Functional independence of the protein translocation machineries in mitochondrial outer and inner membranes

The protein translocation machineries of the outer and inner mitochondrial membranes usually act in concert during translocation of matrix and inner membrane proteins. We considered whether the two machineries can function independently of each other in a sequential reaction. Fusion proteins (pF-CCHL) were constructed which contained dual targeting information, one for the intermembrane space present in cytochrome c heme lyase (CCHL) and the other for the matrix space contained in the signal sequence of the precursor of F1-ATPase beta-subunit (pF1 beta). In the absence of a membrane potential, delta psi, the fusion proteins moved into the intermembrane space using the CCHL pathway. In contrast, in the presence of delta psi they followed the pF1 beta pathway and eventually were translocated into the matrix. The fusion protein pF51-CCHL containing 51 amino acids of pF1 beta, once transported into the intermembrane space in the absence of a membrane potential, could be further chased into the matrix upon re-establishing delta psi. The sequential and independent movement of the fusion protein across the two membranes demonstrates that the translocation machineries act as distinct entities. Our results support a model in which the two translocation machineries can function independently of each other, but generally interact in a dynamic fashion to achieve simultaneous translocation across both membranes. In addition, the results provide information about the targeting sequences within CCHL. The protein does not contain a signal for retention in the intermembrane space; rather, it lacks matrix targeting information, and therefore is unable to undergo delta psi-dependent interaction with the protein translocation apparatus in the inner membrane

Natural abundance 14N and 15N solid-state NMR of pharmaceuticals and their polymorphs

14N ultra-wideline (UW), 1H{15N} indirectly-detected HETCOR (idHETCOR) and 15N dynamic nuclear polarization (DNP) solid-state NMR (SSNMR) experiments, in combination with plane-wave density functional theory (DFT) calculations of 14N EFG tensors, were utilized to characterize a series of nitrogen-containing active pharmaceutical ingredients (APIs), including HCl salts of scopolamine, alprenolol, isoprenaline, acebutolol, dibucaine, nicardipine, and ranitidine. A case study applying these methods for the differentiation of polymorphs of bupivacaine HCl is also presented. All experiments were conducted upon samples with naturally-abundant nitrogen isotopes. For most of the APIs, it was possible to acquire frequency-stepped UW 14N SSNMR spectra of stationary samples, which display powder patterns corresponding to pseudo-tetrahedral (i.e., RR′R′′NH+ and RR′NH2+) or other (i.e., RNH2 and RNO2) nitrogen environments. Directly-excited 14N NMR spectra were acquired using the WURST-CPMG pulse sequence, which incorporates WURST (wideband, uniform rate, and smooth truncation) pulses and a CPMG (Carr-Purcell Meiboom-Gill) refocusing protocol. In certain cases, spectra were acquired using 1H → 14N broadband cross-polarization, via the BRAIN-CP (broadband adiabatic inversion – cross polarization) pulse sequence. These spectra provide 14N electric field gradient (EFG) tensor parameters and orientations that are particularly sensitive to variations in local structure and intermolecular hydrogen-bonding interactions. The 1H{15N} idHETCOR spectra, acquired under conditions of fast magic-angle spinning (MAS), used CP transfers to provide 1H–15N chemical shift correlations for all nitrogen environments, except for two sites in acebutolol and nicardipine. One of these two sites (RR′NH2+ in acebutolol) was successfully detected using the DNP-enhanced 15N{1H} CP/MAS measurement, and one (RNO2 in nicardipine) remained elusive due to the absence of nearby protons. This exploratory study suggests that this combination of techniques has great potential for the characterization of solid APIs and numerous other organic, biological, and inorganic systems

Structures and functions of mitochondrial ABC transporters

A small number of physiologically important ATP-binding cassette (ABC) transporters are found in mitochondria. Most are half transporters of the B group forming homodimers and their topology suggests they function as exporters. The results of mutant studies point towards involvement in iron cofactor biosynthesis. In particular, ABC subfamily B member 7 (ABCB7) and its homologues in yeast and plants are required for iron-sulfur (Fe-S) cluster biosynthesis outside of the mitochondria, whereas ABCB10 is involved in haem biosynthesis. They also play a role in preventing oxidative stress. Mutations in ABCB6 and ABCB7 have been linked to human disease. Recent crystal structures of yeast Atm1 and human ABCB10 have been key to identifying substrate-binding sites and transport mechanisms. Combined with in vitro and in vivo studies, progress is being made to find the physiological substrates of the different mitochondrial ABC transporters

Functional Characterization of the Eukaryotic Cysteine Desulfurase Nfs1p from Saccharomyces cerevisiae

