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Carnitine-acylcarnitine translocase (CACT) deficiency is a rare long-chain fatty acid oxidation disorder (LC-FAOD) with high mortality due to cardiomyopathy or lethal arrhythmia. Triheptanoin (UX007), an investigational drug composed of synthetic medium odd-chain triglycerides, is a novel therapy in development for LC-FAOD patients. However, cases of its safe and efficacious use to reverse severe heart failure in CACT deficiency are limited. Here, we present a detailed report of an infant with CACT deficiency admitted in metabolic crisis that progressed into severe cardiogenic shock who was successfully treated by triheptanoin. The child was managed, thereafter, on triheptanoin until her death at 3 years of age from a cardiopulmonary arrest in the setting of acute respiratory illness superimposed on chronic hypercarbic respiratory failure.

Germination is the first step of plant growth in plant life cycle. An embryonic radicle protruding the seed coat is the first part of plant which has direct contact with external environment including salt-affected soil. In embryo axes, mitochondria are the main energy producer. To understand better salinity impact on mitochondria functioning, this study was focused on the effect of NaCl stress onto mitochondria proteome. Mitochondria were isolated from yellow lupine (Lupine luteus L. ‘Mister’) embryo axes cultured in vitro for 12 h with 250 and 500 mM NaCl. Two-dimensional gel electrophoresis of mitochondrial proteins isolated from NaCl-treated axes demonstrated significant changes in proteins abundances as a response to salinity treatment. Twenty-one spots showing significant changes in protein expression profiles both under 250 and 500 mM NaCl treatment were selected for tandem mass spectrometry identification. This approach revealed proteins associated with different metabolic processes that represent enzymes of tricarboxylic acid cycle, mitochondrial electron transport chain, enzymes and proteins involved in mitochondria biogenesis and stresses response. Among proteins involved in mitochondria biogenesis, mitochondrial import inner membrane translocase, subunit Tim17/22, mitochondrial-processing peptidase subunit alpha-1, mitochondrial elongation factor Tu and chaperonins CPN60 were revealed. Finally, formate dehydrogenase 1 was found to accumulate in lupine embryo axes mitochondria under salinity. The functions of identified proteins are discussed in relation to salinity stress response, including salinity-induced PCD. Electronic supplementary material The online version of this article (doi:10.1007/s11738-013-1273-2) contains supplementary material, which is available to authorized users.

The Uridyl-Peptide Antibiotics (UPAs) are a diverse group of bacterial metabolites that are characterized by a uridyl moiety linked to a peptidic residue. Included in this group are the pacidamycins, liposidomycins, capuramycins, and muraymycins. Most of these antibiotics are produced by Streptomyces species and are naturally found as complexes of closely related congeners. The compounds all bear some resemblance to intermediates involved in cell wall biosynthesis in bacteria and exert their antibacterial action through inhibition of the membrane-bound translocase I. Caprazamycin was the first of the UPAs for which a biosynthetic gene cluster was identified and cloned, which quickly led to the discovery of several others. Preliminary experiments have been reported in which biosynthetic insights were applied to the production of analogs for evaluation of antimicrobial activity. Synthetic chemistry has provided evidence for the core features necessary for target inhibition and antibacterial efficacy. Although many of the UPAs have shown potent antibacterial activity, and are effective in inhibiting a highly selective bacterial target, none have progressed to become commercially viable agents. In this chapter, we will explore recent findings that have clarified the promise and limitations of this class of antibiotics.

