Sup. 51 represents the C-terminal region of Tsc22 and protects yeast cells from the effects of H₂O₂. (a) The amino acid sequence of Sup. 51 is shown aligned to the 144-residue human Tsc22 (GenBank accession #NT_024524). (b) A schematic representation of the domain organization of Tsc22⁽⁸⁶⁾. ‘The conserved TSC-22 domain’ shown in red. The locations of the leucine (L) and valine (V) residues that form the LZ are shown. The domain spans residues Met¹(M1)-Leu⁵⁶(L56), and is succeeded by a variable C-terminal region spanning residues Gln⁵⁷(Q57)-Ala⁸⁶(A86). The predicted ‘LZ’ motif embedded within the TSC22 domain is present between residues V12-L40. (c) Wild Type yeast cells transformed with the indicated constructs were grown to logarithmic phase in galactose-containing minimal media. Aliquots of each culture were normalized for cell number, serially diluted, and then spotted on nutrient-containing agar media (CONTROL). Four (4) millimolar H₂O₂ was added to each culture and aliquots were removed after 2 and 3 h, serially diluted, and spotted on nutrient-containing media. All plates were incubated for 3 days at 30°C.

Analysis and characterization of the alternatively spliced TSC22 transcripts. (a) A schematic representation of the genomic organization of human TSC22. The sequences of all human TSC22 cDNAs available in GenBank were compared to the sequence of the human genome using blast in order to determine the exon composition of the TSC22 gene. The four exons of TSC22 are shown at the top of the Figure as boxes numbered ‘1’–‘4’. The locations of the cryptic exons ‘1a’ and ‘1b’ that are present within exon 1 are indicated. The exon composition of the different TSC22 transcripts is shown boxed. The locations of the translational start (ATG) and stop (STOP) codons are also indicated. The coding sequences are shown in color while the 5′ and 3′ UTRs are shown as empty boxes. Arrows are used to depict the location of the oligos used for RT-PCR. At the bottom are representations of the three predicted Tsc22 proteins produced by the alternatively spliced transcripts. Different regions of the proteins are color-coded and correspond to the similarly colored mRNA sequence from which they are encoded. The region of the conserved TSC22 box is shown in red. The names of the different proteins are shown at the left (‘v’= splice variant), while their sizes are indicated at the right. The 86 aa protein encoded by the TSC22⁽⁸⁶⁾ cDNA is also shown. The location of the start codon (ATG) for this cDNA is shown as ‘^*ATG^*’. (b) RT-PCR analysis of the expression of the three TSC22 transcripts. Total RNA was isolated from a variety of mouse tissues and cell lines (MOUSE), as well as human cells (HUMAN). RT-PCR was used to amplify the TSC22 transcripts using specific oligos. The different mouse tissues are: 1, brain; 2, heart; 3, lung; 4, liver; 5, spleen; 6, kidney; 7, testis; 8, skeletal muscle; 9, HL-1; 10, C2C12+vehicle; 11, C2C12+TNF-α (50 ng mL⁻¹). The human cell lines are: 12, HEK293; 13, fetal brain; 14, skeletal muscle cDNA libraries. β-Actin was also amplified using specific oligos and served as an internal control. (c) Wild Type yeast cells transformed with vectors expressing the indicated Tsc22 proteins were grown to logarithmic phase in galactose-containing minimal media. Aliquots of each culture were normalized for cell number, serially diluted, and then spotted on nutrient-containing agar media before (CTL) and 2 or 3 h after treatment with 4 mM H₂O₂. The plates were incubated for 3 days at 30°C.

