Jonathan G. Van Vranken

A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, Drosophila, and Humans

Letting Pyruvate In Transport of pyruvate is an important event in metabolism whereby the pyruvate formed in glycolysis is transported into mitochondria to feed into the tricarboxylic acid cycle (see the Perspective by Murphy and Divakaruni). Two groups have now identified proteins that are components of the mitochondrial pyruvate transporter. Bricker et al. (p. 96, published online 24 May) found that the proteins mitochondrial pyruvate carrier 1 and 2 (MPC1 and MPC2) are required for full pyruvate transport in yeast and Drosophila cells and that humans with mutations in MPC1 have metabolic defects consistent with loss of the transporter. Herzig et al. (p. 93, published online 24 May) identified the same proteins as components of the carrier in yeast. Furthermore, expression of the mouse proteins in bacteria conferred increased transport of pyruvate into bacterial cells. The genes encoding two components of the pyruvate transporter in mitochondria have been identified. Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast, Drosophila, and humans. Mpc1 and Mpc2 associate to form an ~150-kilodalton complex in the inner mitochondrial membrane. Yeast and Drosophila mutants lacking MPC1 display impaired pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, and silencing of MPC1 or MPC2 in mammalian cells impairs pyruvate oxidation. A point mutation in MPC1 provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Human genetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemia revealed a causal locus that mapped to MPC1, changing single amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of the mitochondrial pyruvate carrier.

A role for the mitochondrial pyruvate carrier as a repressor of the Warburg effect and colon cancer cell growth.

Cancer cells are typically subject to profound metabolic alterations, including the Warburg effect wherein cancer cells oxidize a decreased fraction of the pyruvate generated from glycolysis. We show herein that the mitochondrial pyruvate carrier (MPC), composed of the products of the MPC1 and MPC2 genes, modulates fractional pyruvate oxidation. MPC1 is deleted or underexpressed in multiple cancers and correlates with poor prognosis. Cancer cells re-expressing MPC1 and MPC2 display increased mitochondrial pyruvate oxidation, with no changes in cell growth in adherent culture. MPC re-expression exerted profound effects in anchorage-independent growth conditions, however, including impaired colony formation in soft agar, spheroid formation, and xenograft growth. We also observed a decrease in markers of stemness and traced the growth effects of MPC expression to the stem cell compartment. We propose that reduced MPC activity is an important aspect of cancer metabolism, perhaps through altering the maintenance and fate of stem cells.

The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis

Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4’-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis. DOI: http://dx.doi.org/10.7554/eLife.17828.001

Structure of human Fe–S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP–ISD11 interactions

Significance Prokaryotic and eukaryotic organisms use analogous pathways to synthesize protein cofactors called iron–sulfur clusters. An unexplained difference between pathways is the functional requirements of the respective cysteine desulfurases. In eukaryotes, the cysteine desulfurase NFS1 requires additional accessory subunits for function. The lack of structural information has limited mechanistic insight into the role of these accessory proteins in mitochondrial Fe–S cluster biosynthesis. Here we determined crystallographic and electron microscopic structures of the NFS1–ISD11–ACP subcomplex. These results reveal an unexpected cysteine desulfurase architecture that reconciles mechanistic differences between the prokaryotic and eukaryotic systems, reveals the basis of control of iron–sulfur cluster assembly through fatty acid synthesis, and serves as a structural foundation for investigating human diseases related to iron–sulfur cluster assembly. In eukaryotes, sulfur is mobilized for incorporation into multiple biosynthetic pathways by a cysteine desulfurase complex that consists of a catalytic subunit (NFS1), LYR protein (ISD11), and acyl carrier protein (ACP). This NFS1–ISD11–ACP (SDA) complex forms the core of the iron–sulfur (Fe–S) assembly complex and associates with assembly proteins ISCU2, frataxin (FXN), and ferredoxin to synthesize Fe–S clusters. Here we present crystallographic and electron microscopic structures of the SDA complex coupled to enzyme kinetic and cell-based studies to provide structure-function properties of a mitochondrial cysteine desulfurase. Unlike prokaryotic cysteine desulfurases, the SDA structure adopts an unexpected architecture in which a pair of ISD11 subunits form the dimeric core of the SDA complex, which clarifies the critical role of ISD11 in eukaryotic assemblies. The different quaternary structure results in an incompletely formed substrate channel and solvent-exposed pyridoxal 5′-phosphate cofactor and provides a rationale for the allosteric activator function of FXN in eukaryotic systems. The structure also reveals the 4′-phosphopantetheine–conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, explaining the basis of ACP stabilization. The unexpected architecture for the SDA complex provides a framework for understanding interactions with acceptor proteins for sulfur-containing biosynthetic pathways, elucidating mechanistic details of eukaryotic Fe–S cluster biosynthesis, and clarifying how defects in Fe–S cluster assembly lead to diseases such as Friedreich’s ataxia. Moreover, our results support a lock-and-key model in which LYR proteins associate with acyl-ACP as a mechanism for fatty acid biosynthesis to coordinate the expression, Fe–S cofactor maturation, and activity of the respiratory complexes.

