Trp-ing out on cytosolic [Fe–S]-cluster delivery

Proteins that contain small prosthetic groups comprised of iron and sulfur ([Fe–S]-clusters) were first discovered in the early 1960’s (1). Such proteins, commonly referred to as [Fe–S]-proteins, play essential roles in numerous life-sustaining processes (Fig. 1). In humans, impairment in the formation or activation of [Fe–S]-proteins is associated with an extensive variety of pathological conditions; thus, the complicated process of [Fe–S]-cluster biogenesis and [Fe–S]-protein maturation is of considerable biomedical interest (2). In spite of the important role that [Fe–S]-proteins and their associated clusters play in so many cellular processes, not until the mid-1980s was it appreciated that a consortium of components, perhaps having universal mechanistic features, were involved in the biogenesis of [Fe–S]-clusters and their subsequent delivery to their cognate protein partners (3). Because Fe and S are toxic in their free forms, and because [Fe–S]-clusters are labile in aqueous solution, endogenously formed clusters must be protected within protein matrices and trafficked to their cognate clients through specific protein–protein interactions. In PNAS, Marquez et al. (4) describe an important aspect of how [Fe–S]-clusters are delivered to certain client [Fe–S]-proteins produced in the Eukaryotic cytosol.

Fig. 1.

Eukaryotic processes that require [Fe–S]-proteins and involvement of the TCR signal in apo-client [Fe–S]-protein maturation. The single letter codes for Gln (Q), Asp (D), and Trp (W) are shown as an exemplar of a TCR motif attached to the C-termini of the apo-client and the mature [Fe–S]-protein. The cluster is shown as a typical [4Fe–4S]-cluster with S indicated by a yellow sphere and iron indicated by a red sphere.

There are at least two systems in Eukaryotes involved in the maturation of [Fe–S]-proteins (5). One of them, generally referred to as ISC (iron–sulfur cluster assembly), is responsible for the biogenesis of [Fe–S]-clusters in the mitochondria and their subsequent delivery to apo-forms of client [Fe–S]-protein (5). With the assistance of molecular chaperones, a Leu-Tyr-Arg tripeptide motif (LYR) within client mitochondrial [Fe–S] proteins guides the delivery of nascent [Fe–S]-clusters from the assembly components to immature client [Fe–S]-proteins (6). The other [Fe–S]-cluster assembly/delivery system, designated CIA (cytosol iron–sulfur assembly), is necessary for the maturation of [Fe–S]-proteins located in the cytosol and nucleus. The ISC and CIA systems are connected because the CIA system depends on a probable Fe/S-containing species produced by the ISC system, which is exported out of the mitochondria by another factor designated ATM. In the case of cytosolic [Fe–S]-protein maturation, delivery of nascent clusters from the cytosolic [Fe–S]-cluster assembly complex to client [Fe–S]-proteins involves an intermediate [Fe–S]-cluster carrier and the CIA Targeting Complex designated CTC (7, 8). A key observation ultimately leading to the discovery of a tripeptide motif in the CIA system, which is analogous to the ISC-associated LYR targeting signal, is that a Trp residue is located at the C-termini of several known CTC targets (9–11). Given that Trp is rarely found at the C terminus of proteins produced in nature, Marquez et al. (4) advanced these observations by performing an exhaustive comparative bioinformatic analysis of the C-terminal region of known or suspected [Fe–S]-proteins, including those found in the cytosol, nucleus, mitochondria, and in the Bacteria and Archaea. Remarkably, a signature tripeptide motif, designated the targeting complex recognition (TCR) signal, having Ile, Leu, or Met as the antepenultimate residue, Asp, Glu, or Ser as the penultimate residue, and Trp or Phe as the C-terminal residue was identified in a subpopulation of [Fe–S]-proteins produced in the cytoplasm or, in some cases, “adaptor” proteins associated with an [Fe–S]-protein produced in the cytoplasm. No such tripeptide TCR motif was similarly enriched in mitochondrial localized [Fe–S]-proteins, Bacterial [Fe–S]-proteins, or Archaeal [Fe–S]-proteins.

In PNAS, Marquez et al. describe an important aspect of how [Fe–S]-clusters are delivered to certain client [Fe–S]-proteins produced in the Eukaryotic cytosol.

The TCR motif model for guiding a population of immature client [Fe–S]-proteins to the CTC was tested using a series of elegant biochemical and physiological approaches. Of relevance was the demonstration that candidate proteins containing the TCR motif could be copurified with the heterotrimeric CTC whereas those having elements of the TCR motif removed could not. Complementary studies demonstrated that removal of the TCR motif from a model client protein reduced but did not eliminate either its corresponding activity or its associated physiological function. This observation raises the possibility that, although the TCR motif appears to play a dominant role in the targeting process, other sites or factors may be necessary to achieve maximum [Fe–S]-protein maturation. It was also shown that placement of a three-residue extension to the TCR targeting sequence abrogated its ability to recruit the CTC or to sustain efficient in vivo maturation of a model client protein. This line of experimentation indicated that the location of TCR at the C terminus is an important contributor to CTC recognition. Furthermore, [Fe–S]-cluster occupancy and activity of a model Bacterial [Fe–S]-protein complex that does not normally contain the TCR motif is improved when it is heterologously produced in the cytosol of Yeast and remodeled to contain the TCR motif. Thus, for a specific population of [Fe–S]-proteins produced in the cytosol, a TCR motif is both necessary and sufficient to assist CTC-directed client protein maturation.

Fig. 1 illustrates salient features of the end-stage process of cytosolic client [Fe–S]-protein maturation and provides a framework for highlighting remaining unknown aspects of the process. The CIA targeting complex provides a site for apo-client maturation by mediating [Fe–S]-cluster transfer from an [Fe–S]-cluster carrier protein to the apo-form of the client protein. For the class of identified targets, productive interaction of a client protein with the CTC involves a canonical tripeptide TCR motif located at the C terminus of the client protein. However, certain fundamental aspects of this process remain to be explored. First, the reciprocal site on CTC that interacts with the client-associated TCR motif has not been identified. Also, whether the [Fe–S]-cluster carrier protein delivers its associated [Fe–S]-cluster directly to an apo-client protein as part of a ternary complex that includes CTC, TCR, and the [Fe–S]-cluster carrier protein or whether the cluster is transiently delivered to the CTC by the [Fe–S]-cluster carrier protein and then transferred to the client protein remains an open question. Other intriguing aspects of [Fe–S]-protein maturation have also emerged. A similar approach described by Marquez et al. (4) combining bioinformatic, biochemical, and physiological approaches can now be applied to identify other determinants of client [Fe–S]-protein interactions with the CIA targeting complex and, perhaps, identification of heretofore unknown [Fe–S]-proteins. Also, because defects in the formation or activity of many [Fe–S]-proteins often have pathological consequences (2), it will be of great interest to determine whether substitutions within the targeting sequence of specific client [Fe–S]-proteins are similarly manifested by a disease state. Along these lines, it is particularly interesting that COVID RNA polymerase and helicase have been identified as [Fe–S]-proteins (12). Because viruses do not encode their own [Fe–S]-cluster biogenesis machineries, they must use CIA factors for their maturation thereby making elements necessary for viral-specific [Fe–S]-protein maturation-rich targets for therapeutic intervention. Finally, a demonstration that the maturation of a Bacterial [Fe–S]-protein produced in a Eukaryotic cytosol can be improved by the simple addition of a TCR motif could have practical merit in biotechnological efforts to produce, in Eukaryotes, robust Bacterial pathways having one or more [Fe–S]-proteins.