Like animals and plants, yeast cells contain structures called mitochondria. These structures are commonly referred to as the powerhouses of the cell because they provide much of the energy that cells need to survive. All mitochondria contain a protein called acyl carrier protein (ACP), which cells need in order to live. The ACP protein has a number of known roles including manufacturing the molecules that make up certain fats and helping to organise other proteins that are important for energy production. However, neither of these roles explain why yeast cells require ACP because the other proteins required for these processes are not required for survival.

Mitochondria are also the sites where iron and sulfur atoms are joined together to make the iron sulfur clusters that many proteins need in order to carry out their roles. Van Vranken, Jeong et al. now show that the ACP protein associates with a molecular machine that makes iron sulfur clusters in the mitochondria of budding yeast cells. The experiments show that this interaction is needed to produce iron sulfur clusters, and without it the other proteins involved in the process are not able to work together. Since iron sulfur clusters are essential for life, this could explain why cells cannot survive without ACP. Van Vranken et al. also showed that ACP is only able to efficiently produce iron sulfur clusters when a chemical called a “4-PP-conjugated acyl chain” is attached to it.

It is possible to separate the activity of ACP in making iron sulfur clusters from its previously known roles. Van Vranken et al. suggest that the addition of the 4-PP-conjugated acyl chain to ACP may help to balance the use of ACP between its different activities. Moving forward, Van Vranken et al. hope to determine the structure of ACP in more detail to understand how it contributes to iron sulfur cluster formation, and why this single protein has evolved to perform so many distinct roles.

The mitochondrial acyl carrier protein (ACP; Figure 1—figure supplement 1) plays a critical role in the evolutionarily conserved type II fatty acid biosynthesis pathway (FASII; Figure 1—figure supplement 2A). Unlike the cytosolic fatty acid biosynthesis pathway (FASI), the mitochondrial FASII system, which is homologous to the prokaryotic fatty acid biosynthesis pathway, utilizes a set of monofunctional enzymes that interact transiently with ACP to catalyze the initiation and elongation of nascent acyl chains (Hiltunen et al., 2010). To facilitate FASII, ACP utilizes a 4’-phosphopantetheine prosthetic group (4-PP), which is covalently bound to an invariant Ser residue (Majerus et al., 1965; Stuible et al., 1998). As such, ACP serves as a soluble scaffold for acyl intermediates during the stepwise process of de novo fatty acid synthesis. Currently, it is thought that the primary product of ACP-dependent FASII is octanoate, which is cleaved from ACP and further processed to generate lipoic acid. Lipoic acid is an obligate cofactor of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes as well as the branched chain α-keto acid dehydrogenase and glycine cleavage complex (Hiltunen et al., 2010; Brody et al., 1997). In addition, a FASII-derived acyl chain other than lipoic acid is required for RNase P function in tRNA maturation (Schonauer et al., 2008).

Biochemical analyses of mammalian FASII enzymes demonstrate that this pathway is capable of generating ACP-bound acyl chains as long as fourteen carbons (Zhang et al., 2005). Since lipoic acid biosynthesis requires an acyl chain of just eight carbons, it is likely that these extended FASII-synthesized fatty acids serve an alternative function in mitochondria (Brody et al., 1997). Indeed, proteomic and structural studies have demonstrated that ACP is a stable accessory subunit of mitochondrial respiratory Complex I (CI) (Sackmann et al., 1991; Angerer et al., 2014). Furthermore, the pool of ACP associated with CI contains a 4-PP-conjugated 3-hydroxymyristic acid, however, the functional importance of this 14-carbon acyl chain has never been investigated in the context of CI activity or assembly (Carroll et al., 2003).

In Saccharomyces cerevisiae, ACP1, the gene encoding ACP, is essential for cell viability in multiple strain backgrounds, while the genes required for lipoic acid biosynthesis and ligation are not (Figure 1A, Figure 1—figure supplement 2B,C) (Schonauer et al., 2008; Brody et al., 1997). This is consistent with reports demonstrating that ACP is essential for viability in Yarrowia lipolytica (Dobrynin et al., 2010) and in mammalian cells (Yi and Maeda, 2005; Feng et al., 2009). Moreover, S. cerevisiae mitochondria do not have CI or any structurally similar analog of it. Since neither known function explains the essentiality of ACP1, we reasoned that ACP must perform a distinct, unknown, and essential mitochondrial function.

