The domain structure and the subcellular localisation of Rli1p. (A) Rli1p contains two domains predicted to carry Fe/S clusters at the N-terminus and two C-terminal ABC domains. The multi-sequence alignment of the cysteine-rich regions at the N-terminus of Rli1p-like proteins was created by the Multalin program (Corpet, 1988). The conserved cysteine residues of the two ferredoxin-like motifs are labelled with different arrows. Sc, S. cerevisiae; Sp, Schizosaccharomyces pombe; Hs, Homo sapiens; At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Mj, Methanococcus jannaschii; Mt, Methanobacterium thermoautotrophicum; Pa, Pyrococcus abyssi; Con, consensus sequence. (B) Yeast cells expressing a chromosomal version of Rli1p-HA were subfractionated by differential centrifugation. A post-mitochondrial supernatant (PMS) and a crude mitochondrial (CM) fraction were obtained by centrifugation of the cell lysate for 10 min at 10 000 r.p.m. The PMS fraction was used to separate soluble cytosolic proteins (Cyt) from a microsomal membrane fraction (Mic) by centrifugation at 100 000 r.p.m. for 1 h. Mitochondria (Mito) were purified from the CM fraction by Nycodenz density gradient centrifugation. Equal amounts of protein were analysed by SDS–PAGE and immunostaining for Rli1-HA and the indicated marker proteins (Yfh1p, mitochondrial matrix; Bmh1p, cytosol; Erv2p, endoplasmic reticulum). (C) In situ immunofluorescence labelling of Rli1p-HA and mitochondrial proteins Mge1p and Ssq1p. Wild-type cells (strain PSY581) were transformed with the high-copy expression vector p426 containing RLI1-HA, labelled with antibodies against the HA-tag, Ssq1p or Mge1p, and stained with fluorophore-conjugated secondary antibodies. DNA was counterstained with the fluorescent dye DAPI. Merged images show no co-localisation of Rli1p-HA with Mge1p or Ssq1p. (D) A fraction of Rli1p shuttles between cytosol and nucleus. xpo1–1 cells expressing Rli1p-HA (plasmid p416) were grown in minimal medium at 25°C to OD₆₀₀ of 0.4. Cells were shifted to 37°C for 15 min and examined by fluorescence microscopy.

Maturation of the cytosolic Fe/S protein Rli1p requires the mitochondrial and cytosolic Fe/S protein assembly machineries. (A) Wild-type cells (−) or cells expressing Rli1p-HA (HA) were radiolabelled with ⁵⁵Fe, cell extracts were prepared and an immunoprecipitation was performed using antibodies against the HA-tag or from pre-immune serum (PIS). The binding of ⁵⁵Fe to the immunobeads was estimated by scintillation counting. (B) The assay for maturation of Rli1p-HA was performed as described in (A) using wild-type (WT), Gal-NFS1, Gal-YAH1, and Gal-ATM1 cells that were transformed with a plasmid encoding Rli1p-HA. Cells were incubated in glucose-containing, iron-poor media for the indicated time periods and an immunoprecipitation was performed using antibodies against the HA tag (upper panel). The lower panel shows the iron uptake into the cells measured by scintillation counting of cell extracts. (C) Gal-CFD1 cells transformed with a plasmid encoding Rli1p-HA were used for ⁵⁵Fe incorporation into Rli1-HA after growth of cells in the presence of galactose (Gal) or glucose (Glu) as described in part B. The inset shows an immunostain of Rli1p and Cfd1p using cell extracts.

The Fe/S clusters of Rli1p are essential for cell viability. (A) The RLI1/Δrli1 diploid cells were transformed with the multicopy plasmid p426 harbouring wild-type (WT) or the indicated cysteine to serine mutants of Rli1p. In this plasmid, the expression of RLI1 was driven by part of its own promoter (300 bp upstream of RLI1). Cells with (bottom) and without (top) plasmids were subjected to sporulation and tetrad dissection. Colonies arising from individual spores after growth on glucose-containing rich medium are shown. (B) Fe/S cluster incorporation into mutant Rli1p. Wild-type cells were transformed with plasmid p426 (−) or the vectors containing the RLI1 genes described in (A). Analysis of Fe/S cluster assembly in Rli1p-HA (α-HA) or Leu1p was performed as described in Figure 2. The upper panel shows immunostaining of cell extracts for Leu1p and Rli1p-HA. Data were normalised to the levels of ⁵⁵Fe that was immunoprecipitated in cells carrying plasmid p426 with the WT RLI1 gene.

Rli1p is not involved in Fe/S protein biogenesis in the cytosol. (A) Wild-type (WT) and Tet-RLI1 cells in glucose-containing rich media were treated with the indicated amounts of doxycycline (Dox) for 16 h. Total RNA was isolated, separated in a 1% agarose gel under denaturing conditions and blotted onto Hybond membranes. The probe for Northern hybridisation was ³²P-labelled RLI1 cDNA. (B) Tet-RLI1 cells were grown in iron-poor minimal media in the presence or absence of 2 μg/ml doxycycline for 16 h. A cell lysate was prepared and analysed for maturation of the Fe/S protein Leu1p by immunoprecipitation (left) or for cellular ⁵⁵Fe uptake (right) followed by scintillation counting.

Rli1p is associated with ribosomes. (A) RLI1-TAP cells were grown in glucose-containing rich medium and used to perform a TAP of Rli1p-TAP as described using 100 mM NaCl and 5 mM CaCl₂ in the buffers (Puig et al, 2001). Rli1p-associated proteins were resolved by SDS–PAGE. The identity of major bands was determined by MALDI-TOF mass spectroscopy (see Table II). (B) RLI1-TAP and NBP35-TAP cells were subjected to TAP and aliquots were analysed by immunostaining for the indicated proteins. (C) RLI1-TAP cells were grown in glucose-containing rich medium, and a cell lysate was subjected to sucrose density gradient centrifugation (10–50% sucrose). Fractions were collected and analysed by SDS–PAGE and by immunostaining for the indicated proteins. The OD 260 nm reading (top) shows the positions of the 40S, 60S, and 80S ribosomes and polysomes.

Depletion of Rli1p impairs protein synthesis in cells grown in glucose-containing rich medium. Wild-type (WT) and Tet-RLI1 cells were grown in glucose-containing rich (YPD) and minimal (SD) media and treated with doxycycline (Dox) for 24 h. (A) Cells were radiolabelled with ³⁵S-methionine for 10 min at 30°C. A cell extract was prepared and analysed by SDS–PAGE and autoradiography. (B) Post-mitochondrial supernatants were analysed by immunostaining for the indicated proteins. Pgk1p, cytosolic phosphoglycerate kinase.

Depletion of Rli1p and the cytosolic Fe/S protein assembly component Nar1p results in nuclear accumulation of ribosomal subunits. (A, B) Wild-type and Tet-RLI1 cells were transformed with plasmids carrying the fusion proteins Rpl25p-GFP (A) or Rps2p-GFP (B). Cells were grown in minimal medium and treated with 5 μg/ml doxycycline (Dox). Cells were analysed after 19 h (Rpl25p-GFP) or 24 h (Rps2p-GFP) by immunofluorescence microscopy. Nuclear accumulation of Rpl25p and Rps2p was seen in at least 80 and 50%, respectively, of the cells. (C) Gal-NAR1 cells were transformed with a plasmid coding for Rpl25p-GFP, and were grown in glucose-containing rich medium for depletion of Nar1p (Balk et al, 2004). After 24 h, cells were analysed by immunofluorescence. Wild-type and Nar1p-containing Gal-NAR1 cells are shown as a control.