The yeast scaffold proteins Isu1p and Isu2p are required inside mitochondria for maturation of cytosolic Fe/S proteins

Abstract

Iron-sulfur (Fe/S) proteins are located in mitochondria, cytosol, and nucleus. Mitochondrial Fe/S proteins are matured by the iron-sulfur cluster (ISC) assembly machinery. Little is known about the formation of Fe/S proteins in the cytosol and nucleus. A function of mitochondria in cytosolic Fe/S protein maturation has been noted, but small amounts of some ISC components have been detected outside mitochondria. Here, we studied the highly conserved yeast proteins Isu1p and Isu2p, which provide a scaffold for Fe/S cluster synthesis. We asked whether the Isu proteins are needed for biosynthesis of cytosolic Fe/S clusters and in which subcellular compartment the Isu proteins are required. The Isu proteins were found to be essential for de novo biosynthesis of both mitochondrial and cytosolic Fe/S proteins. Several lines of evidence indicate that Isu1p and Isu2p have to be located inside mitochondria in order to perform their function in cytosolic Fe/S protein maturation. We were unable to mislocalize Isu1p to the cytosol due to the presence of multiple, independent mitochondrial targeting signals in this protein. Further, the bacterial homologue IscU and the human Isu proteins (partially) complemented the defects of yeast Isu protein-depleted cells in growth rate, Fe/S protein biogenesis, and iron homeostasis, yet only after targeting to mitochondria. Together, our data suggest that the Isu proteins need to be localized in mitochondria to fulfill their functional requirement in Fe/S protein maturation in the cytosol.

FIG. 1.

Deficiency of the Isu proteins impairs growth and results in mitochondrial iron accumulation in Gal-ISU1/Δisu2 cells. (A) Wild-type, Gal-NFS1 (23), and Gal-ISU1/Δisu2 cells were grown on agar plates with rich medium containing galactose (YPGal) or glucose (YPD) for 3 days at 30°C. (B) Mitochondria were isolated from wild-type and Gal-ISU1/Δisu2 cells grown in YPGal or lactate medium containing 0.1% glucose (Lac) (11). Equal amounts of protein (50 μg) were analyzed by immunostaining with antisera raised against Isu1p and the indicated mitochondrial proteins. (C) The non-heme, non-Fe/S iron content of isolated mitochondria was measured by the bathophenanthroline method. Standard deviations were derived from three independent experiments.

FIG. 2.

Depletion of Isu1p in Gal-ISU1/Δisu2 cells causes defects in the de novo biogenesis of mitochondrial Fe/S proteins and increases cellular iron uptake. Wild-type and Gal-ISU1/Δisu2 cells were transformed with the plasmid p426GPD carrying the BIO2 gene coding for mitochondrial biotin synthase Bio2p. Cells were grown in iron-poor minimal medium containing galactose (Gal) or glucose (Glu). They were radiolabeled with [⁵⁵Fe]iron chloride in the presence of 1 mM ascorbate, and a cell extract was prepared. The amount of Fe/S cluster incorporation into Bio2p was estimated by immunoprecipitation with Bio2p-specific antibodies (α-Bio2p) and quantitation of coimmunoprecipitated ⁵⁵Fe by liquid scintillation counting. A control immunoprecipitation was performed with preimmune serum (PIS) to detect the background levels of ⁵⁵Fe precipitation. The inset shows the amounts of Bio2p and Isu1p in Gal-ISU1/Δisu2 cells detected by immunostaining of cell extracts (50 μg). (B) The amounts of ⁵⁵Fe uptake by the cells were quantified by liquid scintillation counting. Standard deviations were calculated from three independent experiments.

FIG. 3.

The Isu proteins are required for the maturation of cytosolic Fe/S proteins. Wild-type and Gal-ISU1/Δisu2 cells were transformed with the plasmid p426GPD carrying the gene encoding Rli1p-HA. Cell growth, radiolabeling with ⁵⁵Fe, and immunoprecipitation using antibodies against Leu1p (α-Leu1p) or the HA tag (α-Rli1p-HA) were performed as described in the legend to Fig. 2A. Control immunoprecipitations were performed with preimmune serum (PIS). The insets show the amounts of Leu1p, Rli1p-HA, and Isu1p in Gal-ISU1/Δisu2 cells detected by immunostaining. Standard deviations were estimated from five independent experiments.

FIG. 4.