Less than a billion years ago a protoeukaryote host enslaved a probably photosynthetic α-proteobacterium to form the first mitochondrion. After a phagocytosed α-proteobacterium escaped from the phagosome into the host cytoplasm, host carrier proteins of probable peroxisomal origin entered its periplasm via pre-existing β-barrel outer-membrane (OM) proteins related to the usher proteins of proteobacteria and ancestral to Tom40 – the OM protein translocase. Carrier insertion into the bacterial inner membrane (IM) then extracted photosynthesate for the host, giving it an immediate strong selective advantage for permanently enslaving the bacterium by inserting additional proteins into its envelope to improve carrier insertion, notably the Tim22 complex. Pre-existing bacterial periplasmic chaperones evolved into the periplasmic small Tims, whilst the bacterial Omp85 complex evolved into the Sam50 mitochondrial complex for the insertion of β-barrel proteins into the mitochondrial OM from the periplasm. Concomitant massive transfer of thousands of bacterial protein genes into the nucleus was initially very harmful to the host, especially by inserting many bacterial proteins with signal sequences into its endoplasmic reticulum (ER). This disaster was circumvented by the evolution of positive charges on one face of the signal sequences, preventing their entry into the ER and allowing recognition by Tom22 receptors added to the TOM complex, moving them into the mitochondrial periplasm, and by the origin, by gene duplication of Tim22, of the Tim23/Tim17 complex that translocated them into the mitochondrial matrix, where an additional adaptor Tim44 was added to pass them smoothly to the pre-existing bacterial chaperone Hsp70, ATP-driven, motor to pull them efficiently into the matrix; IM proteins were then inserted via the Oxa1 machinery, derived from proteobacterial YidC or a posibacterial relative. Only then did matrix proteins acquire presequences, allowing massive mitochondrial genome reduction through selection for efficiency. I discuss the molecular mechanisms and selective advantages of these major changes that endowed early eukaryote cells with two novel genetic membranes and a radically improved respiratory machinery. I also discuss the relative contribution of symbiont and host genes to the chimaeric mitochondrion and to its now 8 The Chimaeric Origin of Mitochondria: Photosynthetic Cell Enslavement, Gene-Transfer Pressure, and Compartmentation Efficiency

The Type I and III restriction endonucleases are large, multimeric protein complexes with four enzyme activities; DNA methyltransferase, DNA endonuclease, ATPase and DNA translocase. It has been demonstrated that ATP-dependent protein motion along DNA is necessary for endonuclease activity. Studies have shown that Type I enzymes remain bound to their recognition sites whilst simultaneously translocating adjacent non-specific dsDNA past a stationary complex. This occurs bi-directionally so that two DNAloops are extruded. An equivalent unidirectional mechanism has been suggested for the Type III enzymes. DNA cleavage generally results when the enzymes stall against another restriction enzyme complex. Both the HsdR subunits of the Type I enzymes and the Res subunits of the Type III enzymes carry amino acid motifs characteristic of superfamily 2 helicases. In this review, the structural and mechanistic implications of this relationship are discussed and models suggested for how the ATP-dependent restriction enzymes might couple chemical energy to mechanical motion on DNA.

The elucidation of the pathway for preprotein transit across the plasma membrane of E. coli is a fine example of the synergy between genetic, biochemical, and physiological approaches to study a common problem. Early physiological studies of secretion by Ito and by Randall established important boundary parameters; secretion requires metabolic energy and is not coupled to ongoing polypeptide chain growth. Genetic efforts in the labs of Beckwith and Silhavy were focused on obtaining prl supressor mutants with enhanced capacities to export proteins with mutant leader sequences and sec mutants which were temperature sensitive in the export process itself. In our lab, the biochemical studies of this era focused on the sec-independent Ml3 procoat protein and on the isolation of leader peptidase.