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To begin to define the essential function of Acp1, we purified endogenously expressed and fully functional Acp1-HA (Figure 1A) from purified mitochondria to discover interacting proteins that might explain the acp1Δ phenotype. We were particularly intrigued by the co-purification of three subunits of the ISU complex—Nfs1, Isd11, and Isu1—each of which is required for FeS biogenesis and essential for viability. The cysteine desulfurase (Nfs1) and Isd11 form the core of the ISU complex and catalyze the conversion of cysteine to alanine thereby generating a persulfide intermediate, which is the source of sulfide ions that combine with ferrous iron on the Isu1 scaffold protein to form [2Fe-2S] clusters (Garland et al., 1999; Mühlenhoff et al., 2004; Adam et al., 2006; Wiedemann et al., 2006). To confirm these interactions, we immunoprecipitated Acp1-HA from isolated mitochondria and analyzed the eluates by immunoblot. Indeed, Nfs1, Isd11, and Isu1 all specifically co-immunoprecipitate with Acp1, although Isu1 appears to interact less avidly than Nfs1 or Isd11 (Figure 1B). In addition to SDS-PAGE, the resultant eluates were also resolved by blue native (BN)-PAGE, which demonstrated that Acp1 co-purifies with the intact core Nfs1-Isd11 complex (Figure 1B). Finally, Nfs1-V5 and Isd11-V5 were each immunoprecipitated from isolated mitochondria. As expected, Acp1-HA was detected in the eluates of each immunoprecipitation (Figure 1—figure supplement 3). These results are further supported by proteomics-based interaction studies, which identify human ACP, NFS1, and ISD11 as mutually interacting proteins in mammalian cells (Huttlin et al., 2015). Taken together these data demonstrate that Acp1 is a stable and evolutionarily conserved subunit of the ISU complex with Nfs1, Isd11, and Isu1.

FeS biogenesis is an essential function of mitochondria and is absolutely dependent on the ISU complex with which Acp1 stably interacts (Lill et al., 1999). Thus, we hypothesized that the essential function of Acp1 might relate to FeS biogenesis. We engineered inducible ACP1 knockdown strains (Acp1^KD) using two distinct strategies and strain backgrounds—TetO_7-ACP1 in the BY4741 background and Gal-ACP1 in the DY150 background, in which ACP1 expression is suppressed by doxycycline and galactose withdrawal, respectively. As expected, Acp1^KD cells from each background displayed attenuated growth upon ACP1 shutdown, which is particularly evident on respiration-requiring glycerol medium (Figure 2—figure supplement 1A–C). Importantly, viability could be restored in each of these strains by episomal expression of the Acp1-HA at endogenous levels. In addition to the expected loss of lipoic acid-containing subunits of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase in the Acp1^KD cells, we also observed a specific destabilization of the FeS-containing subunits of Complex II (Sdh2) and Complex III (Rip1) and loss of those assembled respiratory complexes (Figure 2A and Figure 2—figure supplement 2A). A similar destabilization of these complexes occurs in cells depleted for Nfs1 and Isu1 (Adam et al., 2006; Wiedemann et al., 2006). Importantly, Complex III biogenesis stalls in Acp1^KD cells at the final stage of assembly – incorporation of the Rieske FeS protein (Rip1) (Figure 2A; IB: Rip1)–resulting in the accumulation of a stable assembly intermediate of Complex III lacking Rip1 (Figure 2A; III₂*; IB: Qcr7). The presence of the late stage intermediate III₂* indicates that mitochondrial translation of the Cob cytochrome b subunit is normal in Acp1^KD cells (Atkinson et al., 2011; Cui et al., 2012). Likewise, translation of the mitochondrial subunits of ATP synthase is normal as seen by the assembled F₁F₀ complex (Figure 2A). We also observed a loss of activity of aconitase, a mitochondrial enzyme with an obligate FeS cofactor (Figure 2B and Figure 2—figure supplement 2B).

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Source data for Figure 2.