Protein-mediated assembly of succinate dehydrogenase and its cofactors

Abstract Succinate dehydrogenase (or complex II; SDH) is a heterotetrameric protein complex that links the tribarboxylic acid cycle with the electron transport chain. SDH is composed of four nuclear-encoded subunits that must translocate independently to the mitochondria and assemble into a mature protein complex embedded in the inner mitochondrial membrane. Recently, it has become clear that failure to assemble functional SDH complexes can result in cancer and neurodegenerative syndromes. The effort to thoroughly elucidate the SDH assembly pathway has resulted in the discovery of four subunit-specific assembly factors that aid in the maturation of individual subunits and support the assembly of the intact complex. This review will focus on these assembly factors and assess the contribution of each factor to the assembly of SDH. Finally, we propose a model of the SDH assembly pathway that incorporates all extant data.

Vms1p is a release factor for the ribosome-associated quality control complex

Eukaryotic cells employ the ribosome-associated quality control complex (RQC) to maintain homeostasis despite defects that cause ribosomes to stall. The RQC comprises the E3 ubiquitin ligase Ltn1p, the ATPase Cdc48p, Rqc1p, and Rqc2p. Upon ribosome stalling and splitting, the RQC assembles on the 60S species containing unreleased peptidyl-tRNA (60S:peptidyl–tRNA). Ltn1p and Rqc1p facilitate ubiquitination of the incomplete nascent chain, marking it for degradation. Rqc2p stabilizes Ltn1p on the 60S and recruits charged tRNAs to the 60S to catalyze elongation of the nascent protein with carboxy-terminal alanine and threonine extensions (CAT tails). By mobilizing the nascent chain, CAT tailing can expose lysine residues that are hidden in the exit tunnel, thereby supporting efficient ubiquitination. If the ubiquitin–proteasome system is overwhelmed or unavailable, CAT-tailed nascent chains can aggregate in the cytosol or within organelles like mitochondria. Here we identify Vms1p as a tRNA hydrolase that releases stalled polypeptides engaged by the RQC.The ribosome-associated quality control complex (RQC) functions to disassemble stalled ribosomes. Here the authors find that the tRNA hydrolase Vms1 is involved in the release of nascent peptide from stalled ribosomes.

ACP Acylation Is an Acetyl-CoA-Dependent Modification Required for Electron Transport Chain Assembly

The electron transport chain (ETC) is an important participant in cellular energy conversion, but its biogenesis presents the cell with numerous challenges. To address these complexities, the cell utilizes ETC assembly factors, which include the LYR protein family. Each member of this family interacts with the mitochondrial acyl carrier protein (ACP), the scaffold protein upon which the mitochondrial fatty acid synthesis (mtFAS) pathway builds fatty acyl chains from acetyl-CoA. We demonstrate that the acylated form of ACP is an acetyl-CoA-dependent allosteric activator of the LYR protein family used to stimulate ETC biogenesis. By tuning ETC assembly to the abundance of acetyl-CoA, which is the major fuel of the TCA cycle and ETC, this system could provide an elegant mechanism for coordinating the assembly of ETC complexes with one another and with substrate availability.

Structure of human Fe–S assembly sub-complex

aDepartment of Chemistry, Texas A&M University bDepartment of Biochemistry, University of Utah School of Medicine cDepartment of Chemistry, Massachusetts Institute of Technology dHoward Hughes Medical Institute, Massachusetts Institute of Technology eDepartment of Medicine, University of Utah School of Medicine fDepartment of Biology, Massachusetts Institute of Technology gHoward Hughes Medical Institute, University of Utah School of Medicine