Search

Summary Type‐3 effectors (T3E) of phytopathogenic Gram‐negative bacteria fulfill a virulent role, causing disease, or an avirulent role, inducing immunity, following their translocation into plant cells. This study aimed to validate the hypothesis that bacterial T3E translocation requires lipidic compounds in plant cell membranes. Based on genetic, molecular, and biochemical assays, we determined that phosphatidylinositol 4‐phosphate (PI4P) associated with plant cell membranes is essential for the translocation of T3E by bacterial pathogens. Replicate experimental data revealed that PI4P cooperates with the type‐3 translocase HrpF to facilitate the translocation of effectors TAL and Xop from Xanthomonas oryzae and Hop from Pseudomonas syringae into the cells of Oryza sativa and Nicotiana benthamiana, respectively. Genetic and molecular analyses confirmed that, once translocated into plant cells, the distinct effectors induce disease or immunity. Combined genetic and pharmacological analyses revealed that when PI4P content is suppressed via genetic or pharmacological measures, the T3 effector translocation is considerably suppressed, resulting in serious inhibition of bacterial infection. Overall, these findings demonstrate that cooperative functioning of HrpF–PI4P is conserved in bacterial effectors and plants. [ABSTRACT FROM AUTHOR]

ABC toxin complexes are a class of protein toxin translocases comprised of a multimeric assembly of protein subunits. Each subunit displays a unique composition, contributing to the formation of a syringe-like nano-machine with natural cargo carrying, targeting, and translocation capabilities. Many of these toxins are insecticidal, drawing increasing interest in agriculture for use as biological pesticides. The A subunit (TcA) is the largest subunit of the complex and contains domains associated with membrane permeation and targeting. The B and C subunits, TcB and TcC, respectively, package into a cocoon-like structure that contains a toxic peptide and are coupled to TcA to form a continuous channel upon final assembly. In this review, we outline the current understanding and gaps in the knowledge pertaining to ABC toxins, highlighting seven published structures of TcAs and how these structures have led to a better understanding of the mechanism of host tropism and toxin translocation. We also highlight similarities and differences between homologues that contribute to variations in host specificity and conformational change. Lastly, we review the biotechnological potential of ABC toxins as both pesticides and cargo-carrying shuttles that enable the transport of peptides into cells. [ABSTRACT FROM AUTHOR]

ABC toxin complexes are a class of protein toxin translocases comprised of a multimeric assembly of protein subunits. Each subunit displays a unique composition, contributing to the formation of a syringe-like nano-machine with natural cargo carrying, targeting, and translocation capabilities. Many of these toxins are insecticidal, drawing increasing interest in agriculture for use as biological pesticides. The A subunit (TcA) is the largest subunit of the complex and contains domains associated with membrane permeation and targeting. The B and C subunits, TcB and TcC, respectively, package into a cocoon-like structure that contains a toxic peptide and are coupled to TcA to form a continuous channel upon final assembly. In this review, we outline the current understanding and gaps in the knowledge pertaining to ABC toxins, highlighting seven published structures of TcAs and how these structures have led to a better understanding of the mechanism of host tropism and toxin translocation. We also highlight similarities and differences between homologues that contribute to variations in host specificity and conformational change. Lastly, we review the biotechnological potential of ABC toxins as both pesticides and cargo-carrying shuttles that enable the transport of peptides into cells.

The infectious agent for African trypanosomiasis, Trypanosoma brucei, possesses a unique and essential translocase of the mitochondrial inner membrane, known as the TbTIM17 complex. TbTim17 associates with six small TbTims (TbTim9, TbTim10, TbTim11, TbTim12, TbTim13, and TbTim8/13). However, the interaction patterns of these smaller TbTims with each other and TbTim17 are not clear. Through yeast two-hybrid (Y2H) and co-immunoprecipitation analyses, we demonstrate that all six small TbTims interact with each other. Stronger interactions were found among TbTim8/13, TbTim9, and TbTim10. However, TbTim10 shows weaker associations with TbTim13, which has a stronger connection with TbTim17. Each of the small TbTims also interacts strongly with the C-terminal region of TbTim17. RNAi studies indicated that among all small TbTims, TbTim13 is most crucial for maintaining the steady-state levels of the TbTIM17 complex. Further analysis of the small TbTim complexes by size exclusion chromatography revealed that each small TbTim, except for TbTim13, is present in ~70 kDa complexes, possibly existing in heterohexameric forms. In contrast, TbTim13 is primarily present in the larger complex (>800 kDa) and co-fractionates with TbTim17. Altogether, our results demonstrate that, relative to other eukaryotes, the architecture and function of the small TbTim complexes are specific to T. brucei. [ABSTRACT FROM AUTHOR]