TSC22 is a member of a multi-gene family with other antiapoptotic members. Schematic representations of the organization of human TSC22-2 (a), TSC22-3 (b), and TSC22-4 (c) genes. The sequences of all human TSC22-related cDNAs available in GenBank were compared to the sequence of the human genome using blast in order to determine the exon composition of the TSC22 genes. The five exons of each gene are shown at the top of the Figure as boxes numbered ‘1’–‘5’. The locations of the cryptic exons ‘1a’ and ‘1b’ that are located within exon 1 of TSC22-2 and TSC22-3 are indicated. Similarly, Cryptic exon ‘5a’ is indicated in exon 5 of TSC22-2, as well as cryptic exons ‘2a’ and ‘3a’ in exons 2 and 3, respectively of TSC22-4. The exon composition of the different TSC22 transcripts is shown boxed. The locations of the translational start (ATG) and stop (STOP) codons are also indicated. The coding sequences are shown in color while the 5′ and 3′ UTRs are shown as empty boxes. At the bottom are representations of the three predicted Tsc22 proteins produced by the alternatively spliced transcripts. Different regions of the proteins are color-coded and correspond to the similarly colored cDNA sequence from which they are encoded. The region of the conserved TSC22 box is shown in red. The names of the different proteins are shown at the left (‘v’= splice variant), while their sizes are indicated at the right. (d) An alignment of the TSC22 domains of the four Tsc22 family members. Residues identical to Tsc22-1 are shown in red. An asterisk (^*) is used to indicate residues that are identical between all four sequences. (e) The amino acid sequences of the different TSC22 sequences from all four human genes were compared in a pair-wise manner. The percentage of sequence identity between the different family members was determined and is shown boxed. (f) Wild-type yeast cells transformed with the indicated vectors were grown to logarithmic phase in galactose-containing minimal media. Aliquots of each culture were normalized for cell number, serially diluted, and then spotted on nutrient-containing agar media before (CTL) and 2 or 3 h after treatment with 4 mM H₂O₂. The plates were incubated for 3 days at 30°C.

Overexpression of human TSC22⁽⁸⁶⁾ is antiapoptotic in yeast. (a) Wild-type cells harbouring an empty plasmids (VECTORS), a plasmid expressing Bax alone (BAX) or in combination with a plasmid expressing TSC22⁽⁸⁶⁾ were grown to saturation, diluted in fresh galactose-containing media, and incubated for 16 h at 30°C. Viability was determined from aliquots of cells stained with the vital dye trypan blue by visualization with light microscopy. Data are expressed as the percentage of cells that do not stain blue with the dye (% Trypan blue negative) and represent the mean of three independent experiments (±SEM) ^*, Student's t-test P<0.01. (b) Wild-type cells transformed with either an empty vector (VECTOR) or a TSC22⁽⁸⁶⁾-expressing plasmid were grown to logarithmic phase and treated with 5 mM H₂O₂ (grey bars) or left untreated (black bars). Viability was determined using the vital dye trypan blue as in Fig. 4a. Data represent the mean of three independent experiments (±SEM). ^*, Student's t-test P<0.01. (c) Wild-type cells harboring the indicated combinations of empty vectors (VCRT) or YCA1- and TSC22⁽⁸⁶⁾-expressing plasmids were incubated with 0.6 mM H₂O₂ for 16 h. Viability was determined using with the vital dye trypan blue as in Fig. 4a. Data represent the mean of three different experiments (±SEM; Student's t-test: P<0.01 (d) Yeast cells harboring an empty vector (VECTOR) or the TSC22⁽⁸⁶⁾ expressing plasmid were grown in galactose-containing media to early logarithmic phase then incubated with or without 4 mM H₂O₂ for 2 h. The cultures were further incubated for 2 h with 0.1 mg mL⁻¹ of DHR 123 for a further 2 h. Samples of the different cultures were removed and examined by light (DIC) and fluorescent light (DHR 123) microscopy. Representative photographs are shown and the calculated proportion of 300 cells displaying a fluorescent signal (±SEM) is indicated in boxes at the right.

