoding this construct transfected into a cell to modulate cellular expression or activity of protein X. 
     The two-hybrid method in yeast is widely used to characterize or identify protein interactions, see e.g.,  Analysis of Protein Interactions , Chapter 20 , Current Protocols in Molecular Biology  (1989-2007). The classical scheme is based on the reconstitution of a functional transcription factor and relies on protein interactions between (i) a bait consisting of a DNA binding domain of a transcription factor activator fused to the protein of interest (target protein X) and (ii) a prey consisting of an active domain for the transcription factor activator fused to the putative partner(s) of interaction for target protein X. The interaction must occur in the nucleus to lead to activation of the transcription of a gene encoding a selectable marker. As an alternative to this method, the polypeptide constructs of the invention may be used as follows. 
     A bait construct (i) containing target protein X (or a functional portion or domain of X) fused on the N-terminal end of a counterselectable protein whose activity impairs the growth of yeast on a particular medium. An example of such a counterselectable marker is the product of the URA3 gene that is toxic on a medium containing 5-fluoro-orotic acid (5-FOA). Alternatively, other proteins which are toxic when expressed in yeast, such particular bacterial proteins, may be used. A prey (ii) containing an IpaH superfamily E3 ligase domain on its C-terminal end and a putative binding partner for X (protein Y) (or libraries of protein domains or peptides or peptide aptamers) is constructed. 
     The interaction between X and Y places the E3 catalytic domain in close proximity to the counterselectable protein, leading to modification and inactivation or degradation of the counterselectable protein. This permits a positive selection of yeast clones in which there is an interaction between X and Y, since binding of X and Y inactivates the toxic counterselectable protein. An advantage of this system is that the interaction of the bait and prey does not need to occur in the nucleus (as with a SiRNA system). Thus, the interaction between cytoplasmic or even membrane proteins can be investigated. A similar system may be employed to study or identify compounds with pharmacological utilities by selecting lead compounds that would interfere with the interaction of X and Y, using, for example, URA3 as a positive selection marker in conjunction with an appropriate medium. 
     Since ubiquitin-mediated proteolysis has been associated with microbial virulence, the invention also encompasses testing molecules in vitro or in vivo expressing an IpaH superfamily E3 ubiquitin ligase for their ability to inhibit proteolysis of a substrate protein or for their ability to modulate cellular inflammatory responses. The fusion proteins of the present invention which exhibit E3 ligase activity may be employed in such assays. Such methods are incorporated by reference to WO02/16633 A2 or WO 2004/038036 A2. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . Functional analysis of IpaH proteins in sst2Δ yeast and domain organization of IpaHs and homologous proteins. (A) Growth on glucose- or galactose-containing plates of serial dilutions of sst2Δ yeast harbouring plasmids encoding indicated proteins; the lower panel shows plates containing α-factor. (B) Immunoblot analysis using anti-FLAG antibodies of extracts of sst2Δ yeast producing indicated FLAG-tagged IpaH proteins. (C) Schematic representation (not to scale) of  S. flexneri  IpaH9.8 and IpaH7.8,  S. enterica  SspH1 , P. putida  PP2212, and  Yersinia  spp. YopM; LRRs and conserved C-terminal domains are shown by open and solid boxes, respectively, and residue positions are indicated by numbers. 
         FIG. 2 . IpaH9.8 is an E3 ubiquitin ligase. (A) Immunoblot analysis using anti-FLAG, anti-Ste7, and anti-Fus3 antibodies of extracts of cim5-1 yeast producing FLAG-tagged IpaH9.8-C337A (C337A) or IpaH9.8 at 25° C. or 37° C. (B) Immunoblot analysis using streptavidin-peroxidase of reactions performed in the presence of biotinylated ubiquitin, E1, UbcH5B, and either GST-IpaH9.8 (IpaH9.8) or GST-IpaH9.8-C337A (C337A). For the sample loaded in lane 5, GST-IpaH9.8 was added after UbcH5B ubiquitination and the reaction mixture was further incubated for 30 min. (C) Immunoblot analysis using anti-HA and anti-UbcH5 of reactions performed in the presence of HA-ubiquitin, E1, UbcH5B, and either GST-IpaH9.8 (IpaH9.8) or GST-IpaH9.8-C337A (C337A). (D) Immunoblot analysis using anti-Ste7, anti-IpaH, and anti-Kss1 antibodies of reactions performed in the presence of ubiquitin, E1, UbcH5B, GST-IpaH9.8 (IpaH9.8) or GST-IpaH9.8-C337A (C337A), as indicated, and a complex containing Ste7, Ste11-4, and Kss1. Samples were treated or not with DTT prior to loading. 