Previous studies have indicated that the essential protein Nfs1 performs a crucial role in cellular iron-sulfur (Fe/S) protein maturation. The protein is located predominantly in mitochondria, yet low amounts are present in cytosol and nucleus. Here we examined several aspects concerning the molecular function of yeast Nfs1p as a model protein. First, we demonstrated that purified Nfs1p facilitates the in vitro assembly of Fe/S proteins by using cysteine as its specific substrate. Thus, eukaryotic Nfs1 is a functional orthologue of the bacterial cysteine desulfurase IscS. Second, we showed that only the mitochondrial version but not the extramitochondrial version of Nfs1p is functional in generating cytosolic and nuclear Fe/S proteins. Mutation of the nuclear targeting signal of Nfs1p did not affect the maturation of cytosolic and nuclear Fe/S proteins, despite a severe growth defect under this condition. Nfs1p could not assemble an Fe/S cluster on the Isu scaffold proteins when they were located in the yeast cytosol. The lack of function of these central Fe/S cluster assembly components suggests that the maturation of extramitochondrial Fe/S protein does not involve functional copies of the mitochondrial Fe/S cluster assembly machinery in the yeast cytosol. Third, the extramitochondrial version of Nfs1p was shown to play a direct role in the thiomodification of tRNAs. Finally, we identified a highly conserved N-terminal {beta}-sheet of Nfs1p as a functionally essential part of the protein. The implication of these findings for the structural stability of Nfs1p and for its targeting mechanism to mitochondria and cytosol/nucleus will be discussed

Impairing the production of ribosomal RNA activates mammalian target of rapamycin complex 1 signalling and downstream translation factors

Ribosome biogenesis is a key process for maintaining protein synthetic capacity in dividing or growing cells, and requires coordinated production of ribosomal proteins and ribosomal RNA (rRNA), including the processing of the latter. Signalling through mammalian target of rapamycin complex 1 (mTORC1) activates all these processes. Here, we show that, in human cells, impaired rRNA processing, caused by expressing an interfering mutant of BOP1 or by knocking down components of the PeBoW complex elicits activation of mTORC1 signalling. This leads to enhanced phosphorylation of its substrates S6K1 and 4E-BP1, and stimulation of proteins involved in translation initiation and elongation. In particular, we observe both inactivation and downregulation of the eukaryotic elongation factor 2 kinase, which normally inhibits translation elongation. The latter effect involves decreased expression of the eEF2K mRNA. The mRNAs for ribosomal proteins, whose translation is positively regulated by mTORC1 signalling, also remain associated with ribosomes. Therefore, our data demonstrate that disrupting rRNA production activates mTORC1 signalling to enhance the efficiency of the translational machinery, likely to help compensate for impaired ribosome production

Cytosolic Fe-S cluster protein maturation and iron regulation are independent of the mitochondrial Erv1/Mia40 import system

The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways

Zim17/Tim15 links mitochondrial iron–sulfur cluster biosynthesis to nuclear genome stability

Genomic instability is related to a wide-range of human diseases. Here, we show that mitochondrial iron–sulfur cluster biosynthesis is important for the maintenance of nuclear genome stability in Saccharomyces cerevisiae. Cells lacking the mitochondrial chaperone Zim17 (Tim15/Hep1), a component of the iron–sulfur biosynthesis machinery, have limited respiration activity, mimic the metabolic response to iron starvation and suffer a dramatic increase in nuclear genome recombination. Increased oxidative damage or deficient DNA repair do not account for the observed genomic hyperrecombination. Impaired cell-cycle progression and genetic interactions of ZIM17 with components of the RFC-like complex involved in mitotic checkpoints indicate that replicative stress causes hyperrecombination in zim17Δ mutants. Furthermore, nuclear accumulation of pre-ribosomal particles in zim17Δ mutants reinforces the importance of iron–sulfur clusters in normal ribosome biosynthesis. We propose that compromised ribosome biosynthesis and cell-cycle progression are interconnected, together contributing to replicative stress and nuclear genome instability in zim17Δ mutants

Assessing literacy in children and adolescents

This review provides a framework for examining the assessment of literacy underpinned by current theories of reading and writing. Our choice of assessment tools was influenced by a desire to identify tests that are suitable for diagnosing students of all ages. The key dimensions identified were printed word recognition (lexical and non-lexical), comprehension and writing. We conclude that there is no single test currently available that provides a comprehensive profile of literacy difficulties. The assessor will require critical discrimination in choosing tests that are theoretically sound and diagnostically useful. © 2007 Association for Child and Adolescent Mental Health