This file contains raw source data used to make the graphs presented in Figure 2B,C Figure 2—figure supplement 2B and C. PRISM software was used to graph all quantitative data and perform statistical analyses. p values for pairwise comparisons were determined using a Student’s t test.

https://doi.org/10.7554/eLife.17828.008

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The mitochondrial ISU complex is essential for the production of FeS that act in the cytosol as well as ribosome assembly (Kispal et al., 2005). In addition, mitochondrial FeS synthesis is important to attenuate the transcriptional activity of two partially redundant iron-responsive factors Aft1 and Aft2. In Acp1^KD cells the expression of Aft1-target genes FIT2 and FIT3 was elevated consistent with impaired mitochondrial FeS synthesis (Figure 2C) (Chen et al., 2004; Rutherford et al., 2005). These Aft1 target genes are also induced in cells lacking the two mitochondrial iron transporters Mrs3 and Mrs4 (Figure 2C). To further assess the perturbation of cytosolic FeS function, we quantified the activity of the cytosolic FeS-containing enzyme sulfite reductase and observed a diminution in Acp1^KD cells (Figure 2—figure supplement 2C). Combined with the essential nature of ACP1, these data demonstrate that Acp1 is essential for FeS biogenesis.

We next sought to define the mechanism underlying the observed necessity of Acp1 for FeS biogenesis, focusing specifically on the ISU complex. Acp1^KD cells exhibited a marked diminution of the assembled Nfs1-Isd11 complex, similar to depletion of the other ISU complex subunits (Figure 3A, Figure 3—figure supplement 1A,B). Importantly, the Nfs1 and Isd11 protein that remain in these cells is found only in insoluble aggregates in contrast to WT cells where Nfs1 and Isd11 are soluble (Figure 3B). Thus, in the absence of Acp1, the Nfs1-Isd11 complex is destabilized, most likely causing a loss of cysteine desulfurase activity. To determine if mammalian ACP is also necessary for maintaining steady state levels of the FeS biogenesis complex in mammalian cells, C2C12 mouse myoblasts were transfected with a pool of siRNAs targeting NDUFAB1 (the gene encoding mammalian ACP) or a control siRNA. While our methods were unable to detect the mammalian Nfs1-Isd11 complex by BN-PAGE, depletion of ACP in these cells was accompanied by a clear destabilization of the subunits of the mammalian FeS biogenesis machinery, NFS1, ISD11, and ISCU2, which is the mammalian version of Isu1 (Figure 3C). Thus, ACP plays an evolutionarily conserved role in stabilizing the ISU complex thereby enabling FeS biogenesis.

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Source data for Figure 3.

This file contains raw source data used to make the graphs presented in Figure 3—figure supplement 1C. PRISM software was used to graph all quantitative data and perform statistical analyses. p values for pairwise comparisons were determined using a Student’s t test.

https://doi.org/10.7554/eLife.17828.012

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Interestingly, overexpression of the MRS3 iron transporter resulted in robust stabilization of the Nfs1-Isd11 complex in Acp1^KD cells, while not restoring lipoic acid biosynthesis (Figure 3D). Elevated Mrs3 expression also restored Sdh2 and Rip1 protein abundance and complex assembly as well as aconitase activity in Acp1^KD cells (Figure 3D and Figure 3—figure supplement 1C). We tested whether overexpression of other components of the ISU complex stabilized the Nfs1-Isd11 complex in cells depleted of Acp1. Elevated levels of Isu1 yielded a modest stabilization and restored aconitase activity (Figure 3D and Figure 3—figure supplement 1C), while overexpression of Yfh1, the yeast frataxin homologue, had no effect (Figure 3—figure supplement 1D). We speculate that elevated Mrs3 may increase the Fe(II) occupancy of Isu1, which enables it to more effectively stabilize the ISU complex.

Acp1 requires a 4-PP prosthetic group to support mitochondrial fatty acid synthesis. The gene encoding 4-PP transferase, PPT2, is not essential, but the haploid deletion strain exhibits no growth in respiration-requiring medium and impaired growth on glucose, which does not require respiration (Figure 1—figure supplement 2C and Figure 4—figure supplement 1A). The growth impairment on glucose is not explained by any known function of ACP and therefore may relate to defects in FeS biogenesis.