         FIG. 3 . SspH1 is an E3 ubiqitin ligase. (A) Immunoblot analysis using streptavidin-peroxidase (streptavidin-PO) of reactions performed in the presence of biotinylated ubiquitin, E1, UbcH5B, and either GST-IpaH9.8 (IpaH9.8), GST-IpaH9.8-C337A (C337A), or GST-SspH1 (SspH1). The two upper panels correspond to 15% SDS-PAGE and the two lower panels correspond to 8% SDS-PAGE. Samples were treated or not with DTT prior to loading, as indicated. (B) Immunoblot analysis using anti-HA antibodies of reactions performed in the presence of HA-ubiquitin, E1, UbcH5B, and either GST-IpaH9.8 (IpaH9.8), GST-IpaH9.8-C337A (C337A), or GST-SspH1 (SspH1). (C) Immunoblot analysis using anti-PKN1 and anti-GST antibodies of reactions performed in the presence of ubiquitin, E1, UbcH5B, GST-PKN1 (PKN1) and either GST-IpaH9.8 (IpaH9.8), GST-IpaH9.8-C337A (C337A), or GST-SspH1 (SspH1). 
         FIGS. 4A , B and C shows the amino acid sequence of IpAH from  Shigella flexneri  (SEQ ID NO: 4). 
         FIG. 5 . IpaH9.8 can remove ubiquitin from E2 enzymes and possesses autoubiquitination activity. Immunoblot analysis using peroxidase-coupled streptavidin (streptavidin-PO) of reactions performed in the presence of biotinylated ubiquitin, E1, UbcH5B, and either GST-IpaH9.8 (IpaH9.8) or GST-IpaH9.8-C337A (C337A), as indicated. For the sample loaded in lane 5, GST-IpaH9.8 was added after UbcH5B ubiquitination and the reaction mixture was further incubated for 30 min. Before loading, samples were either treated (middle and bottom panels) or untreated (top panel) with DTT. The top and middle panels correspond to a 15% SDS/PAGE and the bottom panel corresponds to a 10% SDS/PAGE. The position of ubiquitinated UbcH5B, ubiquitinated E1, and ubiquitinated IpaH9.8 is indicated on the right side of panels. 
     
    
    
     The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office. 
     DETAILED DESCRIPTION OF THE INVENTION 
     IpaH9.8 and SspH1 have been discovered to exhibit E3 ubiquitin ligase activity. In addition to ubiquitin, a yeast substrate for IpaH9.8 (Ste7) and a mammalian substrate for SspH1 (PKN1) were identified. Ubiquitin was removed from ubiquitinated UbcH5B upon incubation with IpaH9.8 or SspH1; this latter activity corresponding to the hydrolysis of the thioester bond linking ubiquitin to the E2 is not equivalent to the activity of de-ubiquitinating enzymes hydrolyzing the amide bond linking ubiquitin to target proteins (Amerik and Hochstrasser, 2004). 
     It might correspond either to the total consumption of the ubiquitinated E2 by the polyubiquitination activity of IpaH9.8 and SspH1 towards ubiquitin or to the transfer of ubiquitin onto IpaH9.8 and SspH1 prior to its transfer onto the substrate, as described for HECT-domain E3s (Ardley and Robinson, 2005; Scheffner et al., 1995). Although the latter hypothesis is consistent with the observation that the Cys residue conserved in all IpaH family members is required for IpaH9.8 activities both in yeast and in vitro, ubiquitin-IpaH9.8 and ubiquitin-SspH1 intermediates could not be detected. The HECT domain of E3s and the C-terminal domain of IpaH proteins do not share sequence similarities; furthermore, residues surrounding the catalytic Cys residue in HECT-domain E3s and the conserved Cys residue in Ipah proteins are different. Accordingly, IpaH superfamily members constitute a novel class of E3 ubiquitin ligases. 
