Grx5 is a mitochondrial glutaredoxin. (A) N terminus Grx5 amino acid sequence. Arrow marks where the precursor form is processed, as determined by N terminus sequencing of mature Grx5. The length of the two constructed signal peptide deletions (Grx5-Δ8 and Grx5-Δ23) is indicated. (B) Western blot immunodetection of 3HA-tagged Grx5 in extracts from exponential cultures of MML240 cells in YPD and YPG medium at 30°C and in YPD medium plus menadione (Md, 30 min). Left lane (control) corresponds to W303–1A cell extracts. The same amount of total cell protein (40 μg) was run in each lane. (C) MML235 and MML266 (Grx5-Δ8) cells grown exponentially in YPG medium at 30°C were fractionated, and the resulting fractions were analyzed by Western blot. Anti-HA antibodies were used to detect Grx5 (MML235 fractions) and Grx5-Δ8 (MML266 fractions), and anti-lipoic acid antibodies were used to detect the matrix markers pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (α-KGDH). Cytochrome b₂ (cyt b₂) was used as an intermembrane space (IMS) marker. Results for pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and cytochrome b₂ are shown only for MML266; similar results were observed for MML235. Ten micrograms of protein were loaded in each lane for “Total” cell extracts and postmitochondrial supernatant (PMS) fractions, and 3 μg were loaded for the mitochondrial (Mito), intermembrane space, and matrix fractions. (D) Sensitivity to menadione in strains MML240 (GRX5), MML100 (Δgrx5), and MML290 (grx5-Δ8) growing exponentially at 30°C in YPD medium. (E) Protein carbonylation in extracts from exponentially growing MML240, MML100, and MML290 cells in YPD medium at 30°C.

Effect of Grx5 depletion on the amount of heme covalently bound to cytochrome c. Expression of GRX5 was interrupted (time 0) by the addition of doxycycline (2 μg/ml) to MML313 cells growing exponentially in YPG medium at 30°C. Proteins from whole cell lysates (samples taken at the indicated times after antibiotic addition) were separated by nonreducing SDS-PAGE, blotted onto a polyvinylidene difluoride membrane, and analyzed for heme-carrying proteins. In these conditions, heme bound to cytochrome c was the most prominent band detected (marked with an arrow). As a standard, 0.1 μg of cytochrome c from bovine heart was loaded on the left-most line.

Activity of Fe/S enzymes is inhibited in grx5 cells independently of intracellular iron concentration. (A) CML235 (wt) or MML19 (Δgrx5) cells were grown exponentially in YPD medium (also in the presence of 80 μM BPS in the case of MML19 cells), and total cellular iron concentration and aconitase and malate dehydrogenase activity were determined. (B) The same parameters as in A were determined in exponential YPD cultures at 30°C of W303–1A (wt), MML100 (Δgrx5), MML348 (Δaft1), and MML345 (Δgrx5 Δaft1) cells.

Suppression of phenotypic defects of null grx5 mutants. (A) CML235 wild-type (GRX5) cells and MML19 (Δgrx5) cells nontransformed or transformed with the multicopy plasmids pMM62 (GRX5), pMM70 (SSQ1), or pMM74 (ISA2) were tested for growth on SD medium plates (left) and for sensitivity to 10 mM menadione (Md) treatment at 30°C (right). Serial dilutions of exponential cultures (treated with menadione for the indicated times) were plated on YPD plates. (B) CML235 and MML19 cells transformed with the pCM190 (tetO₇ promoter)-derived plasmids pCM317 (GRX3), pCM316 (GRX4), or pCM318 (GRX5) were tested for growth on SD medium plates (left) and for menadione sensitivity (right) in overexpression conditions (minus doxycycline).

Lack of Grx5 negatively affects Fe/S enzyme activity. (A) MML313 cells growing exponentially in YPG medium at 30°C (1 × 10⁷ cells/ml) were added at time 0 with doxycycline (2 μg/ml) to inhibit GRX5 expression. Enzyme activity in cell extracts was determined at the indicated times. (B) Western blot analysis of aconitase (Aco1) and succinate dehydrogenase (Sdh2) in the same samples as in A. (C) Growth of wild-type CML235 and mutant MML19 (Δgrx5) cells containing the integrative LEU2 plasmid YIplac128 (Gietz and Sugino, 1988) in SC medium with all the supplements or deprived of leucine, lysine, or glutamic acid.

Iron accumulates in the cell in the absence of Grx5. (A) W303–1A (wt) and MML100 (Δgrx5) cells were grown exponentially in YPD medium at 30°C to determine iron concentration in the cell and also aconitase and citrate synthase activities in total cell extracts. (B) MML313 cells growing exponentially in YPG medium at 30°C were added (time 0) with doxycycline (2 μg/ml), and total cell iron concentration was determined at different intervals after antibiotic addition. (C) Distribution of iron (relative to total protein in the fraction) between postmitochondrial fraction (PMF) and mitochondria (Mito), in MML313 cells untreated (−doxy) or treated for 24 h with doxycycline at 2 μg/ml (+doxy).

Growth of grx5 cells is affected in anaerobiosis or in the presence of external reductants. (A) CML235 (wt) and MML19 (Δgrx5) cells were grown exponentially in SC liquid medium at 30°C to exponential phase, in anaerobic or aerobic conditions, and total iron concentration in the cell was determined (top). The same strains were inoculated on SD agar plates, and growth was recorded after 3 d (+ O₂) or 4 d (−O₂) of incubation at 30°C. (B) CML235 (wt) and MML19 (Δgrx5) cells were inoculated on SD agar plates containing different DTT concentrations, and growth was recorded after 3 d (wt) or 4 d (Δgrx5) of incubation at 30°C.