Restriction maps and subclones of plasmids with (A) SIT4 and (B) ISF1. All the inserts were cloned in multicopy plasmids. The locations of restriction sites EcoRI, (E); HindIII, (H); BglII, (G); KpnI, (K); SphI, (S); XbaI, (X); XmaI, (Xm) are marked. The genes are depicted by the solid arrows and the disruptors by the open bars. Growth of the transformants on rich glycerol/ethanol medium is indicate by the + (wild type growth), +/− (two times slower than wild type), − (mutant growth).

The altered catabolite repression response of the sit4 mutant described in this study is evidenced by the derepressed cytochrome spectra and respiratory activities of mitochondria from cells grown in high glucose. The escape from glucose repression was confirmed by microarray analysis of total cellular mRNAs in cells grown under repressed and derepressed conditions (www.columbia.edu/cu/biology/faculty/tzagoloff/sit4data). Average values of mRNAs functioning in pathways previously reported to be subject to glucose repression through the Ssn6p/Tup1p/Mig1p co-repressor complex (sugar transport, carbohydrate metabolism), were higher in the mutant than in wild type grown on glucose (Fig. 4), indicating that the effect of the sit4 mutation is general. This finding points to a novel Sit4p-dependent transduction pathway involved in catabolite regulation.

Effect of glucose on expression of mitochondrial respiratory chain components. (A) Spectra of mitochondrial cytochromes in the wild type D273-10B/A1 and the sit4 mutant aE153/U2. Mitochondria of cells grown in YPGal to early stationary phase were extracted with deoxycholate at a final protein concentration of 5 mg/ml and difference spectra of reduced vs oxidized extracts were recorded at room temperature as described previously [21]. The positions of the α absorption bands of cytochromes a,a₃, cytochrome b, and cytochromes c and c₁ are identified. The specific activities of NADH oxidase (NADH->O₂) are expressed in μmoles O₂/min/mg proteins (B) The wild type strain D273-10B/A1 and the sit4 mutant E153 were grown to log phase (A₆₀₀ about 1) either on 10% glucose or 2% galactose. Spectra of mitochondrial cytochrome were obtained as above. (C) Expression of COX5a-lacZ fusions in wild type W303-1A and the sit4 mutant E153/A grown on different carbon sources. Yeast strains were inoculated from fresh YPGal plates into 10 ml of YPD, YPGal or YPEG and grown at 30°C for 24 hours. The YPEG medium required a larger inoculum of sit4 mutant because of its slow growth on glycerol. Cells were then transferred to 50 ml of the same medium so that after overnight growth the A₆₀₀ were approximately 1. The cultures were harvested and β-galactosidase activities were assayed [23]. The specific activities (ΔA₄₂₀/min/A₆₀₀) reported are averages of three independent experiments.