     Co-opting the ubiquitination pathway, either to promote or prevent ubiquitination of host proteins, is emerging as a common strategy employed by pathogens using T3S systems to down regulate host responses. The effector AvrPtoB from the tomato pathogen  P. syringae  is structurally similar to U-box and RING-finger E3s, possesses autoubiquitination and presumably ubiquitin ligase activities towards host proteins, and blocks signalling cascades that limit infection by activating the cell death program (Abramovitch et al., 2006; Janjusevic et al., 2006). Through an unknown mechanism, the  P. syringae  effector HopM1 promotes the proteasome-dependent degradation of the  Arabidopsis  protein AtMIN7 and inhibits vesicle trafficking required to mount a cell wall-based defense to infection (Nomura et al., 2006). The  Salmonella  effector SopA has recently been shown to be a HECT-like E3 endowed with an autoubiquitination activity (Zhang et al., 2006). The  Shigella  effector OspG, encoded in the same operon as ipaH9.8, is a kinase that binds ubiquitinated E2s, prevents ubiquitination of phospho-I□B□ and dampens inflammation in the host (Kim et al., 2005). As shown here, the  Salmonella  effector SspH1 is an E3 ubiquitin ligase for PKN1, a protein kinase involved in the NF-κB pathway and activated upon cell infection (Haraga and Miller, 2006). The  S. flexneri  chromosomally-encoded IpaH proteins have been reported to play a role in dampening inflammation (Ashida et al., 2007). The LRR-containing N-terminal domain of IpaHs is likely involved in protein-protein interactions and substrate recognition; the  Yersinia  effector YopM containing only LRRs related to those of IpaHs ( FIG. 2C ) can act as a scaffolding protein that brings host kinases together (McDonald et al., 2003) and the LRR domain of SspH1 interacts with PKN1 (Haraga and Miller, 2006). In HeLa cells infected by  S. flexneri  for 90 min, a significant decrease in the amount of the MAPKK Mek1, Mek3, and IKKα was not observed, suggesting that these proteins are not degraded upon invasion of epithelial cells. The substrates of IpaH proteins in human cells remain to be identified. 
     The demonstration that IpaH9.8 and SspH1 are E3 ubiquitin ligases permits determination of the function of these proteins and their homologues during infection by identifying their target(s), possibly protein kinases, in host cells.  Shigella  produces multiple IpaHs that differ in their LRR domain and, likewise, some other pathogens contain several genes encoding IpaH homologues. This diversity suggests that each of these pathogens uses a repertoire of E3 ubiquitin ligases to promote degradation of several host proteins. 
     EXAMPLES 
     Many bacteria pathogenic for plants or animals, including  Shigella  spp. responsible for shigellosis in humans, use a type III secretion apparatus to inject effector proteins into host cells. Effectors alter cell signalling and host responses induced upon infection, however, their activities have been elucidated in very few cases. Utilizing  Saccharomyces cerevisiae  as a surrogate host, the Examples below show that the  Shigella  effector protein IpaH9.8 interrupts pheromone response signalling by promoting the proteasome-dependent destruction of the MAPKK Ste7. In vitro, IpaH9.8 displayed ubiquitin ligase activity towards ubiquitin and Ste7. 
     Replacement of a Cys residue invariant among IpaH homologues of plant and animal pathogens abolished IpaH9.8 activities. 
     The Examples also show that the IpaH homologue SspH1 from  Salmonella enterica  can ubiquitinate ubiquitin and PKN1, a previously identified partner of interaction of SspH1. These results demonstrate that IpaH superfamily members constitute a novel class of E3 ubiquitin ligases. 
     Example 1 
     IpaH9.8 Inhibits the Pheromone Response MAPK Pathway 
     To gain insight to IpaH9.8 activity,  Saccharomyces cerevisiae  was employed as a surrogate model. Yeast producing FLAG-tagged IpaH9.8 under the control of the GAL promoter were not impaired in their ability to grow at elevated temperatures, in the presence of a variety of ions, or under high and low osmotic stresses. Detection of mating pheromone by a G protein-coupled receptor activates an archetypal MAPK signalling cascade, inducing both arrest of the cell cycle and transcription of mating genes. The pheromone α-factor diffusing from a disk causes cell cycle arrest in MATα cells, resulting in a halo of inhibited growth (Hoffman et al., 2002). Upon exposure to α-factor, wild-type yeast producing IpaH9.8 failed to form a halo and to induce expression of a pheromone-responsive FUS1-lacZ reporter gene, indicating that IpaH9.8 interferes with the pheromone response pathway and acts on or upstream of the MAPK Fus3. 
