scholarly journals Mutation in the Fe–S scaffold protein Isu bypasses frataxin deletion

2011 ◽  
Vol 441 (1) ◽  
pp. 473-480 ◽  
Author(s):  
Heeyong Yoon ◽  
Ramesh Golla ◽  
Emmanuel Lesuisse ◽  
Jayashree Pain ◽  
Jason E. Donald ◽  
...  
Keyword(s):  
Unique Feature ◽  
Mitochondrial Protein ◽  
Scaffold Protein ◽  
Yeast Cells ◽  
Single Amino Acid ◽  
Cluster Assembly ◽  
Iron Availability ◽  
Direct Effects ◽  
Cysteine Desulfurase ◽  
Mitochondrial Iron

Frataxin is a conserved mitochondrial protein deficient in patients with Friedreich's ataxia. Frataxin has been implicated in control of iron homoeostasis and Fe–S cluster assembly. In yeast or human mitochondria, frataxin interacts with components of the Fe–S cluster synthesis machinery, including the cysteine desulfurase Nfs1, accessory protein Isd11 and scaffold protein Isu. In the present paper, we report that a single amino acid substitution (methionine to isoleucine) at position 107 in the mature form of Isu1 restored many deficient functions in Δyfh1 or frataxin-depleted yeast cells. Iron homoeostasis was improved such that soluble/usable mitochondrial iron was increased and accumulation of insoluble/non-usable iron within mitochondria was largely prevented. Cytochromes were returned to normal and haem synthesis was restored. In mitochondria carrying the mutant Isu1 and no frataxin, Fe–S cluster enzyme activities were improved. The efficiency of new Fe–S cluster synthesis in isolated mitochondria was markedly increased compared with frataxin-negative cells, although the response to added iron was minimal. The M107I substitution in the highly conserved Isu scaffold protein is typically found in bacterial orthologues, suggesting that a unique feature of the bacterial Fe–S cluster machinery may be involved. The mechanism by which the mutant Isu bypasses the absence of frataxin remains to be determined, but could be related to direct effects on Fe–S cluster assembly and/or indirect effects on mitochondrial iron availability.

scholarly journals Frataxin-bypassing Isu1: characterization of the bypass activity in cells and mitochondria

2014 ◽  
Vol 459 (1) ◽  
pp. 71-81 ◽  
Author(s):  
Heeyong Yoon ◽  
Simon A. B. Knight ◽  
Alok Pandey ◽  
Jayashree Pain ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Change ◽  
Scaffold Protein ◽  
Cluster Assembly ◽  
Cysteine Desulfurase ◽  
In Cells

The Fe–S cluster assembly scaffold protein Isu1 with the amino acid change M107I is able to bypass lack of the frataxin by stimulating cysteine desulfurase activity in mitochondria, although a kinetic defect in Fe–S cluster assembly persists.

scholarly journals Mitochondrial and Nucleolar Localization of Cysteine Desulfurase Nfs and the Scaffold Protein Isu in Trypanosoma brucei

2013 ◽  
Vol 13 (3) ◽  
pp. 353-362 ◽  
Author(s):  
Julie Kovářová ◽  
Eva Horáková ◽  
Piya Changmai ◽  
Marie Vancová ◽  
Julius Lukeš
Keyword(s):  
Trypanosoma Brucei ◽  
Morphological Changes ◽  
Scaffold Protein ◽  
Complex Life Cycle ◽  
Insect Vector ◽  
Cluster Assembly ◽  
Cysteine Desulfurase ◽  
Content Type ◽  
Dual Localization ◽  
Localization Signal

ABSTRACT Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.

scholarly journals Overlapping Binding Sites of the Frataxin Homologue Assembly Factor and the Heat Shock Protein 70 Transfer Factor on the Isu Iron-Sulfur Cluster Scaffold Protein

2014 ◽  
Vol 289 (44) ◽  
pp. 30268-30278 ◽  
Author(s):  
Mateusz Manicki ◽  
Julia Majewska ◽  
Szymon Ciesielski ◽  
Brenda Schilke ◽  
Anna Blenska ◽  
...  
Keyword(s):  
Transfer Factor ◽  
Binding Sites ◽  
Scaffold Protein ◽  
Sequence Motif ◽  
Cluster Assembly ◽  
Assembly Process ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Cluster Transfer ◽  
Assembly Factor

In mitochondria FeS clusters, prosthetic groups critical for the activity of many proteins, are first assembled on Isu, a 14-kDa scaffold protein, and then transferred to recipient apoproteins. The assembly process involves interaction of Isu with both Nfs1, the cysteine desulfurase serving as a sulfur donor, and the yeast frataxin homolog (Yfh1) serving as a regulator of desulfurase activity and/or iron donor. Here, based on the results of biochemical experiments with purified wild-type and variant proteins, we report that interaction of Yfh1 with both Nfs1 and Isu are required for formation of a stable tripartite assembly complex. Disruption of either Yfh1-Isu or Nfs1-Isu interactions destabilizes the complex. Cluster transfer to recipient apoprotein is known to require the interaction of Isu with the J-protein/Hsp70 molecular chaperone pair, Jac1 and Ssq1. Here we show that the Yfh1 interaction with Isu involves the PVK sequence motif, which is also the site key for the interaction of Isu with Hsp70 Ssq1. Coupled with our previous observation that Nfs1 and Jac1 binding to Isu is mutually exclusive due to partially overlapping binding sites, we propose that such mutual exclusivity of cluster assembly factor (Nfs1/Yfh1) and cluster transfer factor (Jac1/Ssq1) binding to Isu has functional consequences for the transition from the assembly process to the transfer process, and thus regulation of the biogenesis of FeS cluster proteins.