Critical for transcription initiation and bulky lesion DNA repair, TFIIH provides an exemplary system to connect molecular mechanisms to biological outcomes due to its strong genetic links to different specific human diseases. Recent advances in structural and computational biology provide a unique opportunity to re-examine biologically relevant molecular structures and develop possible mechanistic insights for the large dynamic TFIIH complex. TFIIH presents many puzzles involving how its two SF2 helicase family enzymes, XPB and XPD, function in transcription initiation and repair: how do they initiate transcription, detect and verify DNA damage, select the damaged strand for incision, coordinate repair with transcription and cell cycle through Cdk-activating-kinase (CAK) signaling, and result in very different specific human diseases associated with cancer, aging, and development from single missense mutations? By joining analyses of breakthrough cryo-electron microscopy (cryo-EM) structures and advanced computation with data from biochemistry and human genetics, we develop unified concepts and molecular level understanding for TFIIH functions with a focus on structural mechanisms. We provocatively consider that TFIIH may have first evolved from evolutionary pressure for TCR to resolve arrested transcription blocks to DNA replication and later added its key roles in transcription initiation and global DNA repair. We anticipate that this level of mechanistic information will have significant impact on thinking about TFIIH, laying a robust foundation suitable to develop new paradigms for DNA transcription initiation and repair along with insights into disease prevention, susceptibility, diagnosis and interventions.

Mycobacteria, such as the pathogen M. tuberculosis, utilize up to five paralogous type VII secretion systems to transport proteins across their cell envelope. Since these proteins associate in pairs that depend on each other for transport to a different extent, the secretion pathway to the bacterial surface remained challenging to address. Structural characterization of the inner-membrane embedded secretion machineries along with recent advances on the substrates' co-dependencies for transport allow for the first time more detailed and testable models for secretion. [ABSTRACT FROM AUTHOR]

Abstract Background The nuclease NurA and the ATPase/translocase HerA play a vital role in repair of double-strand breaks (DSB) during the homologous recombination in archaea. A NurA-HerA complex is known to mediate DSB DNA end resection, leading to formation of a free 3′ end used to search for the homologous sequence. Despite the structures of individual archaeal types of NurA and HerA having been reported, there is limited information regarding the molecular mechanisms underlying this process. Some bacteria also possess homologs of NurA and HerA; however, the bacterial type of this complex, as well as the detailed mechanisms underlying the joining of NurA-HerA in DSB DNA end resection, remains unclear. Results We report for the first time the crystal structures of Deinococcus radiodurans HerA (drHerA) in the nucleotide-free and ADP-binding modes. A D. radiodurans NurA-HerA complex structure was constructed according to a low-resolution cryo-electron microscopy map. We performed site-directed mutagenesis to map the drNurA-HerA interaction sites, suggesting that their interaction is mainly mediated by ionic links, in contrast to previously characterized archaeal NurA-HerA interactions. The key residues responsible for the DNA translocation activity, DNA unwinding activity, and catalytic activities of the drNurA-HerA complex were identified. A HerA/FtsK-specific translocation-related motif (TR motif) that guarantees the processivity of double-stranded DNA (dsDNA) translocation was identified. Moreover, a mechanism for the translocation-regulated resection of the 5′ tail of broken dsDNA and the corresponding generation of a recombinogenic 3′ single-stranded DNA tail by the drNurA-HerA complex was elucidated. Conclusions Our work provides new insights into the mechanism underlying bacterial NurA-HerA-mediated DSB DNA end resection, and the way this complex digests the 5′ tail of a DNA duplex and provides long 3′ free end for strand invasion in the bacterial homologous recombination process.