     To identify the target of IpaH9.8, yeast strains were used that were altered in the signalling cascade. Overproduction of the G protein α subunit Ste4 activates the signalling pathway and promotes growth arrest (Cole et al., 1990); production of IpaH9.8 rescued this phenotype, i.e. allowed growth, indicating that IpaH9.8 acts downstream of Ste4. The constitutively active variant of the MAPKKK Ste11 encoded by the allele STE11-4 promotes elevated transcription of pheromone responsive genes, even in the absence of pheromone (Stevenson et al., 1992). Growth of the strain SY2625 harbouring a FUS1-HIS3 pheromone-inducible reporter is dependent on signalling through the pheromone response pathway on a medium lacking histidine and containing 3-amino triazole (Evangelista et al., 1997). SY2625 containing a plasmid encoding Ste11-4, but not those containing the vector, were His + , consistent with activation of the pathway by Ste11-4 and transcription of FUS1-HIS3. In contrast, yeast containing plasmids encoding Ste11-4 and IpaH9.8 were His − , indicating that IpaH9.8 interrupts signalling at or downstream of Ste11, on either the MAPK Fus3 or the MAPKK Ste7. Immunoblot analysis indicated that the amount of Ste7, but not of Ste11 and Fus3, was drastically reduced in wild-type yeast producing IpaH9.8, regardless of stimulation by α-factor. Upon phosphorylation by Ste11, Ste7 is ubiquitinated and, following removal of ubiquitin chains by the specific deubiquitinase Ubp3, is degraded by the proteasome (Wang et al., 2003). In both ubp3           , and ste11Δ cells, production of IpaH9.8 still resulted in the disappearance of Ste7, indicating that IpaH9.8-mediated disappearance of Ste7 is independent of the known Ste7 degradation pathway.
     Example 2 
     Structure and Function of IpaH Family Members 
     Blockage of signalling downstream of Ste11 suggested that IpaH9.8 should rescue sst2Δ cells defective for the GTPase activating protein encoded by SST2; these cells are unable to dampen signalling and can not grow in the presence of pheromone (Dohlman et al., 1996). Indeed, production of IpaH9.8 allowed sst2Δ cells to grow in the presence of pheromone ( FIGS. 1A and 1B ). IpaH7.8, another IpaH family member from  Shigella , also rescued the pheromone-induced growth arrest of sst2Δ cells ( FIG. 1A ), indicating that IpaH9.8 and IpaH7.8 have similar activities in yeast. 
     The strong phenotype of sst2Δ cells producing IpaH proteins was used to perform a functional analysis of IpaH domains. The nine different IpaHs encoded by the virulence plasmid and the chromosome (Yang et al., 2005) consist of a ≈250-residue variable N-terminal domain containing six to eight 20-residue leucine-rich repeats (LRR) and a ≈300-residue conserved C-terminal domain. Production of neither IpaH9.8-Nter nor IpaH9.8-Cter ( FIG. 1C ) rescued growth of sst2Δ cells exposed to pheromone ( FIG. 1A ), indicating that both domains of IpaH9.8 are required for the function in yeast. 
     Sequence comparisons revealed that the IpaH C-terminal domain shares 25-40% identity with two groups of proteins of bacteria that contain a T3S system and are pathogens of plants, fish, and mammals ( FIG. 4 ). One group includes eighteen ≈600-residue proteins from  Shigella  spp.,  Yersinia pestis  (and  Y. pseudotuberculosis ),  Salmonella enterica, Edwardsiella ictaluri, Bradyrhizobium japonica , and  Rhizobium  sp. strain NGR234 and the other includes fifteen ≈1500-residue proteins from  Pseudomonas putida, P. entomophila, P. fluorescens , and  P. syringae . In both groups, the conserved domain is C-terminal and preceded by LRRs ( FIG. 2C ). 