Mechanisms Involved in Co- and Posttranslational Protein Transport.- Glycosylation Mapping of the Interaction Between Topogenic Sequences and the ER Translocase.- The Various Roles of Invariant Chain in the Act of Antigen Presentation.- Progress Towards the Identification of Secretion Signals in a Protein Transported in a Folded State Across a Lipid Bilayer.- E. coli Preprotein Translocase: a 6 Stroke Engine with 2 Fuels and 2 Piston Rods.- The Tol/PAL and TonB Systems: two Envelope-Spanning Protein Complexes Involved in Colicin Import in E. coli.- In vitro Assembly of Outer Membrane Protein PhoE of E. coli.- Protein Folding the Cell: the Role of Molecular Chaperons.- Thermodynamics of the Membrane Insertion Process of the Ml3 Procoat Protein, a Lipid Bilayer Traversing Protein Comprising a Leader Sequence.- Lipid-Protein Interactions in Chloroplast Protein Import.- Protein Transport Into and Across the Mitochondrial Outer Membrane: Recognition, Insertion and Translocation of Preproteins.- Protein Import Across the Inner Mitochondrial Membrane.- How Mitochondria Recognize and Bind Precursor Proteins at the Surface.- The General Features of Membrane Traffic During Endocytosis in Polarized and Non-Polarized Cells.- Mitotic Fragmentation of the Golgi Apparatus.- Kinetic Measurements of Fusion Between Vesicles Derived from the Endoplasmic Reticulum.- The Sorting of Membrane Proteins During the Formation of ER-Derived Transport Vesicles.- Isolation and Characterization of Yeast Mutants Defective in the Dolichol Pathway for N-Glycosylation.- The Importance of Lipid-Protein Interactions in Signal Transduction Through the Calcium-Phospholipid Second Messenger System.- Covalently Attached Lipid Bilayers on Planar Waveguides for Measuring Protein Binding to Functionalized Membranes.- The Effect of Sterol Side Chain Conformation on Lateral Lipid Domain Formation in Monolayer Membranes.- The Kinetics, Specificities and Structural Features of Lipases.- Phospholipases A2 and the Production of Bioactive Lipids.- Phospholipases in the Yeast Saccharomyces cerevisiae.- Functional Analysis of Phosphatidylinositol Tranfer Protiens.- Phosphatidylcholine Biosynthesis in Saccharomyces cerevisiae: Effects on Regulation of Phospholipid Synthesis and Respiratory Competence.- Resynthesis of the Cell Surface Pool of Phosphatidylinositol.- The GlcNac-PI de-N-Acetylase of Glycosylphosphatidylinositol (GPI) Biosynthesis in Trypanosoma brucei.- Phospholipid Flippases: Neither Exclusively, nor only Involved in Maintaining Membrane Phospholipid Asymmetry.- A New Efficient Strategy to Reconstitute Membrane Proteins into Liposomes: Application to the Study of Ca++-ATPases.- Interaction of Pulmonary Surfactant-Associated Proteins with Phospholipid Vesicles.

Many integral membrane proteins span the hydrophobic core of a membrane with one or more a-helical segments each consisting of about 20 hydrophobic amino acids. The orientation of the hydrophobic stretch in the membrane is determined by its flanking amino acids. In general there are more positively charged residues present in cytoplasmic loops than in extra-cytoplasmic ones (von Heijne, 1986). In both prokaryotic and eukaryotic cells, proteins are targeted for secretion by N-terminal signal sequences with a common basic design: a positively charged N-terminus, a central hydrophobic stretch, and a C-terminal cleavage region that serves as a recognition site for the signal peptidase enzyme (von Heijne, 1985). Signal sequences from prokaryotes and eukaryotes look very similar and are often functionally interchangeable. They are essential for the efficient and selective targeting of the nascent protein chains either to the endoplasmic reticulum (in eukaryotes) or to the cytoplasmic membrane (in prokaryotes) (Gierasch, 1989). Signal sequences also play a central role in the interaction with the translocation machinery of the cell and in the translocation of the polypeptide chains across the membrane. Proteins are co-translationally translocated across the endoplasmic reticulum (ER) membrane. In eukaryotes two kinds of topogenic sequences are important for assembly in ER membrane: Those that initiate translocation such as signal peptides (SPs) and signalanchor sequences (SAs) and those that halt translocation, stop-transfer signals (STs). Signal peptides and signal anchor sequences differ in that SA sequences tend to have longer hydrophobic cores and lack a signal peptidase cleavage site (von Heijne, 1988).

Our understanding of cellular membranes has rapidly progressed from the point of view of the lipid bilayer being only a simple permeability barrier and matrix for membrane proteins to one in which membrane lipids are viewed as dynamic components capable of initiating and regulating various cellular functions. Only recently, however, has it become clear that the distribution of lipids between bilayer leaflets plays an important role in many cellular processes. While most of these phenomena are directly associated with what is considered to be normal membrane lipid asymmetry, the “atypical” display of phosphatidylserine (PS) on the cell’s outer leaflet has significant physiological consequences. PS participates, for example, in various cell-cell interactions (Schroit et al., 1985; Schlegel et al., 1985), cell activation and hemostasis (Bevers et al., 1983; Rosing et al., 1985; Sims et al., 1989), cell aging (Shukla and Hanahan, 1982; Herrmann and Devaux, 1990), membrane fusion events (Farooqui et al., 1987; Song et al., 1992; Schewe et al., 1992), and apoptosis (Fadok et al., 1992a, 1992b).