Background: The nuclease NurA and the ATPase/translocase HerA play a vital role in repair of double-strand breaks (DSB) during the homologous recombination in archaea. A NurA-HerA complex is known to mediate DSB DNA end resection, leading to formation of a free 3′ end used to search for the homologous sequence. Despite the structures of individual archaeal types of NurA and HerA having been reported, there is limited information regarding the molecular mechanisms underlying this process. Some bacteria also possess homologs of NurA and HerA; however, the bacterial type of this complex, as well as the detailed mechanisms underlying the joining of NurA-HerA in DSB DNA end resection, remains unclear. Results: We report for the first time the crystal structures of Deinococcus radiodurans HerA (drHerA) in the nucleotide-free and ADP-binding modes. A D. radiodurans NurA-HerA complex structure was constructed according to a low-resolution cryo-electron microscopy map. We performed site-directed mutagenesis to map the drNurA-HerA interaction sites, suggesting that their interaction is mainly mediated by ionic links, in contrast to previously characterized archaeal NurA-HerA interactions. The key residues responsible for the DNA translocation activity, DNA unwinding activity, and catalytic activities of the drNurA-HerA complex were identified. A HerA/FtsK-specific translocation-related motif (TR motif) that guarantees the processivity of double-stranded DNA (dsDNA) translocation was identified. Moreover, a mechanism for the translocation-regulated resection of the 5′ tail of broken dsDNA and the corresponding generation of a recombinogenic 3′ single-stranded DNA tail by the drNurA-HerA complex was elucidated. Conclusions: Our work provides new insights into the mechanism underlying bacterial NurA-HerA-mediated DSB DNA end resection, and the way this complex digests the 5′ tail of a DNA duplex and provides long 3′ free end for strand invasion in the bacterial homologous recombination process. [ABSTRACT FROM AUTHOR]

Summary: Mitochondrial diseases are a heterogeneous group of genetic disorders caused by pathogenic variants in genes encoding gene products that regulate mitochondrial function. These genes are located either in the mitochondrial or in the nuclear genome. The TOMM7 gene encodes a regulatory subunit of the translocase of outer mitochondrial membrane (TOM) complex that plays an essential role in translocation of nuclear-encoded mitochondrial proteins into mitochondria. We report an individual with a homozygous variant in TOMM7 (c.73T>C, p.Trp25Arg) that presented with a syndromic short stature, skeletal abnormalities, muscle hypotonia, microvesicular liver steatosis, and developmental delay. Analysis of mouse models strongly suggested that the identified variant is hypomorphic because mice homozygous for this variant showed a milder phenotype than those with homozygous Tomm7 deletion. These Tomm7 mutant mice show pathological changes consistent with mitochondrial dysfunction, including growth defects, severe lipoatrophy, and lipid accumulation in the liver. These mice die prematurely following a rapidly progressive weight loss during the last week of their lives. Tomm7 deficiency causes a unique alteration in mitochondrial function; despite the bioenergetic deficiency, mutant cells show increased oxygen consumption with normal responses to electron transport chain (ETC) inhibitors, suggesting that Tomm7 deficiency leads to an uncoupling between oxidation and ATP synthesis without impairing the function of the tricarboxylic cycle metabolism or ETC. This study presents evidence that a hypomorphic variant in one of the genes encoding a subunit of the TOM complex causes mitochondrial disease.