     The presence of one Cys residue among the nine residues that are identical in all members of the IpaH family suggested that its thiol group might be involved in catalysis. To test this hypothesis, Cys-337 of IpaH9.8 was replaced by Ala in IpaH9.8-C337A. Although IpaH9.8-C337A was produced in similar amounts to IpaH9.8 ( FIG. 1B ), it did not allow sst2Δ cells to grow in the presence of pheromone ( FIG. 1A ). Circular dichroism measurements in the far-UV and near-UV regions on purified GST-IpaH9.8 and GST-IpaH9.8-C337A showed that these two proteins have similar secondary and tertiary structure contents, suggesting that the Cys residue conserved in all IpaH homologues is involved in function rather than in structure. 
     Example 3 
     IpaH-Mediated Disappearance of Ste7 is Proteasome Dependent 
     To test if the IpaH-mediated disappearance of Ste7 required proteasome function, a yeast strain carrying the cim5-1 allele encoding a component of the 26-S proteasome that is functional at 25° C. but not at 37° C. (Ghislain et al., 1993) was used. Ste7 was present in cim5-1 yeast producing IpaH9.8-C337A at both temperatures and IpaH9.8 at 37° C. but was not present in yeast producing IpaH9.8 at 25° C. ( FIG. 3A ). Moreover, the proteasome inhibitor MG132 prevented the disappearance of Ste7 provoked by IpaH9.8 in a MG132-permeable erg6            mutant (Lee and Goldberg, 1998). Thus, the disappearance of Ste7 promoted by IpaH9.8 is proteasome-dependent.
     Example 4 
     IpaH9.8 is an E3 Ubiquitin Ligase for Ste7 
     The observation that the degradation of Ste7 promoted by IpaH9.8 is proteasome-dependent led us to test in vitro if IpaH9.8 might be involved in an ubiquitination pathway. It was discovered that the E2 enzyme UbcH5B was apparently not ubiquitinated by E1 in the presence of GST-IpaH9.8 ( FIG. 2B ). Furthermore, after ubiquitination of UbcH5B, addition of GST-IpaH9.8 to the reaction mixture promoted the removal of ubiquitin from UbcH5B ( FIG. 3B ). This activity was not observed when GST-IpaH9.8-C337A was added to the reaction mixture ( FIG. 2B ), indicating that it required the Cys residue of IpaH9.8. 
     The amide linkage, but not the thioesther linkage, of ubiquitin to ubiquitinated proteins is resistant to dithiothreitol (DTT). In reactions containing IpaH9.8, but not in those containing IpaH9.8-C337A or lacking UbcH5B, a DTT-resistant ubiquitinated protein of the size of GST-IpaH9.8 ( FIG. 3 ) was detected. Since some E3 ubiquitin ligases possess an autoubiquitination activity (Beaudenon et al., 2005), these results indicate that IpaH9.8 is an E3 ubiquitin ligase. 
     Ubiquitin biotinylated on Lys residues can not support polyubiquitination reactions. To test if IpaH9.8 could polyubiquitinate proteins, reactions were performed using HA-tagged ubiquitin, instead of biotinylated ubiquitin. Anti-HA antibodies detected a ladder of ubiquitinated proteins, from 24 to &gt;200 kDa, in reactions performed in the presence of GST-IpaH9.8, but not in the presence of GST-IpaH9.8-C337A ( FIG. 3C ). Anti-UbcH5B antibodies detected a single species corresponding to UbcH5B (18 kDa), indicating that UbcH5B was not polyubiquitinated ( FIG. 2C ). The sizes of species detected by anti-HA antibodies were multiples of the size of HA-ubiquitin (9 kDa), indicating that the molecule that was polyubiquitinated is ubiquitin. Using K48R and K63R ubiquitin variants, it was found that IpaH9.8 catalyzed the formation of polyubiquitin chains on Lys-48, but not Lys-63. Polyubiquitinated proteins using the E2 UbcH7 were not detected. These results demonstrate that IpaH9.8 is endowed with ubiquitin ligase activity towards ubiquitin and uses UbcH5B, but not UbcH7, as an E2. 