A transversal, asymmetric distribution of phospholipids is characteristic for most membrane systems investigated so far. Following a period in which an inventory was made of the asymmetry in various membrane systems, research was focused on questions regarding the biogenesis, the maintenance and the functional aspects of lipid asymmetry. As a result, a crucial factor involved in the maintenance of the asymmetry — the ATP dependent aminophospholipid translocase (flippase) — was detected. Both the existence of phospholipid asymmetry and the translocase were detected first in the erythrocyte membrane. However, if one wants to study dynamic characteristics of asymmetry and its maintenance, the biogenesis of this phenomenon, and in particular the influence that metabolic and physiological processes might have on the specific localization of membrane phospholipids, the erythrocyte provides a rather limited model system. Therefore, more recently emphasis was laid on studies with complex cells which can undergo severe physiological modifications. The cultured neonatal cardiomyocyte appeared to be a suitable model system and the study of its plasma membrane phospholipid asymmetry, its maintenance and changes therein upon metabolic alterations, can provide detailed information about the role that a specific phospholipid organization can play in proper cell functioning.

The preprotein translocase of Escherichia coli (Wickner et al.,1991) guards preproteins from the site of synthesis at the ribosome in the cytosol to the processed form to be released into the periplasm. SecA (Schmidt et al.,1988) is the ATP-hydrolysing, peripheral subunit of the translocase (Brundage et al.,1990; Wickner et al., 1991), and plays an essential role in preprotein translocation (Lill et al., 1989). The low endogenous ATPase activity of SecA is stimulated by interactions with acidic phospholipids, preproteins and the SecY/E protein (Lill et al.,1989, 1990; Brundage et al., 1990). This stimulated ATPase activity initiates translocation which is further driven by ATP hydrolysis and Ap (Schiebel et al., 1991; Driessen, 1992). Biochemical studies suggest that SecA possess three ATP binding sites (Lill et al., 1989; Oliver, 1993). Only one domain shows a significant level of sequence similarity to the Walker A- and B-motifs for a NTP-binding site (Walker et al., 1982) (Fig. 1). Both regions are highly conserved among different bacterial and algal SecA homologues. To analyze the function of this putative ATP binding site, we started a site-directed mutagenesis approach and changed critical residues of the A-domain (Klose et al., 1983; van der Wolk et al., 1993) of the Bacillus subtilis SecA homolog (Overhoff et al., 1991). Now we report on the localization of the B-domain and further characterized ATP- and preprotein-binding activities of the mutants.

The questions. Classical studies by Palade, deDuve, and colleagues established that membranes divide cells into distinct compartments, each with a unique set of resident proteins catalyzing distinct functions. Each compartment is either a membrane, with its own set of embedded proteins, or a soluble space surrounded by a membrane. A typical eukaryotic cell may have over 20 compartments, while a bacterium such as E. coli has four the cytoplasm, inner [plasma] membrane, periplasm, and outer membrane. In contrast, almost all protein synthesis begins in the cytosol in all cells, in a basically spatially undifferentiated manner. The first question then is how proteins are targeted, either to remain in the cytosol or to the appropriate membrane for translocation. Having arrived there, the second question is one of translocation mechanism. Is it by radically changing its structure to pass from the aqueous cytosol to the hydrocarbon-like interior of a membrane, or by a proteinaceous transport system (“translocase”)? In either case, what is the energy source for this transfer? Is it the energy of protein synthesis pushing the chain out of the ribosome, a pulling force on the other side, electrophoresis, or is metabolic energy coupled to protein translocation by translocase? In Fig. 1, these questions are illustrated, with a fig leaf both conveying the attractive quality of the hidden solution and covering our ignorance about the ultimate answers.