A cyclic hexapeptide, RA-VII isolated from the Rubiaceae family of plants, has high cytotoxic activity. Although RA-VII has been shown to inhibit protein synthesis in eukaryotic cells, the molecular mode of its action is not clear. Here we investigate the mechanism of the RAVII action on the translation apparatus. Biochemical functional assays showed that RA-VII inhibits poly(U)-dependent polyphenylalanine synthesis in the presence of animal elongation factors eEF1A and eEF2. Furthermore, RAVII prevented eEF2/ribosome-dependent GTPase activity, but not eEF-1A/ribosome-dependent activity. A filter binding assay demonstrated that RA-VII markedly enhances the binding affinity of eEF2 for GTP, but not for GDP, and prevents exchange of GTP in the eEF2-GTP complex, even after addition of a large excess of GTP/GDP. Limited proteolysis experiments indicated that RA-VII prevents the digestion of eEF2 in the presence of either GTP or GMPPCP, but not with GDP. Further footprint analysis and a translocation assay showed that the eEF2•GMPPNP•RA-VII complex binds to the conserved rRNA regions at the factor-binding center of the ribosome and retains the ability to translocate the A site-bound tRNA to the P-site. These results suggest that RA-VII tightly stabilizes the GTP•eEF2 complex structure, which is able to bind to the ribosomal functional site, but seems to suppress normal turnover of eEF2 after translocation. The properties of RA-VII make it a novel ligand for probing the action of eEF2 in the process of translocation on the ribosome. [Display omitted] • A cyclic hexapeptide, RA-VII inhibits translation elongation. • RA-VII inhibits eEF2/ribosome-dependent GTPase turnover. • RA-VII tightly stabilizes an eEF2.•GTP complex structure. • RA-VII-stabilized eEF2.•GTP retains ability to bind to the ribosomal functional site. • RA-VII seems to stall eEF2 on the ribosome. [ABSTRACT FROM AUTHOR]

FANCM is a multifunctional DNA repair enzyme that acts as a sensor and coordinator of replication stress responses, especially interstrand crosslink (ICL) repair mediated by the Fanconi anaemia (FA) pathway. Its specialised ability to bind and remodel branched DNA structures enables diverse genome maintenance activities. Through ATP-powered "branchpoint translocation", FANCM can promote fork reversal, facilitate replication traverse of ICLs, resolve deleterious R-loop structures, and restrain recombination. These remodelling functions also support a role as sensor of perturbed replication, eliciting checkpoint signalling and recruitment of downstream repair factors like the Fanconi anaemia FANCI:FANCD2 complex. Accordingly, FANCM deficiency causes chromosome fragility and cancer susceptibility. Other recent advances link FANCM to roles in gene editing efficiency and meiotic recombination, along with emerging synthetic lethal relationships, and targeting opportunities in ALT-positive cancers. Here we review key properties of FANCM's biochemical activities, with a particular focus on branchpoint translocation as a distinguishing characteristic. • FANCM is a DNA repair enzyme that resolves branched DNA structures. • ATP-powered branchpoint translocation enables fork reversal and crosslink traverse. • FANCM also restrains recombination and elicits checkpoint signaling. • FANCM deficiency causes fragile chromosomes, ICL sensitivity and cancer susceptibility. [ABSTRACT FROM AUTHOR]

The translocon SecYEG and the associated ATPase SecA form the primary protein secretion system in the cytoplasmic membrane of bacteria. The secretion is essentially dependent on the surrounding lipids, but the mechanistic understanding of their role in SecA : SecYEG activity is sparse. Here, we reveal that the unsaturated fatty acids (UFAs) of the membrane phospholipids, including tetraoleoyl‐cardiolipin, stimulate SecA : SecYEG‐mediated protein translocation up to ten‐fold. Biophysical analysis and molecular dynamics simulations show that UFAs increase the area per lipid and cause loose packing of lipid head groups, where the N‐terminal amphipathic helix of SecA docks. While UFAs do not affect the translocon folding, they promote SecA binding to the membrane, and the effect is enhanced up to fivefold at elevated ionic strength. Tight SecA : lipid interactions convert into the augmented translocation. Our results identify the fatty acid structure as a notable factor in SecA : SecYEG activity, which may be crucial for protein secretion in bacteria, which actively change their membrane composition in response to their habitat. [ABSTRACT FROM AUTHOR]

Ring ATPases perform a variety of tasks in the cell. Their function involves complex communication and coordination among the often identical subunits. Translocases in this group are of particular interest as they involve both chemical and mechanical actions in their operation. We study the DNA packaging motor of bacteriophage φ29, and using single-molecule optical tweezers and single-particle cryo-electron microscopy, have discovered a novel translocation mechanism for a molecular motor. [ABSTRACT FROM AUTHOR]