     To test whether IpaH9.8 might ubiquitinate Ste7, purified active MAPK complexes containing Ste7, as well as Ste11-4 and the MAPK Kss1, were incubated with native ubiquitin, E1, UbcH5B, and GST-IpaH9.8 or GST-IpaH9.8-C337A. Both the non- and mono-ubiquitinated forms of IpaH9.8 were detected using anti-IpaH antibodies ( FIG. 2D ), confirming the autoubiquitination activity of IpaH9.8. In addition to Ste7, larger species that formed only in the presence of IpaH9.8 were detected by anti-Ste7 antibodies ( FIG. 2D ). Thus, IpaH9.8 is an E3 ubiquitin ligase for Ste7; the proteasome-dependent disappearance of Ste7 in yeast was likely due to the degradation of Ste7 following its polyubiquitination by IpaH9.8 and IpaH7.8. 
     Example 5 
     The Ipah Homologue SspH1 is an E3 Ubiquitin Ligase for PKN1 
     SspH1, one of the  Salmonella enterica  Typhimurium homologues of IpaH, has been shown to interact with the mammalian protein kinase PKN1 (Haraga and Miller, 2006). To test whether SspH1 shares activities with IpaH9.8, a GST-SspH1 recombinant protein was purified. As described above for IpaH9.8, SspH1 was endowed with the activities (i) to remove ubiquitin from ubiquitinated UbcH5B, (ii) to autoubiquitinate, (iii) and to polyubiquitinate HA-tagged ubiquitin ( FIG. 3 ). Ubiquitination of PKN1 by SspH1; SspH1, IpaH9.8, or IpaH9.8-C337A was incubated with E1, native ubiquitin, UbcH5B, and GST-PKN1 was tested. When the reaction was performed in the presence of SspH1, anti-PKN1 antibodies detected an additional species migrating at a size &gt;250 kDa ( FIG. 3C ). These results demonstrated that SspH1 is an E3 ubiquitin ligase that can use both ubiquitin and PKN1 as substrates. 
     Experimental Procedures 
     Materials 
     Plasmids encoding FLAG-tagged IpaH9.8, IpaH9.8-C337A, IpaH9.8-Cter, IpaH9.8-Nter, and IpaH7.8 were derivatives of the vector pFL38CII/pGal1 containing the GAL promoter (Badis et al., 2004). YCp50-STE11-4 carrying STE11-4 under the control of its own promoter and pRS316-GAL-STE4 carrying STE4 under the control of the GAL promoter have been described (Dohlman et al., 1995; Stevenson et al., 1992). Yeast strains are described in Table S1. UbcH7, E1, ubiquitin, HA-ubiquitin, biotinylated ubiquitin, ubiquitin-K48R and -K63R, horseradish peroxidase-coupled avidin, MG132, and anti-UbcH5 antibodies were purchased from Boston Biochem. Anti-IκBα, -ubiquitin, -Ste7, -Fus3, -Mek3, -I_Kα, -Kss1, and -PKN1 antibodies were purchased from Santa Cruz Biotechnology. Purified GST-PKN1 was purchased from Invitrogen. Anti-Mek1 antibodies and purified active Mek1 were purchased from Upstate Cell Signaling Solutions. The mating pheromone α-factor was purchased from Sigma. His-tagged UbcH5B was prepared as described (Kim et al., 2005). GST-IpaH9.8, GST-IpaH9.8-C337A, and GST-SspH1 were prepared as described (Mavris et al., 2002). Complexes containing Ste11-4, Ste7, and Kss1 were prepared as described (Breitkreutz et al., 2001) and eluted from FLAG M2-agarose affinity gel (Sigma) using a FLAG peptide. 
