Manuel Fischer

Protein import and oxidative folding in the mitochondrial intermembrane space of intact mammalian cells

Oxidative folding facilitates protein import into the mitochondrial intermembrane space. An analysis of the process in intact mammalian cells reveals the contributions of Mia40, ALR, glutathione, and the membrane potential. Proteins that rely on oxidative folding remain stable and reduced in the cytosol for several minutes.

The Ca2+-Dependent Release of the Mia40-Induced MICU1-MICU2 Dimer from MCU Regulates Mitochondrial Ca2+ Uptake

The essential oxidoreductase Mia40/CHCHD4 mediates disulfide bond formation and protein folding in the mitochondrial intermembrane space. Here, we investigated the interactome of Mia40 thereby revealing links between thiol-oxidation and apoptosis, energy metabolism, and Ca(2+) signaling. Among the interaction partners of Mia40 is MICU1-the regulator of the mitochondrial Ca(2+) uniporter (MCU), which transfers Ca(2+) across the inner membrane. We examined the biogenesis of MICU1 and find that Mia40 introduces an intermolecular disulfide bond that links MICU1 and its inhibitory paralog MICU2 in a heterodimer. Absence of this disulfide bond results in increased receptor-induced mitochondrial Ca(2+) uptake. In the presence of the disulfide bond, MICU1-MICU2 heterodimer binding to MCU is controlled by Ca(2+) levels: the dimer associates with MCU at low levels of Ca(2+) and dissociates upon high Ca(2+) concentrations. Our findings support a model in which mitochondrial Ca(2+) uptake is regulated by a Ca(2+)-dependent remodeling of the uniporter complex.

Oxidation-driven protein import into mitochondria: Insights and blind spots☆

The intermembrane space of mitochondria contains a dedicated machinery for the introduction of disulfide bonds into proteins. In this case, oxidative protein folding is believed to drive the vectorial translocation of polypeptides after their synthesis in the cytosol across the mitochondrial outer membrane. Substrates of this system are recognized by a hydrophobic binding cleft of the oxidoreductase Mia40 which converts them into an oxidized stably folded conformation. Mia40 is maintained in an oxidized, active conformation by the sulfhydryl oxidase Erv1, a homodimeric flavoenzyme, which can form disulfide bonds de novo. Erv1 passes electrons on to cytochrome c and further to the respiratory chain. The components of this system, their structures and the mechanisms of disulfide bond formation were analyzed only very recently. This review discusses our knowledge about this system as well as open questions which still wait to be addressed. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

The mitochondrial disulfide relay system: roles in oxidative protein folding and beyond.

Disulfide bond formation drives protein import of most proteins of the mitochondrial intermembrane space (IMS). The main components of this disulfide relay machinery are the oxidoreductase Mia40 and the sulfhydryl oxidase Erv1/ALR. Their precise functions have been elucidated in molecular detail for the yeast and human enzymes in vitro and in intact cells. However, we still lack knowledge on how Mia40 and Erv1/ALR impact cellular and organism physiology and whether they have functions beyond their role in disulfide bond formation. Here we summarize the principles of oxidation-dependent protein import mediated by the mitochondrial disulfide relay. We proceed by discussing recently described functions of Mia40 in the hypoxia response and of ALR in influencing mitochondrial morphology and its importance for tissue development and embryogenesis. We also include a discussion of the still mysterious function of Erv1/ALR in liver regeneration.

Engineering fungal de novo fatty acid synthesis for short chain fatty acid production

Fatty acids (FAs) are considered strategically important platform compounds that can be accessed by sustainable microbial approaches. Here we report the reprogramming of chain-length control of Saccharomyces cerevisiae fatty acid synthase (FAS). Aiming for short-chain FAs (SCFAs) producing bakers yeast, we perform a highly rational and minimally invasive protein engineering approach that leaves the molecular mechanisms of FASs unchanged. Finally, we identify five mutations that can turn bakers yeast into a SCFA producing system. Without any further pathway engineering, we achieve yields in extracellular concentrations of SCFAs, mainly hexanoic acid (C6-FA) and octanoic acid (C8-FA), of 464 mg l−1 in total. Furthermore, we succeed in the specific production of C6- or C8-FA in extracellular concentrations of 72 and 245 mg l−1, respectively. The presented technology is applicable far beyond bakers yeast, and can be plugged into essentially all currently available FA overproducing microorganisms.

