(A) Schematic representation of glutaredoxin homologues of S. cerevisiae. The conserved glutaredoxin domain is depicted as black box, in which the conserved cysteine and serine residues are indicated. The numbers refer to positions of amino acid residues in the sequences. Hydrophobic sequences are indicated by hatched boxes and charged residues N-terminal to these sequences are indicated by minus and plus signs. MTS indicates the mitochondrial-targeting sequence of Grx5, the position at which proteins are cleaved by the mitochondrial-processing peptidase (MPP) is shown. The intracellular locations of the proteins are depicted on the right. (B) Phylogenetic analysis of the yeast glutaredoxin sequences. Bootstrap numbers are indicated. The tree was calculated using the DNAman software package (Lynnon, Quebec, Canada).

Grx6 and Grx7 localize to the cis-Golgi by immunofluorescence. A GFP-tagged version of cis-Golgi marker Sed5 was expressed in Grx6-HA and Grx7-HA cells. The cells were grown in YPD and processed for immunofluorescence using mouse anti-HA and rabbit anti-GFP antibodies. The anti-mouse antibody was coupled to CY3 and the anti-rabbit to FITC. Overlay images of the glutaredoxin and the GFP signals are shown on the right. The DNA was visualized with DAPI.

Immunofluorescence with Grx7-specific antibodies reveals a punctate staining consistent with the localization in the Golgi apparatus. (A) Protein extracts (100 μg) of the strains indicated were analyzed by Western blotting using antibodies against Grx6, Grx7, Ero1, and Pdi1. Positions of molecular-weight markers are indicated. (B) The strains indicated were processed for immunofluorescence using Grx7-specific antibodies. The DNA was visualized with DAPI.

Grx7 fractionates with proteins of the cis-Golgi on sucrose gradients. Cells expressing Grx7-HA were converted into spheroplasts. The spheroplasts were lysed, and the lysate was separated on a sucrose step gradient. Fourteen fractions were collected from the top and analyzed by Western blotting using different marker proteins and Grx7-HA. Grx7-HA cofractionated with the cis-Golgi markers Emp47 and Anp1. Grx7-HA was absent from fractions containing ER (BiP), medium/late Golgi (Mnn1), mitochondria (porin), or plasma membrane (Pma1).

Grx7 is a membrane-anchored protein exposing its soluble domain to the Golgi lumen. (A) Hydrophobicity profiles of Grx6 and Grx7 were generated using the DNAman software. N-terminal hydrophobic regions of the proteins are indicated in black. (B) Crude microsome fractions of a wild-type (wt) and Grx7-HA cells were isolated. 100 μg of the Grx7-HA fraction were incubated in the presence or absence of 100 μg/ml proteinase K (PK) or 0.2% Triton X-100 (TX) for 30 min on ice. For the extraction of membrane proteins, 100 μg of membranes were incubated with unbuffered sodium carbonate on ice. Membrane-containing (P) and soluble fractions (S) were separated by centrifugation. Proteins were precipitated with trichloroacetic acid and applied to SDS-PAGE and Western blotting. Controls show the integral membrane protein Erp1, the membrane-associated protein Ero1, and the soluble protein Pdi1. (C) Radiolabeled Grx6 or Grx7 were incubated with a crude microsome fraction for 20 min at 30°C. During this reaction, 2 mM ATP (lanes 1 and 2), 2 mM NADH (lanes 1 and 3), or 10 μM valinomycin (lanes 2 and 4) were present. To remove nonimported material after the translocation reaction, the samples were treated with proteinase K. Microsomes were reisolated and subjected to SDS-PAGE and autoradiography. (D) Scheme of the Grx7 topology concluded from our results.

Grx6 and Grx7 show glutaredoxin activity in vitro. (A) Scheme of the glutaredoxin activity assay using HEDS as electron acceptor (Holmgren and Aslund, 1995). (B) Recombinant PfGrx, Grx6, Grx6^C136S, and Grx7 were expressed and purified to homogeneity from E. coli cells. Their activity was monitored at 340 nm by oxidation of NADPH in a coupled enzyme assay. Glutaredoxin proteins were present in the reactions at a final concentration of 52 nM. For control, reactions with PfGrx were carried out in the absence of glutathione (GSH) or HEDS (left columns). All reactions were carried out in a two-beam photometer in which a glutaredoxin-free sample served as reference. (C) The NADPH consumption in the HEDS assay over time was analyzed for different concentrations of purified Grx7. (D) The activity of Grx7 in the experiment shown in C was plotted against the Grx7 concentration showing a linear correlation up to 1 mg Grx7 in the assay.

The deletion of Grx6 and Grx7 leads to increased sensitivity toward oxidizing reagents. (A) The indicated strains were grown in liquid medium over night. Equal amounts of cells were plated on rich media plates containing 2% glucose (YPD). Filter disks were placed in the middle of each plate soaked with 10 μl of either 500 mM diamide, 9.8 M hydrogen peroxide, or 3 M DTT. Plates were incubated at 30°C for 2 d. (B) The indicated strains were grown to midlog phase. Serial dilutions of the culture were dropped on YPD plates lacking or containing 5 mM hydrogen peroxide and incubated for 3 d at 30°C.

Relevance of Grx6 and Grx7 for oxidative protein folding in the secretory pathway. (A) The indicated strains were grown to midlog phase. Serial dilutions of the cultures were dropped on SD plates lacking tryptophan and incubated at 15, 30, or 37°C for 7, 3, or 6 d, respectively. (B) RNA was isolated from strains lacking Grx6, Grx7, or both and the corresponding wild type. For control, one wild-type culture was treated with 10 μg/ml tunicamycin (TM) for 30 min before RNA was extracted. The splicing of HAC1 mRNA was analyzed by Northern blotting. On induction of the UPR pathway, HAC1 mRNA is spliced leading to its increased mobility in the gel (arrowhead). (C) Scrambled RNase was incubated with 1 mM reduced glutathione and 0.2 mM oxidized glutathione in the presence or absence of 1 μM purified Pdi1 and/or 18 μM purified Grx7. The RNase activity was photometrically measured and is depicted in comparison of the spontaneous RNase refolding which was set as one.