Archaebacterial and eukaryotic elongation factor 2 (EF-2) and bacterial elongation factor G (EF-G) are five domain GTPases that catalyze the ribosomal translocation of tRNA and mRNA. In the classical mechanism of activation, GTPases are switched on through GDP/GTP exchange, which is accompanied by the ordering of two flexible segments called switch I and II. However, crystal structures of EF-2 and EF-G have thus far not revealed the conformations required by the classical mechanism. Here, we describe crystal structures of Methanoperedens nitroreducens EF-2 (MnEF-2) and MnEF-2-H595N bound to GMPPCP (GppCp) and magnesium displaying previously unreported compact conformations. Domain III forms interfaces with the other four domains and the overall conformations resemble that of SNU114, the eukaryotic spliceosomal GTPase. The gamma phosphate of GMPPCP is detected through interactions with switch I and a P-loop structural element. Switch II is highly ordered whereas switch I shows a variable degree of ordering. The ordered state results in a tight interdomain arrangement of domains I-III and the formation of a portion of a predicted monovalent cation site involving the P-loop and switch I. The side chain of an essential histidine residue in switch II is placed in the inactive conformation observed for the “on” state of elongation factor EF-Tu. The compact conformations of MnEF-2 and MnEF-2-H595N suggest an “on” ribosome-free conformational state.

Summary: Protein machines undergo conformational motions to interact with and manipulate polymeric substrates. The Sec translocase promiscuously recognizes, becomes activated, and secretes >500 non-folded preprotein clients across bacterial cytoplasmic membranes. Here, we reveal that the intrinsic dynamics of the translocase ATPase, SecA, and of preproteins combine to achieve translocation. SecA possesses an intrinsically dynamic preprotein clamp attached to an equally dynamic ATPase motor. Alternating motor conformations are finely controlled by the γ-phosphate of ATP, while ADP causes motor stalling, independently of clamp motions. Functional preproteins physically bridge these independent dynamics. Their signal peptides promote clamp closing; their mature domain overcomes the rate-limiting ADP release. While repeated ATP cycles shift the motor between unique states, multiple conformationally frustrated prongs in the clamp repeatedly “catch and release” trapped preprotein segments until translocation completion. This universal mechanism allows any preprotein to promiscuously recognize the translocase, usurp its intrinsic dynamics, and become secreted.

Autosomal recessive mutations in the PINK1 gene are causal for Parkinson's disease (PD). PINK1 encodes a mitochondrial localized protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function; however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain is unknown. We have employed mutagenesis studies to define the minimal region of human PINK1 required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal α-helical extension (NTE) domain forming an intramolecular interaction with the C-terminal extension (CTE), which we corroborate using hydrogen/deuterium exchange mass spectrometry of recombinant insect PINK1 protein. Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE : CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228) and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE and CTE domain mutants disrupt PINK1 stabilization at the mitochondrial Translocase of outer membrane complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE : CTE interface towards PINK1 stabilization and activation and show that loss of NTE : CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.

Dexamethasone augments mitochondrial protein abundance. The translocase of the outer membrane (Tom) of mitochondria plays a major role in importing largely cytosolically synthesized proteins into mitochondria. We hypothesize that dexamethasone upregulates the Tom transport system, leading to increase of mitochondrial protein localization. Tom20 and Tom70 are the two major subunits. Dexamethasone increased Tom20 and Tom70 mRNA levels by 53 ± 11% and 25 ± 9% and mitochondrial protein abundance by 27 ± 7% and 25 ± 4% (p < 0.05 for all), respectively, in HEK293 cells. In parallel, dexamethasone elevated the SGK1 mRNA by 79 ± 17% and activity by 190 ± 42%, and mitochondrial protein level by 41 ± 2% (all p < 0.05) without significantly affecting the cytosol counterpart. The discovery of the effect of dexamethasone on SGK1 protein restricted in the mitochondria attracted us to examine the effect of the hormone on MnSOD, an enzyme with known mitochondrial localization and function. Similarly, dexamethasone significantly increased MnSOD transcripts by 67 ± 15% and protein level only in the mitochondria dose-dependently. Inhibition of SGK1 by GSK650394 and RNAi significantly attenuated the effects of the hormone on Tom20, Tom70, and MnSOD, indicating that SGK1 relays the effects of dexamethasone. Catalase inhibited the effects of dexamethasone on SGK1 and the subsequent effects of SGK1 on Tom20, Tom70, and MnSOD. Finally, knock-down of Tom20 and Tom70 by their siRNAs reduced dexamethasone-induced increases in the mitochondrial localization of SGK1 and MnSOD proteins. In conclusion, dexamethasone upregulates Tom20, Tom70, and MnSOD, and these effects are dependent on reactive oxygen species and SGK1. Dexamethasone-induced increases of SGK1 and MnSOD mitochondrial localization requires Tom20 and Tom70. [ABSTRACT FROM AUTHOR]

