Biogenesis of the mitochondrial phosphate carrier

The mitochondrial phosphate carrier (PiC) is a member of the family of inner-membrane carrier proteins which are generally synthesized without a cleavable presequence. Surprisingly, the cDNA sequences of bovine and rat PiC suggested the existence of an amino-terminal extension sequence in the precursor of PiC. By expressing PiC in vitro, we found that PiC is indeed synthesized as a larger precursor. This precursor was imported and proteolytically processed by mitochondria, whereby the correct amino-terminus of the mature protein was generated. Import of PiC showed the characteristics of mitochondrial protein uptake, such as dependence on ATP and a membrane potential and involvement of contact sites between mitochondrial outer and inner membranes. The precursor imported in vitro was correctly assembled into the functional form, demonstrating that the authentic import and assembly pathway of PiC was reconstituted when starting with the presequence-carrying precursor. These results are discussed in connection with the recently postulated role of PiC as an import receptor located in the outer membrane

Cyclosporin A-binding protein (cyclophilin) of Neurospora crassa

Cyclophilin (cyclosporin A-binding protein) has a dual localization in the mitochondria and in the cytosol of Neurospora crassa. The two forms are encoded by a single gene which is transcribed into mRNAs having different lengths and 5' termini (approximately 1 and 0.8 kilobases). The shorter mRNA specifies the cytosolic protein consisting of 179 amino acids. The longer mRNA is translated into a precursor polypeptide with an amino-terminal extension of 44 amino acids which is cleaved in two steps upon entry into the mitochondrial matrix. Neurospora cyclophilin shows about 60% sequence homology to human and bovine cyclophilins

Biogenesis of mitochondrial porin

We review here the present knowledge about the pathway of import and assembly of porin into mitochondria and compare it to those of other mitochondrial proteins. Porin, like all outer mitochondrial membrane proteins studied so far is made as a precursor without a cleavble lsquosignalrsquo sequence; thus targeting information must reside in the mature sequence. At least part of this information appears to be located at the amino-terminal end of the molecule. Transport into mitochondria can occur post-translationally. In a first step, the porin precursor is specifically recognized on the mitochondrial surface by a protease sensitive receptor. In a second step, porin precursor inserts partially into the outer membrane. This step is mediated by a component of the import machinery common to the import pathways of precursor proteins destined for other mitochondrial subcompartments. Finally, porin is assembled to produce the functional oligomeric form of an integral membrane protein wich is characterized by its extreme protease resistance

Visualizing active membrane protein complexes by electron cryotomography.

This is the final version of the article. Available from Nature Publishing Group via the DOI in this record.Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future.We thank Drs Ulrike Endesfelder and Mike Heilemann (Institute of Physical and Theoretical Chemistry, University of Frankfurt) for help with confocal microscopy, Deryck Mills (MPI of Biophysics, Frankfurt) for maintenance of the EM facility, and Paolo Lastrico (Graphics Department, MPI of Biophysics, Frankfurt) for assistance with Supplementary Movies and Fig. 1a. We thank Drs Bertram Daum and Karen Davies for helpful discussions on tomography. The plasmids pMAL-c2x-MT2 and pMAL-c2x-MT3 were a gift from Dr Christina Risco (CNB-CSIC, Madrid). This work was supported by the Max Planck Society, Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 746), Excellence Initiative of the German Federal & State Governments (EXC 294 BIOSS) and by an EMBO Long-Term Fellowship to V.A.M.G. (ALTF 1035-2010)

Are low tolerable upper intake levels for vitamin a undermining effective food fortification efforts?

Vitamin A deficiency (VAD) is a major health problem, particularly in low-resource countries, putting an estimated 125-130 million preschool-aged children at increased risk of morbidity and mortality from infectious diseases. Vitamin A supplementation reduces VAD and increases child survival; it is complemented by fortifying foods with vitamin A. Concern over increased risk of bone fracture associated with vitamin A intakes below the tolerable upper intake level (UL) among populations in affluent countries conflicts with the need to increase intakes in less developed countries, where populations are at greater risk of VAD and intakes are unlikely to reach the UL as diets include fewer foods containing retinol while vitamin A from carotenoids poses no risk of overdose. With the implementation of recently developed risk management tools, vitamin A can be used safely in food fortification, including point-of-use fortification in the context of supplementation among specific target groups in low-resource countrie

Relationship of homestead food production with night blindness among children below 5 years of age in Bangladesh

Abstract Objective To examine the relationship between homestead food production and night blindness among pre-school children in rural Bangladesh in the presence of a national vitamin A supplementation programme. Design A cross-sectional study. Setting A population-based sample of six rural divisions of Bangladesh assessed in the Bangladesh Nutrition Surveillance Project 2001-2005. Subjects A total of 158 898 children aged 12-59 months. Results The prevalence rates of night blindness in children among those who did and did not receive vitamin A capsules in the last 6 months were 0·07 % and 0·13 %, respectively. Given the known effect of vitamin A supplementation on night blindness, the analysis was stratified by children's receipt of vitamin A capsules in the last 6 months. Among children who did not receive vitamin A capsules in the last 6 months, the lack of a home garden was associated with increased odds of night blindness (OR = 3·16, 95 % CI 1·76, 5·68; P = 0·0001). Among children who received vitamin A capsules in the last 6 months, the lack of a home garden was not associated with night blindness (OR = 1·28, 95 % CI 0·71, 2·31; P = 0·4). Conclusions Homestead food production confers a protective effect against night blindness among pre-school children who missed vitamin A supplementation in rural Banglades

Mitochondrial targeting adaptation of the hominoid-specific glutamate dehydrogenase driven by positive Darwinian selection

Many new gene copies emerged by gene duplication in hominoids, but little is known with respect to their functional evolution. Glutamate dehydrogenase (GLUD) is an enzyme central to the glutamate and energy metabolism of the cell. In addition to the single, GLUD-encoding gene present in all mammals (GLUD1), humans and apes acquired a second GLUD gene (GLUD2) through retroduplication of GLUD1, which codes for an enzyme with unique, potentially brain-adapted properties. Here we show that whereas the GLUD1 parental protein localizes to mitochondria and the cytoplasm, GLUD2 is specifically targeted to mitochondria. Using evolutionary analysis and resurrected ancestral protein variants, we demonstrate that the enhanced mitochondrial targeting specificity of GLUD2 is due to a single positively selected glutamic acid-to-lysine substitution, which was fixed in the N-terminal mitochondrial targeting sequence (MTS) of GLUD2 soon after the duplication event in the hominoid ancestor ~18–25 million years ago. This MTS substitution arose in parallel with two crucial adaptive amino acid changes in the enzyme and likely contributed to the functional adaptation of GLUD2 to the glutamate metabolism of the hominoid brain and other tissues. We suggest that rapid, selectively driven subcellular adaptation, as exemplified by GLUD2, represents a common route underlying the emergence of new gene functions