A genome-wide two-hybrid screen for TSC22⁽⁸⁶⁾-interacting proteins. (a) Tsc22⁽⁸⁶⁾ interacts specifically with Nkp1p and Erg6p by a yeast two-hybrid assay. Strains expressing Gal4 DNA-binding domain (Gal4DBD) or Gal4DBD fused to Rad17 or Tsc22⁽⁸⁶⁾ were mated with yeast strains of the opposite mating type expressing Gal4p activation domain (Gal4AD) or Gal4AD fused to Mec3p, Nkp1p or Erg6p. Diploids were selected and pinned on media lacking tryptophan, leucine and histidine, and containing 1.5 mM 3-aminotriazole. The Rad17-Mec3 pair serves as a positive control in this assay. (b) Wild-type (WT), Δnkp1, and Δtrm7 cells were incubated in minimal media to logarithmic phase before treatment with 4 mM H₂O₂ for 2 h. Samples of each culture were removed, normalized for cell number, and spotted on nutrient-containing agar media and incubated for 2–3 days. (c) WT (black bars) and Δnkp1 mutant (grey bars) cells were incubated in minimal media to logarithmic phase. Aliquots of each sample were removed prior to, as well as after 2 and 3 h of, treatment with 4 mM H₂O₂. At each time point, an equal number of cells (250) from each sample were plated on nutrient-containing agar media. The number of colonies formed was counted after 2 days of incubation at 30°C. The data are expressed as the percentage (%) cells that were plated that are capable of forming colonies (CFU). The data are representative of two different experiments. (d) Δnkp1 mutant cells transformed with either an empty vector (VECTOR) or a TSC22⁽⁸⁶⁾-encoding vector were grown to logarithmic phase in galactose-containing minimal media. Separate cultures of each sample were left untreated (CTL) or treated with 4 mM H₂O₂ (+H₂O₂). After 3 h of further incubation, aliquots were removed and normalized for cell number, serially diluted, then spotted on nutrient-containing agar media and incubated for 2–3 days. Photographs of the plates are shown. (e) Δnkp1 mutant cells transformed with either an empty vector (black bars) or a TSC22⁽⁸⁶⁾-encoding plasmid (grey bars) were grown to logarithmic phase and treated with 4 mM H₂O₂. Aliquots were removed at the times indicated, and 250 cells from each sample were plated on nutrient-containing agar media. The number of colonies formed was counted after 2 days of incubation at 30°C. The data re expressed as the percentage (%) cells that were plated that are capable of forming colonies (CFU). Data represent the mean of three independent experiments (±SEM). (f) Galactose-inducible ERG6-overexpressing strain KTY3 (P_GAL1-ERG6) and parental wild-type strain KTY1 (WT-ERG6) were grown to early logarithmic phase in media containing glucose, galactose and raffinose each to 1%. Cells were either left untreated (CTL) or treated with H₂O₂ to 1.2 mM (+H₂O₂) for 2 h before spotting different serial dilutions of aliquots normalized for cell number on nutrient-containing agar media. Photographs of the plates after 3 days of incubation at 30°C are shown. (g) Cells of the galactose-inducible ERG6-overexpressing strain KTY3 (P_GAL1-ERG6) transformed individually with either empty vector (VECTOR) or with the vectors expressing the indicated proteins were grown to early logarithmic phase in media containing glucose, galactose and raffinose each to 1%. Cells were either left untreated (CTL) or treated with 1.2 mM H₂O₂ (+H₂O₂) for 2 h before spotting different serial dilutions of aliquots normalized for cell number on nutrient-containing agar media. Photographs of the plates after 3 days of incubation at 30°C are shown. (H) Δnkp1 mutant cells transformed with either an empty vector (VECTOR) or a TSC22⁽⁸⁶⁾-encoding vector were grown to early logarithmic phase in galactose-containing minimal media. Separate cultures of each sample were left untreated (CTL) or treated with H₂O₂ to 4 mM (+H₂O₂). After 2 h, aliquots were removed and normalized for cell number, serially diluted, then spotted on nutrient-containing agar media. Photographs of the plates after 3 days of incubation at 30°C are shown. (i) Δerg6 cells transformed with either an empty vector (black bars) or a TSC22⁽⁸⁶⁾-encoding plasmid (grey bars) were grown to early logarithmic phase in treated with 4 mM H₂O₂. Aliquots were removed at the times indicated, and an equal number of cells (250) from each sample were spotted on nutrient-containing media. The number of colonies formed was counted after 2 days of incubation at 30°C. The data are expressed as the percentage (%) cells that were plated that are capable of forming colonies (CFU). Data represent the mean of three independent experiments (±SEM).

Analysis of the Bax suppressive properties of Tsc22⁽⁸⁶⁾ in yeast mutants lacking genes encoding LZ motif-containing transcription factors. (a) Strains of yeast deleted for the indicated genes were grown overnight to saturation, serially diluted, and spotted on galactose-containing nutrient agar minimal media and incubated for 3 days at 30°C. (b) Yeast mutants lacking the indicated genes were transformed with plasmids expressing Bax alone (BAX) or in combination with plasmids expressing TSC22⁽⁸⁶⁾ (TSC22) or Bax suppressor 32 (Bax Sup.32) were grown overnight to saturation in glucose-containing media, serially diluted and spotted onto both glucose- and galactose-containing media (GLU and GAL, respectively). A legend is shown at the top of the panel. (c) Yeast strain DSY-1 expressing Gal4 DNA-binding domain (Gal4DBD) fused to TSC22⁽⁸⁶⁾ or SMS1 was cotransformed with different vectors encoding Gal4 activation domain (Gal4AD) fusions of the indicated LZ-containing transcription factors or WIPI49. Overnight cultures of each transformant were serially diluted, and spotted on both histidine-containing (+HIS) and histidine-deficient (−HIS) nutrient-containing agar media. The Gal4DBD-fused SMS1 and Gal4AD-fused WIPI49 pair serves as a positive control in this assay.