Effect of the sit4 mutation on expression and phosphorylation of Mig1p. (A) The wild type strain W303-1A and the sit4 mutant aE153/U2 were each transformed with pM9 containing HA-tagged MIG1. The two transformants were grown in minimal selective media in the presence of glucose (Glu) or galactose (Gal) to log phase (A600 = 0.5 – 1). Cells were harvested, cooled with ice and mixed with an equal volume of ice cold 2M NaOH containing 1% β-mercaptoethanol (β-ME). After 10 min lysis on ice, the suspension was neutralized with 6M HCl and centrifuged at 20,000 × g for 15 min at 4°C. The denatured protein pellets were suspended in 1% SDS, 25 mM Tris-Cl, pH 7.5, 1% β-ME, heated at 95 °C for 5 min and clarified by centrifugation at 20,000 × g for 15 min. Supernatants (30 μg protein) were separated on an 8% SDS-PAGE gel, transferred to nitrocellulose, and processed with rabbit anti HA antibody (Covance Inc.) as in Fig. 3A. The migration of the unphosphorylated (HA-Mig1p), phosphorylated (HA-Mig1p-P) protein, and of molecular weight standards are indicated in the margins. (B) W303-1A and aE153/U2 were grown to log phase in 10% glucose and 2% galactose media. PolyA- enriched RNA (3 μg) was separated on a 1% agarose gel, blotted to DBM paper [28], and probed with a ³²P-labeled DNA fragment containing the entire MIG1 reading frame. The blot was stripped of the probe and rehybridized to a ³²P-labeled actin probe. (C) Overnight cultures of D273-10B/A1 (WT) and aE153/U2 were diluted to an A₆₀₀ 0.1 in 100 ml liquid YPD medium and grown at 30°C with vigorous shaking to a density of A₆₀₀ =1.0. Cycloheximide was added to a final concentration of 50 μg/ml, and the suspension was poured into a centrifuge bottle containing an equivalent volume of ice. Cell were harvested, washed twice with lysis buffer (10 mM Tris-Cl, pH7.4, 100 mM NaCl, 30 mM MgCl₂, 200 μg/ml heparin, 50 mg/ml cycloheximide and 2 mM PMSF) and suspended in the same buffer. The washed cells were mixed with 0.75 volumes of glass beads (0.45 mm) and the mixture was vortexed 6 times for 20 seconds, with 1 min ice cooling intervals. The homogenates were cleared by centrifugation at 3,000 × g for 5 min followed by 10,000 × g for 10 min. Cell extract equivalent to 25 A₂₅₄ units were loaded on 5 ml of a linear 7–47 % sucrose gradient prepared in the presence of 50 mM Tris-acetate pH 7.0, 50 mM NH₄Cl, 12 mM MgCl₂, 1 mM DTT, 50 mg/ml cycloheximide, and 200 μg/ml heparin. The gradients were centrifuged at 39,000 rpm for 90 min in a Beckman SW65 rotor at 4°C. Fourteen equal fractions were collected with a Brandel Model 184 Fractionator connected to Pharmacia UV MII monitor equipped with a A254 filter. Samples (135 μl) of each fraction were diluted with 346 μl H₂O and 60 μl 10% SDS, extracted with 520 μl water-saturated phenol and the aqueous phase (500 μl) was precipitated in the presence of 30 μl 5M NaCl and 1.5 ml ethanol. The RNA pellets were dried, suspended in loading buffer and one half used for Northern analysis as in part (B). The 80S monosome and polysome peaks are marked. The distribution of the MIG1 mRNA is shown under the A₂₅₄ tracing of each gradient. Fractions 4 and 5 have a high background but no specific hybridization signals.

The Ssn6p/Tup1p complex has been shown to recruit Mig1p to the promoter regions of glucose repressed genes by interacting with a GC-rich consensus site in their promoter regions [29]. The almost complete absence of Mig1p in the sit4 mutant helps to explain the lack of repression of the mitochondrial respiratory chain constituents as well as other gene products regulated by this pathway. The association of MIG1 mRNA with the polysomes fraction of the mutant favors turnover of Mig1p as the chief mechanism for its depletion. This mechanism appears to operate independent of the regulatory pathway involving Snf1p kinase-dependent phosphorylation of Mig1p (Fig. 5) [9, 27]. Mig1p could be degraded by protease regulated by Sit4p or Mig1p itself may be more susceptible to proteolysis in the sit4 mutant [30]. Dephosphorylation of cytoplasmic Mig1p has been proposed to be catalyzed by the Glc7p/Reg1p phosphatase [27] making it unlikely that Sit4p is the catalytic subunit of the phosphatase responsible for the activation of Mig1p. Despite the low amounts of Mig1p in the mutant, the ratio of phosphorylated and unphosphorylated Mig1p was about the same as in wild type. Although this suggests that both forms of Mig1p are degraded, it is not excluded that only one form is susceptible and the loss of the other occurs as a result of its phosphorylation or dephosphorylation.