Background: The translocase of the mitochondrial inner membrane (TIM) imports most of the nucleus‐encoded proteins that are destined for the matrix, inner membrane (IM) and the intermembrane space (IMS). Trypanosoma brucei, the infectious agent for African trypanosomiasis, possesses a unique TIM complex consisting of several novel proteins in association with a relatively conserved protein TbTim17. Tandem affinity purification of the TbTim17 protein complex revealed TbTim54 as a potential component of this complex. Results: TbTim54, a trypanosome‐specific IMS protein, is peripherally associated with the IM and is present in a protein complex slightly larger than the TbTim17 complex. TbTim54 knockdown (KD) reduced the import of TbTim17 and compromised the integrity of the TbTim17 complex. TbTim54 KD inhibited the in vitro mitochondrial import and assembly of the internal signal‐containing mitochondrial carrier proteins MCP3, MCP5 and MCP11 to a greater extent than TbTim17 KD. Furthermore, TbTim54 KD, but not TbTim17 KD, significantly hampered the mitochondrial targeting of ectopically expressed MCP3 and MCP11. These observations along with our previous finding that the mitochondrial import of N‐terminal signal‐containing proteins like cytochrome oxidase subunit 4 and MRP2 was affected to a greater extent by TbTim17 KD than TbTim54 KD indicating a substrate‐specificity of TbTim54 for internal‐signal containing mitochondrial proteins. In other organisms, small Tim chaperones in the IMS are known to participate in the translocation of MCPs. We found that TbTim54 can directly interact with at least two of the six known small TbTim proteins, TbTim11 and TbTim13, as well as with the N‐terminal domain of TbTim17. Conclusion: TbTim54 interacts with TbTim17. It also plays a crucial role in the mitochondrial import and complex assembly of internal signal‐containing IM proteins in T. brucei. Significance: We are the first to characterise TbTim54, a novel TbTim that is involved primarily in the mitochondrial import of MCPs and TbTim17 in T. brucei. [ABSTRACT FROM AUTHOR]

Protein translocation is a highly dynamic process and, in addition, mitochondrial protein import is especially complicated as the majority of nuclear encoded precursor proteins must engage with multiple translocases before they are assembled in the correct mitochondrial subcompartment. In this chapter, we describe assays for engineered disulfide bond formation and cysteine specific crosslinking to analyze the rearrangement of translocase subunits or to probe protein-protein interactions between precursor proteins and translocase subunits. Such assays were used to characterize the translocase of the outer membrane, the presequence translocase of the inner membrane and the sorting and assembly machinery for the biogenesis of β-Barrel proteins. Moreover, these approaches were also employed to determine the translocation path of precursor proteins (identification of import receptors and specific domains required for translocation) as well as the analysis, location and translocase subunit dependence for the formation of β-Barrel proteins. Here we describe how engineered disulfide bond formation and cysteine specific crosslinking assays are planned and performed and discuss important aspects for its application to study mitochondrial protein translocation., (Copyright © 2024. Published by Elsevier Inc.)

Mitochondrial protein import is a complex process governing the delivery of the organelle's proteome. This process, in turn, is essential for maintaining mitochondrial function and cellular homeostasis. Initiated by protein synthesis in the cytoplasm, precursor proteins destined for the mitochondria possess targeting signals that guide them to the mitochondrial surface. At mitochondria, the translocation of proteins across the mitochondrial membranes involves an intricate interplay between translocases, chaperones, and receptors. The mitochondrial import assay offers researchers the opportunity to recapitulate the process of protein import in vitro. The assay has served as an indispensable tool in helping decipher the intricacies of protein translocation into mitochondria, first in fungal models, and subsequently in higher eukaryotic models. In this chapter, we will describe how protein import can be assayed using mammalian mitochondria and provide insight into the types of questions that can be addressed in mammalian mitochondrial biology using this experimental approach., (Copyright © 2024. Published by Elsevier Inc.)