Isu1p lacking its N-terminal presequence is targeted efficiently to mitochondria. (A) Schematic description of various yeast Isu1p mutant proteins either carrying another N-terminal mitochondrial targeting sequence from the F₀-ATPase subunit 9 of N. crassa (pSu9-Isu1pΔ30), lacking its N-terminal presequence (Isu1pΔ30), or containing a short negatively charged tripeptide (DAE-Isu1pΔ30) instead of the presequence. Numbers indicate the amino acid positions in the proteins. The white boxes represent wild-type mature Isu1p, and the light gray boxes represent the various N-terminal extensions. (B) Plasmids (p426Met25) carrying the indicated ISU1 mutants (described for panel A) were transformed into Gal-ISU1/Δisu2 cells, and cells were grown in glucose-containing minimal medium to deplete endogenous Isu1p. Mitochondria (M) and postmitochondrial supernatants (PMS) were isolated from wild-type cells and from Gal-ISU1/Δisu2 cells expressing the different ISU1 mutants (11). Equal amounts of protein (50 μg) were analyzed by immunostaining with specific antisera against Isu1p, the mitochondrial marker Aco1p, and the cytosolic marker Bmh1p. (C) Wild-type cells and Gal-ISU1/Δisu2 (Gal-ISU) cells transformed with plasmids encoding various Isu1p mutant proteins (indicated in parentheses and described for panel A) were incubated on synthetic complete minimal medium with either galactose (SGal) or glucose (SD) for 3 days at 30°C.

FIG. 5.

Targeting of E. coli IscU to mitochondria partially restores the defects of Isu protein deficiency in Gal-ISU1/Δisu2 cells. (A) The gene encoding IscU from E. coli was inserted into the yeast vector p416Met25 either without (IscU) or with an N-terminal mitochondrial targeting sequence of N. crassa F₀-ATPase subunit 9 (pSu9-IscU). Plasmids were transformed into Gal-ISU1/Δisu2 (Gal-ISU) cells, and cell growth was compared to that of wild-type cells or Gal-ISU1/Δisu2 cells with a plasmid lacking an insert (none) by incubation on agar plates containing synthetic complete medium with either galactose (SGal) or glucose (SD) for 3 days at 30°C. (B) Mitochondria (M) and postmitochondrial supernatants (PMS) were isolated from Gal-ISU1/Δisu2 cells described for panel A after growth in minimal medium containing galactose (Gal) or glucose (Glu). Subcellular localization of IscU was analyzed by immunostaining (50 μg of protein per lane) with antisera raised against yeast Isu1p, which exhibits cross-reactivity against E. coli IscU. The mitochondrial proteins Por2p and Yfh1p and cytosolic Bmh1p served as controls. (C) The cells described for panel A were transformed with the plasmid p424GPD carrying the BIO2 or the HA-tagged RLI1 genes. Cells were examined for de novo Fe/S protein maturation as described for Fig. 2A. The levels of Rli1p-HA and Bio2p were visualized by immunostaining (insets). (D) The cellular ⁵⁵Fe uptake of the various cell types was quantified by liquid scintillation counting. Standard deviations were calculated from five independent experiments.

FIG. 6.

Upon targeting to mitochondria, human Isu proteins restore the defects of Isu protein-depleted Gal-ISU1/Δisu2 cells. (A) Schematic description of human cytosolic hIsu1 and mitochondrial hIsu2 with and without mitochondrial targeting sequences. hIsu1 was used in native form or with the N-terminal mitochondrial targeting sequence of F₁β-ATPase (pF₁β-hIsu1). The presequence of hIsu2 was either deleted (hIsu2Δ33) or replaced with the presequence of F₁β-ATPase (pF₁β-hIsu2Δ33). (B) Gal-ISU1/Δisu2 (Gal-ISU) cells weretransformed with plasmids carrying either no gene or the genes for human hIsu1 and hIsu2 proteins indicated in panel A. These cells and wild-type cells were grown on agar plates containing synthetic complete medium with either galactose (SGal) or glucose (SD) for 3 days at 30°C. (C) Cells from panel B were transformed with the plasmid p426GPD carrying the BIO2 gene. After growth in the presence of galactose (Gal) or glucose (Glu), cell extracts were examined for de novo ⁵⁵Fe/S cluster incorporation into mitochondrial Bio2p and cytosolic Leu1p as described for Fig. 2A. (D) The cellular ⁵⁵Fe uptake of the various cell types was quantified by liquid scintillation counting. Data were normalized to the amounts of iron uptake into Isu1p-containing Gal-ISU1/Δisu2 cells (grown with galactose). Standard deviations were calculated from four independent experiments. (E) Plasmid p424GPD carrying the genes for HA-tagged versions of pF₁β-hIsu2Δ33, hIsu2Δ33, or hIsu1 was transformed into Gal-ISU1/Δisu2 cells. After growth in minimal medium containing glucose, mitochondria (M) and postmitochondrial supernatants (PMS) were prepared. Equal amounts of protein (50 μg) were analyzed by immunostaining for the HA-tagged human Isu proteins and Bmh1p.