Although phosphatidylserine (PS) is an essential component for the formation of membrane bilayers, it has been shown to contribute to many regulatory processes in biological responses (Kaibuchi et al., 1981, Zwaal et al., 1986, Madsen et al., 1989, Inoue et al., 1989). Pharmacological effects of PS have also been reported (Bruni et al., 1976, 1989). Some PS may exhibit biological activity through interacting with specific binding proteins. Some of the proteins were shown to interact with PS in a highly specific manner and precise configurations of PS molecule are required for the interaction. The typical examples are the receptor for lyso-PS on rat mast cells (Horigome et al., 1986, Chang et al., 1988), aminophospholipid translocase (Morrot et al., 1989) and protein kinase C (Lee and Bell, 1989). The PS-binding proteins so far reported are listed in Table 1. Although much effort has been focused on understanding the molecular mechanism involved in the interactions between PS and PS-binding proteins, the difficulties of handling these proteins because of their limited aqueous solubility and scarcity in the biological systems have hampered progress in this area.

The first stage in the eukaryotic secretory pathway is the translocation of polypeptides across the endoplasmic reticulum (ER) membrane. This process has been extensively studied in mammalian systems using an in vitro assay which faithfully reproduces the cotranslational translocation of specific precursor proteins into the ER lumen (Blobel, & Dobberstein, 1975). Such biochemical analyses have revealed that translocation requires both cytosolic and membrane-associated factors, including the cytosolic ribonucleoprotein complex, signal recognition particle (SRP), and its cognate membrane-bound receptor, SRP-receptor (or “Docking protein”; Walter & Blobel, 1980: Meyer et al., 1982). Evidence suggests that SRP binds to the signal sequence of a nascent secretory protein as it emerges from the ribosome, and in doing so effects a reduction in the rate of polypeptide chain elongation (“elongation arrest”, Walter and Blobel, 1981). The arrested complex is then targeted to the ER membrane via an interaction between SRP and the integral membrane protein SRP-receptor (Gilmore et al., 1982; Meyer et al., 1982). SRP-receptor then mediates the GTP-dependent displacement of SRP from the signal sequence/ribosome complex (Connolly & Gilmore, 1989), thus releasing elongation arrest, and enabling the co-translational translocation of the targeted precursor across the.ER membrane. Whilst the SRP-dependent targeting cycle is relatively well characterised, the mechanism by which a targeted polypeptide actually penetrates the lipid bilayer remains obscure. There exists a wealth of data implicating integral membrane proteins in the translocation process, however, the identification and characterisation of these proteins has proven problematic. Several candidates for components of the mammalian ER translocase have been identified in a series of crosslinking studies in which a translocating polypeptide is first trapped in the membrane, and then cross-linked to those proteins in closest proximity. Such analyses have identified an ER membrane protein termed signal sequence receptor (SSR), which interacts directly with the signal peptide of nascent pre-proteins (Weidmann et al., 1987). Current evidence suggest that SSR exists as an oligomeric complex comprising at least two integral membrane glycoproteins, namely SSRα (34K), and SSRβ (22K) (Rapoport, 1990). Moreover, SSRα remains in close proximity to the mature portion of a translocating pre-protein, and may therefore represent a constituent of the translocon per se (Weidmann et al., 1989; Prehn et al., 1990; Rapoport, 1990). More recent cross-linking studies have identified a 37K ER protein (P37) which can be cross-linked to the signal anchor sequence of a type I membrane protein during its insertion into the membrane (High et al., 1991). However, despite the identification of these putative translocon components, their actual contribution to the translocation process remains speculative. A more potent case can be made for the recently identified integral membrane glycoprotein TRAM (for translocating chain-associating membrane protein; Gorlich et al., 1992). TRAM represents the major species found crosslinked to short nascent chains, and may therefore be involved in the early stages of translocation. TRAM is an abundant ER-protein, present at a level at least equivalent to the number of membrane-bound ribosomes, raising the possibility that TRAM represents a constitutive component of the active translocase. A direct role for TRAM in translocation is supported by the observation that addition of the purified protein stimulates the translocation of preprolactin into reconstituted microsomes to levels approaching those of native ER microsomes (Gorlich et al., 1992).