Analysis of the antiapoptotic properties of Tsc22⁽⁸⁶⁾ deletion mutants. (a) A schematic representation of Tsc22⁽⁸⁶⁾, as well as the deletion mutants that were generated by PCR. The PCR products were cloned into the yeast expression vector p426GAL1. (b) Wild-type yeast cells harboring empty plasmid (VECTOR) or different plasmids expressing TSC22⁽⁸⁶⁾ deletion mutants were grown to early logarithmic phase and treated with 4 mM H₂O₂ for 2.5 h. Aliquots of untreated (CTL) and treated (+H₂O₂) cells were diluted and spotted on nutrient-containing agar media. (c) The levels of ROS were determined using the fluorescent dye DHR123 in yeast cells expressing the TSC22⁽⁸⁶⁾ deletion mutants indicated. Representative photographs of the light (DIC) and the corresponding fluorescent image (DHR123) for the four samples are shown. The percentage of cells (±SEM) displaying a fluorescent signal was determined and is indicated in boxes at the right of rows corresponding to each of the transformants.

Analysis of the antiapoptotic properties of the yeast SNO1 and FYV10 genes an alignment of the amino acid sequence of Tsc22⁽⁸⁶⁾ with the sequences of Sno1p (a) and Fyv10p (b). clustalw was used to align the sequences of Tsc22⁽⁸⁶⁾ with Sno1p (GenBank accession #: NP_013813) and Fyv10p (GenBank accession #: NP_012169). The regions corresponding to the 16-residue Tsc22⁽⁸⁶⁾ sequence responsible for its anti-apoptotic function are boxed. An asterisk (^*) is used to indicate identical residues. Gaps denoted by dashes (‘-’) are introduced to maximize the possible similarity between the aligned sequences. (c) Wild-type cells transformed with vectors expressing GFP alone (VECTOR) or GFP fused to the C-terminus of Sno1p or Fyv10p were grown to early logarithmic phase in galactose-containing minimal media. Separate cultures of each sample were left untreated (CTL) or treated with 4 mM H₂O₂ (+H₂O₂) for 3 h. Aliquots were removed and normalized for cell number, serially diluted, then spotted on nutrient-containing agar media and incubated for 3 days. (d) Wild-type cells expressing GFP (VECTOR) with either an empty vector, or vectors encoding C-terminal GFP fusions of Sno1p or Fyv10p were grown to logarithmic phase in treated with 4 mM H₂O₂. Aliquots were removed at the times indicated, and an equal number of cells from each sample were spotted on nutrient-containing media and incubated for 2 days. CFU were then scored and the data expressed as ‘% of plated cells’. Data represent the mean of 3 independent experiments (±SEM). (e) Wild-type (WT), Δsno1, and Δfyv10 cells were grown to early logarithmic phase in minimal media before cultures of each sample were left untreated (CTL) or treated with 2.5 mM H₂O₂ for 2 h (+H₂O₂). Aliquots were removed and normalized for cell number, serially diluted, then spotted on nutrient-containing agar media and incubated for 2–3 days. Untreated samples were also spotted on plates that were subsequently irradiated with UV light at 100 mJ cm⁻² (+UV), or incubated at elevated temperature (38°C), each with corresponding untreated control plates (CTL). (F) Wild-type (WT Δsno1, and Δfyv10 cells were grown to early logarithmic phase in minimal media before treatment with 2.5 mM H₂O₂ for 2 h. Aliquots were removed at the times indicated, and an equal number of cells from each sample were spotted on nutrient-containing media and incubated for 2 days. CFU were then scored and the data expressed as ‘% of plated cells’. Data represent the mean of three independent experiments (±SEM).