Biology subjects

Schlame, M.

Publications and source records attributed to Schlame, M..

5 recordsLinked to original sources

bioRxiv2026

Lipids are essential for potassium transport by KdpFABC from E. coli

KdpFABC is a hetero-tetrameric potassium pump that uses ATP to import potassium and thereby maintain homeostasis in bacteria under stress conditions. KdpA is a channel-like subunit with a selectivity filter that binds potassium from the periplasm. K+ then moves through a [~]40[A]-long intramembrane tunnel to reach a canonical binding site in KdpB. KdpB is a P-type ATPase that orchestrates conformational changes associated with the Post-Albers reaction cycle, involving E1 and E2 conformations and formation of an aspartyl phosphate intermediate as a way of coupling ATP hydrolysis to K+ transport. To elucidate the associated structural changes in a lipid environment, we reconstituted wild-type KdpFABC into lipid nanodiscs and used cryo-EM to image the complex under active turnover. The resulting six high resolution (2.1-2.7 [A]) structures provide new insight into the sequence of allosteric changes that produce (1) occlusion of K+ at the canonical binding site and (2) expulsion of K+ from this site and into a low-affinity release site. The structures also reveal two types of lipids bound to the complex. Specifically, two structural lipids bind at subunit interfaces and [~]20 annular lipids are seen at the periphery of the complex. In addition, we tested functional effects of mutations to residues at the KdpA/KdpB interface. ATPase and transport assays were used to document functional defects that reflect delipidation of structurally compromised complexes. We conclude that lipids play an integral role in structure and function of the KdpFABC complex. SignificanceKdpFABC uses ATP to transport potassium across the plasma membrane of E. coli. To further our understanding of its mechanism, we put purified KdpFABC molecules into membrane bilayers and used cryo-EM to capture structures during active transport. We have thereby produced structures representing all major states of the transport cycle with a high degree of precision. Analysis of these structures reveals new details about two key steps in this cycle and shows lipid molecules bound to the protein. We then introduced mutations at the interface between the two main subunits, which controls passage of potassium across the membrane. Activity measurements reveal how the protein depends on lipid to stabilize the structure and facilitate transport.

biophysics↗

bioRxiv2024

Upregulation of the AMPK-FOXO1-PDK4 pathway is a primary mechanism of pyruvate dehydrogenase activity reduction and leads to increased glucose uptake in tafazzin-deficient cells

Barth syndrome (BTHS) is a rare disorder caused by mutations in the TAFAZZIN gene. Previous studies from both patients and model systems have established metabolic dysregulation as a core component of BTHS pathology. In particular, features such as lactic acidosis, pyruvate dehydrogenase (PDH) deficiency, and aberrant fatty acid and glucose oxidation have been identified. However, the lack of a mechanistic understanding of what causes these conditions in the context of BTHS remains a significant knowledge gap, and this has hindered the development of effective therapeutic strategies for treating the associated metabolic problems. In the current study, we utilized tafazzin-knockout C2C12 mouse myoblasts (TAZ-KO) and cardiac and skeletal muscle tissue from tafazzin-knockout mice to identify an upstream mechanism underlying impaired PDH activity in BTHS. This mechanism centers around robust upregulation of pyruvate dehydrogenase kinase 4 (PDK4), resulting from hyperactivation of AMP-activated protein kinase (AMPK) and subsequent transcriptional upregulation by forkhead box protein O1 (FOXO1). Upregulation of PDK4 in tafazzin-deficient cells causes direct phospho-inhibition of PDH activity accompanied by increased glucose uptake and elevated intracellular glucose concentration. Collectively, our findings provide a novel mechanistic framework whereby impaired tafazzin function ultimately results in robust PDK4 upregulation, leading to impaired PDH activity and likely linked to dysregulated metabolic substrate utilization. This mechanism may underlie previously reported findings of BTHS-associated metabolic dysregulation.

cell biology↗

bioRxiv2023

Knockout of cardiolipin synthase disrupts postnatal cardiac development by inhibiting the maturation of mitochondrial cristae

BackgroundCardiomyocyte maturation requires a massive increase in respiratory enzymes and their assembly into long-lived complexes of oxidative phosphorylation (OXPHOS). The molecular mechanisms underlying the maturation of cardiac mitochondria have not been established. MethodsTo determine whether the mitochondria-specific lipid cardiolipin is involved in cardiac maturation, we created a cardiomyocyte-restricted knockout (KO) of cardiolipin synthase (Crls1) in mice and studied the postnatal development of the heart. We also measured the turnover rates of proteins and lipids in cardiolipin-deficient flight muscle from Drosophila, a tissue that has mitochondria with high OXPHOS activity like the heart. ResultsCrls1KO mice survived the prenatal period but failed to accumulate OXPHOS proteins during postnatal maturation and succumbed to heart failure at the age of 2 weeks. Turnover measurements showed that the exceptionally long half-life of OXPHOS proteins is critically dependent on cardiolipin. ConclusionsCardiolipin is essential for the postnatal maturation of cardiomyocytes because it allows mitochondrial cristae to accumulate OXPHOS proteins to a high concentration and to shield them from degradation.

developmental biology↗

bioRxiv2021

Structural basis for Potassium transport by KdpFABC

KdpFABC is an oligomeric K+ transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the Superfamily of K+ Transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryo-EM. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC and KdpF remain static. We observe a series of spherical densities that we assign as K+ or water and which define a pathway for K+ transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K+ transfer between alternative sites in the membrane domain of KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K+ to the cytoplasm.

biophysics↗

bioRxiv2020

An unexpected lack of difference in superoxide/H2O2 production rates in isolated heart and skeletal muscle mitochondria from a mouse model of Barth Syndrome

Barth Syndrome (BTHS) is a rare X-linked genetic disorder caused by mutations in tafazzin and characterized by loss of cardiolipin and severe cardiomyopathy. Mitochondrial superoxide/H2O2 production has been implicated in the cardiomyopathy observed in different BTHS models. There are at least 11 mitochondrial sites that produce superoxide/H2O2 at significant rates. Which of these sites generate oxidants at excessive rates in BTHS is unknown. Here, we measured the maximum capacity of superoxide/H2O2 production from each site in mitochondria isolated from heart and skeletal muscle of the tafazzin knockdown mice (tazkd) at 3, 7 and 12 months of age. Strikingly, the superoxide/H2O2 production capacities of these sites were overall indistinguishable between tazkd mice and their wildtype littermates across the time points analyzed. The only exception was site GQ in glycerol phosphate dehydrogenase, which was increased in the skeletal muscle of 7 months old tazkd mice. Mitochondrial superoxide/H2O2 production was also measured ex vivo during the oxidation of a complex mixture of substrates mimicking either heart or skeletal muscle cytosol and was found to be indistinguishable between wildtype and tazkd mice. However, we consistently measured decreased FAD-linked respiration in mitochondria isolated from tazkd mice. We conclude that the maximum capacity and ex vivo rates of superoxide/H2O2 production were not increased in mitochondria isolated from heart and skeletal muscle of tazkd mice, despite reduced oxidative capacity. Therefore, it seems unlikely that mitochondrial oxidants contribute to the development of cardiomyopathy in tazkd mice. These observations raise questions about the involvement of mitochondrial oxidants in BTHS pathology.

biochemistry↗