Cytosolic iron-sulfur protein assembly system identifies clients by a C-terminal tripeptide

Abstract

The eukaryotic cytosolic Fe-S protein assembly (CIA) machinery inserts iron-sulfur (Fe-S) clusters into cytosolic and nuclear proteins. In the final maturation step, the Fe-S cluster is transferred to the apo-proteins by the CIA-targeting complex (CTC). However, the molecular recognition determinants of client proteins are unknown. We show that a conserved [LIM]-[DES]-[WF]-COO^- tripeptide present at the C-terminus of clients is necessary and sufficient for binding to the CTC in vitro and directing Fe-S cluster delivery in vivo. Remarkably, fusion of this TCR (target complex recognition) signal enables engineering of cluster maturation on a non-native protein via recruitment of the CIA machinery. Our study significantly advances our understanding of Fe-S protein maturation and paves the way for bioengineering applications.

Fig. 1.. A conserved [LIM]-[DES]-[WF] tripeptide is found at the C-terminus of CIA clients.

(A) Overview of the CIA pathway (yeast nomenclature). In the final step, the CIA targeting complex (CTC) identifies the CIA clients through direct, or adaptor mediated, protein-protein interactions and delivers the Fe-S cluster. (B) The enrichment of C-terminal amino acids (frequency in dataset/frequency in C-terminal proteome) in cytosolic and nuclear Fe-S proteomes of plants (Arabidopsis thaliana, yellow), yeast (Saccharomyces cerevisiae, pink), and in humans (Homo sapiens, purple). (C) The C-terminal residue of Fe-S proteins, CIA factors, and adaptors terminating in W/F in H. sapiens (Hs), S. cerevisiae (Sc), Aspergillus nidulans (An), Neurospora crassa (Nc), Danio rerio (Dr), Xenopus laevis (Xl), Drosophilia melonogaster (Dm), and A. thaliana (At). (D) Pie charts showing frequency of amino acids found at the C-terminus (n=360 sequences), and the −1 (penultimate) and −2 positions (n=95 sequences, those from the C-terminal dataset terminating in W/F). Amino acids indicated are enriched >2-fold (light green or blue) or >3-fold (dark green or blue) relative to their frequency in the human proteome. (E) WebLogos depicting conservation of C-termini for [4Fe-4S] proteins (Leu1, Pol3, Rev3, POLR3F, PUR1, Ctu1), [2Fe-2S] proteins (Neverland and Apd1), and the CIA factors/adaptors (Nar1, Lto1, Pri1, Elp4).

Fig. 2.. Deletion of the C-terminal tryptophan of CIA clients results in defective Fe-S cluster delivery.

(A-C) The indicated yeast strains, wild-type W303, apd1 deletion, or Gal-regulable POL3 or NAR1, were transformed with a centromeric plasmid for expression of Pol3 (A), Apd1 (B), or Nar1 (C) from their natural promoters. The Δ indicates the number of C-terminal amino acids that were removed, ø indicates the empty vector control, and WT indicates the full length wild-type insert. Yeast cells were grown overnight in glucose (Glc) containing medium, then spotted after serial dilution onto Glc medium with the indicated concentrations of methylmethane sulfonate (MMS) or gallobenzophenone (Gallo). (D) Plasmid-borne Leu1 was expressed from the weak (RET2) promoter in a Δleu1 strain with a functional LEU2 allele. Yeast were grown as in (A-C), but in the absence of leucine. (E and F) Determination of Leu1 enzymatic activity in yeast crude extracts and ⁵⁵Fe incorporation to quantify Fe-S cluster insertion into Leu1. W303 or Δleu1 strains harboring the indicated Leu1 variant (expressed from its natural promoter) were grown as in (A-C). For de novo ⁵⁵Fe incorporation cells were grown in Fe-free medium overnight, followed by incubation with ⁵⁵Fe and immunoprecipitation of Leu1. (G) Fe-S cluster insertion by the E. coli ISC machinery (cell extract, dark gray) or by chemical reconstitution of Leu1’s Fe-S cluster following purification (light gray) are unaffected by C-terminal truncation up to 17 amino acids.

Fig. 3.. CTC binding in vitro and Fe-S cluster maturation in vivo depend on the C-terminal tryptophan and its carboxy terminus.

(A) Apd1 (wild-type; WT; W316A variant, or none; -), was mixed with yeast CTC subunits (Met18, Strep-tagged Cia1, and truncated Cia2^D102). Apd1 associated with the CTC was evaluated via SDS-PAGE analysis after Streptactin affinity purification. (B) Variants (red) of wild type (WT) Leu1 tested in panels (C-E). Non-bold numbers refer to the positions in the amino acid sequence. (C) The Leu1 variants (bolded numbers) were mixed with the yeast CTC (Met18, Cia1, and Strep-tagged Cia2). Leu1 associated with the CTC was evaluated by SDS-PAGE after Streptactin affinity purification. (D) Leucine-independent growth of yeast (as in Fig. 2D) depends on the C-terminal W of Leu1. (E) Determination of Leu1 variant enzymatic activity in yeast cell extract as in Fig. 2E. (F) Affinity copurification of C-terminal Leu1 truncations as in (B). (G) The last 18 amino acids of S. cerevisiae Leu1 (gray, 10) were replaced with Leu1 tails from S. pombe (green, 11) or A. nidulans (purple, 12). The specific activities of the resulting tail transplant variants were analyzed as in Fig. 2E.

Fig. 4.. An engineered TCR signal is sufficient to recruit non-native proteins to the CTC and for Fe-S cluster delivery.

(A) Cartoon of SUMO peptide carrier (SPC) fusions used in copurification, as in Fig. 3C. Streptactin elution fractions were analyzed by SDS-PAGE and immunoblotting against the SPC N-terminal His-tag. (B) A non-native Fe-S protein, E. coli LeuCD, was engineered for cluster delivery from the CIA machinery. The E. coli subunits were fused with a linker (yellow) and the TCR-tail (salmon) of yeast Leu1 was attached. Protein structures are AlphaFold models. (C) In vivo cytosolic [4Fe-4S] cluster insertion into engineered E. coli LeuCD depends on the TCR signal with an appropriate tail length. Specific activities in cell extracts were determined as in Fig. 2E. Each LeuCD variant was expressed in a Δleu1 yeast strain (light gray), or a Δleu1 strain with galactose-regulatable genes (dark gray). Growth on Glc medium for 40 h led to depletion (↓) of the indicated Fe-S cluster biosynthesis protein. (D) Modelling of the PRIM1-PRIM2 (AlphaFold, orange and sand) and CTC (6TC0, blue hues) into the CryoEM density of the Primase-CTC complex (32). This model shows that the TCR signal (KDF, salmon) interacts with the proposed client binding site on blade 3 of CIAO1.