To directly test the role of the 4-PP prosthetic group and the acyl chain that is conjugated to it, we investigated the ability of apo-Acp1 lacking 4-PP to support FeS biogenesis. Apo-Acp1 can be generated in vivo by mutating the invariant Ser (S82) to which 4-PP is conjugated (Stuible et al., 1998). While re-expression of WT Acp1 could fully restore the steady state abundance of the Nfs1-Isd11 complex in Acp1^KD cells, expression of Acp1^S82A had only modest effects (Figure 4A). These modest effects were sufficient to enable the Acp1^KD cells expressing Acp1^S82A to retain viability, albeit with impaired grow rate, and to exhibit a modest recovery of aconitase activity compared to Acp1^KD cells (Figure 4B, Figure 4—figure supplement 1B). Thus, the apparent absence of the Nfs1-Isd11 complex on BN gels is likely the result of a severely destabilized complex that is not capable of surviving the stringent detergent conditions of BN-PAGE and not the complete loss of the complex in vivo.

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Source data for Figure 4.

This file contains raw source data used to make the graphs presented in Figure 4B, Figure 4D, and Figure 4E. PRISM software was used to graph all quantitative data and perform statistical analyses. p values for pairwise comparisons were determined using a Student’s t test.

https://doi.org/10.7554/eLife.17828.015

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To further investigate the role of the 4-PP-conjugated acyl chain in FeS biogenesis we interrogated the effects of PPT2 deletion on the function of Acp1 in FeS biogenesis. Like Acp1^KD cells expressing Acp1^S82A, the Nfs1-Isd11 complex was severely depleted in ppt2Δ cells (Figure 4C). Furthermore, these cells exhibit a clear diminution in activity of FeS-containing enzymes in both the mitochondria and cytosol as represented by aconitase and sulfite reductase activity, respectively (Figure 4D,E). We also interrogated the ability of Acp1 to interact with Nfs1 in WT and ppt2Δ cells. While the steady state levels of Acp1 are not affected in ppt2Δ cells (Figure 4C), we observed a clear defect in the ability of Acp1 to interact with Nfs1 in this strain (Figure 4—figure supplement 1C). Therefore, the Acp1-conjugated 4-PP plays an important role in the interaction of Acp1 with the core Nfs1-Isd11 complex and in FeS biogenesis. Importantly, lip5Δ cells, which cannot synthesize lipoic acid but remain competent for Acp1-dependent fatty acid synthesis (Hiltunen et al., 2010), maintain normal abundance of the Nfs1-Isd11 complex and aconitase and sulfide reductase activity (Figure 4C–E). Thus, the defects in FeS biogenesis observed in cells expressing apo-Acp1 are a result of the inability to generate an acyl-conjugated Acp1 species and not a defect in lipoic acid biosynthesis.

The data presented herein define a new and unexpected role of ACP in FeS biogenesis. ACP functions as a stable subunit of the ISU complex where it acts to stabilize the complex in part by exploiting a 4-PP-conjugated acyl chain. Unlike ACP, however, the acyl chain is not absolutely required for FeS biogenesis and viability, which raises the intriguing possibility that ACP is not simply an obligate subunit, but may exploit this unique interaction modality to provide additional structural or regulatory functions on FeS biogenesis. It is particularly intriguing to speculate that ACP may serve to coordinate mitochondrial fatty acid synthesis and FeS biogenesis, which represent two critical biosynthetic processes performed by mitochondria.

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Author details

1. Jonathan G Van Vranken

   Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States

   Contribution

   JGV, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Mi-Young Jeong

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-8931-852X

2. Mi-Young Jeong

   1. Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States
   2. Department of Medicine, University of Utah School of Medicine, Salt Lake City, United States

   Contribution

   M-YJ, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Jonathan G Van Vranken

   Competing interests

   The authors declare that no competing interests exist.

3. Peng Wei

   Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States

   Contribution

   PW, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Yu-Chan Chen

   Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States

   Present address

   Department of Biology, Stanford University, Stanford, United States

   Contribution

   Y-CC, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

5. Steven P Gygi

   Department of Cell Biology, Harvard University School of Medicine, Boston, United States

   Contribution

   SPG, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

6. Dennis R Winge

   1. Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States
   2. Department of Medicine, University of Utah School of Medicine, Salt Lake City, United States

   Contribution

   DRW, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   dennis.winge@hsc.utah.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-1160-1189

7. Jared Rutter

   1. Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States
   2. Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, United States

   Contribution

   JR, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   rutter@biochem.utah.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-2710-9765

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