scholarly journals Thio Modification of Yeast Cytosolic tRNA Is an Iron-Sulfur Protein-Dependent Pathway

2007 ◽  
Vol 27 (8) ◽  
pp. 2841-2847 ◽  
Author(s):  
Yumi Nakai ◽  
Masato Nakai ◽  
Roland Lill ◽  
Tsutomu Suzuki ◽  
Hideyuki Hayashi
Keyword(s):  
Scaffold Proteins ◽  
Yeast Cells ◽  
Cluster Assembly ◽  
Cysteine Desulfurase ◽  
S Protein ◽  
Iron Sulfur ◽  
Severe Impairment ◽  
Sulfur Protein ◽  
Iron Sulfur Protein ◽  
Dependent Pathway

ABSTRACT Defects in the yeast cysteine desulfurase Nfs1 cause a severe impairment in the 2-thio modification of uridine of mitochondrial tRNAs (mt-tRNAs) and cytosolic tRNAs (cy-tRNAs). Nfs1 can also provide the sulfur atoms of the iron-sulfur (Fe/S) clusters generated by the mitochondrial and cytosolic Fe/S cluster assembly machineries, termed ISC and CIA, respectively. Therefore, a key question remains as to whether the biosynthesis of Fe/S clusters is a prerequisite for the 2-thio modification of the tRNAs in both of the subcellular compartments of yeast cells. To elucidate this question, we asked whether mitochondrial ISC and/or cytosolic CIA components besides Nfs1 were involved in the 2-thio modification of these tRNAs. We demonstrate here that the three CIA components, Cfd1, Nbp35, and Cia1, are required for the 2-thio modification of cy-tRNAs but not of mt-tRNAs. Interestingly, the mitochondrial scaffold proteins Isu1 and Isu2 are required for the 2-thio modification of the cy-tRNAs but not of the mt-tRNAs, while mitochondrial Nfs1 is required for both 2-thio modifications. These results clearly indicate that the 2-thio modification of cy-tRNAs is Fe/S protein dependent and thus requires both CIA and ISC machineries but that of mt-tRNAs is Fe/S cluster independent and does not require key mitochondrial ISC components except for Nfs1.

scholarly journals Single-amino-acid substitutions within the signal sequence of yeast prepro-alpha-factor affect membrane translocation.

1988 ◽  
Vol 8 (5) ◽  
pp. 1915-1922 ◽  
Author(s):  
D S Allison ◽  
E T Young
Keyword(s):  
Amino Acid ◽  
Signal Sequence ◽  
Mitochondrial Protein ◽  
Point Mutations ◽  
Yeast Cells ◽  
Single Amino Acid ◽  
Hydrophobic Core ◽  
Free System ◽  
Alpha Factor

We used a genetic approach to identify point mutations in the signal sequence of a secreted eucaryotic protein, yeast alpha-factor. Signal sequence mutants were obtained by selecting for cells that partially mistargeted into mitochondria a fusion protein consisting of the alpha-factor signal sequence fused to the mature portion of an imported mitochondrial protein (Cox IV). The mutations resulted in replacement of a residue in the hydrophobic core of the signal sequence with either a hydrophilic amino acid or a proline. After reassembly into an intact alpha-factor gene, the substitutions were found to decrease up to 50-fold the rate of translocation of prepro-alpha-factor across microsomal membranes in vitro. Two of three mutants tested produced lower steady-state levels of alpha-factor in intact yeast cells, although the magnitude of the effect was less than that in the cell-free system.

Single-amino-acid substitutions within the signal sequence of yeast prepro-alpha-factor affect membrane translocation

1988 ◽  
Vol 8 (5) ◽  
pp. 1915-1922
Author(s):  
D S Allison ◽  
E T Young
Keyword(s):  
Amino Acid ◽  
Signal Sequence ◽  
Mitochondrial Protein ◽  
Point Mutations ◽  
Yeast Cells ◽  
Single Amino Acid ◽  
Hydrophobic Core ◽  
Free System ◽  
Alpha Factor

We used a genetic approach to identify point mutations in the signal sequence of a secreted eucaryotic protein, yeast alpha-factor. Signal sequence mutants were obtained by selecting for cells that partially mistargeted into mitochondria a fusion protein consisting of the alpha-factor signal sequence fused to the mature portion of an imported mitochondrial protein (Cox IV). The mutations resulted in replacement of a residue in the hydrophobic core of the signal sequence with either a hydrophilic amino acid or a proline. After reassembly into an intact alpha-factor gene, the substitutions were found to decrease up to 50-fold the rate of translocation of prepro-alpha-factor across microsomal membranes in vitro. Two of three mutants tested produced lower steady-state levels of alpha-factor in intact yeast cells, although the magnitude of the effect was less than that in the cell-free system.

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