     In Vitro Assays 
     Ubiquitination of UbcH5B-His by E1 was performed in a 40-μl reaction mixture containing buffer A (25 mM Tris.HCl (pH 7.5), 50 mM NaCl, 5 mM ATP, 10 mM MgCl 2 , 0.1 mM DTT), 2 μg of biotinylated ubiquitin, 0.5 μg of E1, and 2 μg of E2 in the presence, or not, of 1 μg of GST-IpaH9.8, GST-IpaH9.8-C337A, or GST-SspH1. Reactions were incubated at 37° C. for 1 h and stopped by the addition of an equal volume of Laemmli sample buffer (62.5 mM Tris-Hcl, pH 6.8, 10% glycerol, 2% SDS, 0.0005% bromophenol blue) containing, or not, 100 mM DTT. Ubiquitination reactions were carried out in the same manner except that 2 μg of HA-tagged ubiquitin, ubiquitin, ubiquitin-48R or -3R were used instead of biotinylated ubiquitin. Approximately 1 μg of Ste11-4:Ste7:Kss1 complexes, or 0.4 μg of GST-PKN1, was incubated in buffer A with 5 μg of ubiquitin, 0.5 μg of E1, and 2 μg of E2 in the presence, or not, of 0.6 μg of GST-IpaH9.8, GST-IpaH9.8-C337A, or GST-SspH1. Reaction mixtures were separated by SDS/PAGE, transferred onto a nitrocellulose membrane and probed with specific antibodies or peroxidase-coupled streptavidin when biotinylated ubiquitin was used. 
     Additional Experimental Procedures 
     Plasmids 
     DNA fragments encoding IpaH9.8, IpaH9.8-Nter, IpaH9.8-Cter, and IpaH7.8 were amplified by PCR and cloned as XbaI-NotI fragments into the vector pFL38CII/pGal1 containing the GAL promoter (Badis et al., 2004) to create plasmids pJR001, pJR002, pJR003, and pJR004, respectively. The 3′ oligonucleotides encoded the FLAG epitope followed by a stop codon and a XbaI site. YCp50-STE11-4 carrying STE11-4 under the control of its own promoter and pRS316-GAL-STE4 carrying STE4 under the control of the GAL promoter have been described (Dohlman et al., 1995; Stevenson et al., 1992). An EcoRI-SpeI fragment from pJR001 encompassing the GAL promoter and the sequence encoding IpaH9.8 and the FLAG tag was cloned into plasmid pRS425 (Sikorski and Hieter, 1989) to create pJR005. Site directed mutagenesis of ipaH9.8 codon 337 (TGT encoding Cys) carried by plasmid pRT7 (Mavris et al., 2002) encoding GST-IpaH9.8 was performed using a Stratagene Quick Change II kit to create plasmid pJR006 encoding the IpaH9.8-C337A variant (GCT encoding Ala). To construct pJR007 encoding IpaH9.8-FLAG, a NdeI-PvuII fragment from pJR006 was transformed into yeast BY4741 along with BsaBI-BsiWI digested pJR001 and transformants were plated on Ura-medium; plasmids were rescued from Ura+ prototrophs. To construct pJR008 encoding GST-SspH1, a the sspH1 gene was amplified by PCR from  Salmonella typhimurium  ATCC 14028 and cloned as a BamHI-XhoI fragment into the vector pGEX-6P1. All plasmid insertions were confirmed by DNA sequencing. 
     Strains 
     Invasive wild-type  Shigella flexneri  5 M90T-Sm (Allaoui et al., 1992) and its mxiE derivative SF1060 (Mavris et al., 2002) were used for infection. Plasmids were propagated in  Escherichia coli  DH5α (end A1 hsdR17 sup E44 thi1 recA1 gyrA relA1 lacZYA-argF). To create JRY101, the integrating plasmid pFC23 (O&#39;Rourke and Herskowitz, 1998) containing a FUS-lacZ transcriptional fusion was digested with SphI and transformed into BY4741; Leu+ colonies were tested for pheromone-responsive β-galactosidase production. JRY100 was constructed by PCR-mediated gene disruption in strain BY4741, replacing the entire open reading frame of UBP3 by the Nat marker, as described (Goldstein and McCusker, 1999; Longtine et al., 1998); the gene replacement was confirmed by PCR. 
     Yeast Manipulations 
     Standard media and techniques were used for transformation, maintenance, and growth of yeast (Guthrie C, 1991). Halo assays were performed as described (Hoffman et al., 2002) using disks impregnated with 15 μg of α-factor. erg6            yeast were transformed with plasmids encoding IpaH9.8 or IpaH9.8-C337A and grown in selective media containing 1% raffinose to mid log phase. Following addition of MG132 (100 μM) and galactose (2%), cultures were incubated for 2 h and cell extracts were prepared as described (Wang et al., 2003). β-Galactosidase assays were performed as described (Guarente, 1983).
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