The past several decades have witnessed tremendous growth in the protein targeting, transport and translocation field. Major advances were made during this time period. Now the molecular details of the targeting factors, receptors and the membrane channels that were envisioned in Blobel's Signal Hypothesis in the 1970s have been revealed by powerful structural methods. It is evident that there is a myriad of cytosolic and membrane associated systems that accurately sort and target newly synthesized proteins to their correct membrane translocases for membrane insertion or protein translocation. Here we will describe the common principles for protein transport in prokaryotes and eukaryotes. [ABSTRACT FROM AUTHOR]

The mitochondrial ATP‐Mg/Pi carrier is responsible for the calcium‐dependent regulation of adenosine nucleotide concentrations in the mitochondrial matrix, which allows mitochondria to respond to changing energy requirements of the cell. The carrier is expressed in mitochondria of fungi, plants and animals and belongs to the family of mitochondrial carriers. The carrier is unusual as it consists of three separate domains: (i) an N‐terminal regulatory domain with four calcium‐binding EF‐hands similar to calmodulin, (ii) a loop domain containing an amphipathic α‐helix and (iii) a mitochondrial carrier domain related to the mitochondrial ADP/ATP carrier. This striking example of three domains coming together from different origins to provide new functions represents an interesting quirk of evolution. In this review, we outline how the carrier was identified and how its physiological role was established with a focus on human isoforms. We exploit the sequence and structural information of the domains to explore the similarities and differences to their closest counterparts; mitochondrial ADP/ATP carriers and proteins with four EF‐hands. We discuss how their combined function has led to a mechanism for calcium‐regulated transport of adenosine nucleotides. Finally, we compare the ATP‐Mg/Pi carrier with the mitochondrial aspartate/glutamate carrier, the only other mitochondrial carrier regulated by calcium, and we will argue that they have arisen by convergent rather than divergent evolution. © 2018 The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(12):1222–1232, 2018 [ABSTRACT FROM AUTHOR]

DNA helicases are a class of molecular motors that catalyze processive unwinding of double stranded DNA. In spite of much study, we know relatively little about the mechanisms by which these enzymes carry out the function for which they are named. Most current views are based on inferences from crystal structures. A prominent view is that the canonical ATPase motor exerts a force on the ssDNA resulting in “pulling” the duplex across a “pin” or “wedge” in the enzyme leading to a mechanical separation of the two DNA strands. In such models, DNA base pair separation is tightly coupled to ssDNA translocation of the motors. However, recent studies of the Escherichia coli RecBCD helicase suggest an alternative model in which DNA base pair melting and ssDNA translocation occur separately. In this view, the enzyme‐DNA binding free energy is used to melt multiple DNA base pairs in an ATP‐independent manner, followed by ATP‐dependent translocation of the canonical motors along the newly formed ssDNA tracks. Repetition of these two steps results in processive DNA unwinding. We summarize recent evidence suggesting this mechanism for RecBCD helicase action. [ABSTRACT FROM AUTHOR]

DNA translocases are a diverse group of molecular motors responsible for a wide variety of cellular functions. The goal of this review is to identify common aspects in the mechanisms for how these enzymes couple the binding and hydrolysis of ATP to their movement along DNA. Not surprisingly, the shared structural components contained within the catalytic domains of several of these motors appear to give rise to common aspects of DNA translocation. Perhaps more interesting, however, are the differences between the families of translocases and the potential associated implications both for the functions of the members of these families and for the evolution of these families. However, as there are few translocases for which complete characterizations of the mechanisms of DNA binding, DNA translocation, and DNA-stimulated ATPase have been completed, it is difficult to form many inferences. We therefore hope that this review motivates the necessary further experimentation required for broader comparisons and conclusions.