Human copper chaperone for superoxide dismutase 1 mediates its own oxidation-dependent import into mitochondria

Oxidative stress is counteracted by various cellular systems, including copper-zinc superoxide dismutase 1 (SOD1) and its activating chaperone, that is, the copper chaperone for SOD1 (CCS1). Both enzymes are structurally related, and both localize to the cytosol and the mitochondrial intermembrane space where they specifically counteract mitochondria-derived superoxide. The mechanism by which human CCS1 is transported into mitochondria is largely unclear. Here we show that CCS1 import depends on the presence of mature CCS1 in the mitochondria. During import, a disulphide bond is formed in CCS1 in a CCS1-dependent reaction. We demonstrate that oxidation and import depend on the presence of cysteine residues at positions 227 and 141/144 in CCS1. Notably, CCS1 import parallels SOD1 import that also depends on CCS1. Our observations suggest that CCS1 serves as a specialized import receptor in mitochondria that facilitates the import and folding of SOD1 and CCS1, thereby extending the substrate spectrum of oxidation-dependent protein import in the mitochondrial intermembrane space.

Cryo‐EM structure of fatty acid synthase (FAS) from Rhodosporidium toruloides provides insights into the evolutionary development of fungal FAS

Fungal fatty acid synthases Type I (FAS I) are up to 2.7 MDa large molecular machines composed of large multifunctional polypeptides. Half of the amino acids in fungal FAS I are involved in structural elements that are responsible for scaffolding the elaborate barrel‐shaped architecture and turning fungal FAS I into highly efficient de novo producers of fatty acids. Rhodosporidium toruloides is an oleaginous fungal species and renowned for its robust conversion of carbohydrates into lipids to over 70% of its dry cell weight. Here, we use cryo‐EM to determine a 7.8‐Å reconstruction of its FAS I that reveals unexpected features; its novel form of splitting the multifunctional polypeptide chain into the two subunits α and β, and its duplicated ACP domains. We show that the specific distribution into α and β occurs by splitting at one of many possible sites that can be accepted by fungal FAS I. While, therefore, the specific distribution in α and β chains in R. toruloides FAS I is not correlated to increased protein activities, we also show that the duplication of ACP is an evolutionary late event and argue that duplication is beneficial for the lipid overproduction phenotype.

Molecular mechanisms in fungal fatty acid synthase (FAS) assembly

The fungal fatty acid synthase (fFAS) multienzyme is a barrel-shaped 2.6 MDa complex comprising six times eight catalytic domains. Upon barrel-formation, up to several hundred kDa large polypeptides intertwine to bury about 170,000 Å2 of protein surface. Functional, regulatory and structural data as well as evolutionary aspects of fFAS have been elucidated during the last decades. Notwithstanding a profound knowledge of this protein family, the biogenesis of the elaborate structure remained elusive. Remarkably, experimental data have recently demonstrated that fFAS self-assembles without the assistance of specific factors. Considering the infinitesimal probability that the barrel-shaped complex forms simply by domains approaching in the correct orientation, we were interested in understanding the sequence of events that have to orchestrate fFAS assembly. Here, we show that fFAS attains its quaternary structure along a pathway of successive domain-domain interactions, which is strongly related to the evolutionary development of this protein family. The knowledge on fFAS assembly may pave the way towards antifungal therapy, and further develops fFAS as biofactory in technological applications.

Strategies in megasynthase engineering – fatty acid synthases (FAS) as model proteins

Megasynthases are large multienzyme proteins that produce a plethora of important natural compounds by catalyzing the successive condensation and modification of precursor units. Within the class of megasynthases, polyketide synthases (PKS) are responsible for the production of a large spectrum of bioactive polyketides (PK), which have frequently found their way into therapeutic applications. Rational engineering approaches have been performed during the last 25 years that seek to employ the “assembly-line synthetic concept” of megasynthases in order to deliver new bioactive compounds. Here, we highlight PKS engineering strategies in the light of the newly emerging structural information on megasynthases, and argue that fatty acid synthases (FAS) are and will be valuable objects for further developing this field.