A morphological view on mitochondrial protein targeting

Mitochondrial protein targeting includes both intramitochondrial sorting of proteins encoded by the organellar genome and import and subsequent sorting of nuclear encoded precursor proteins. Only a few proteins are encoded by the mitochondrial genome and synthesized in the organellar matrix. These include predominantly inner membrane proteins that are perhaps co-translationally inserted into this membrane. Biochemical data suggest that insertion into the inner membrane may be confined to the inner boundary membrane. Ultrastructurally, however, a preferential association of ribosomes with either inner boundary or cristae membranes has not been established. The majority of the mitochondrial proteins are nuclear encoded and synthesized as precursors in the cytosol. Electron microscopic studies revealed that import of precursor proteins is generally confined to sites where both mitochondrial envelope membranes are closely apposed. In line with these observations, biochemical studies indicated that precursor proteins destined for the inner membrane or matrix have to interact with the energized inner membrane to allow complete passage of the precursor through the outer membrane. As a consequence, the mitochondrial envelope membranes have to be in close proximity at protein import sites. In isolated mitochondria distinct sites (designated as contact sites) exist where both envelope membranes are closely apposed and presumably stably associated. In situ, however, mitochondrial boundary membranes are in close proximity over large areas that cover almost the entire mitochondrial periphery. Consequently, the relative area of the mitochondrial surface, where both boundary membranes are in sufficient proximity for allowing protein translocation, is generally larger in situ compared to that in isolated organelles. Immunocytochemical localization studies showed a rather random distribution of components of the mitochondrial protein translocation machinery over the entire mitochondrial surface and not confined to contact sites. Based on these ultrastructral data and recent biochemical findings we propose that mitochondrial protein import sites are dynamic in nature and include relatively labile regions of close association of the boundary membranes. In vitro, however, mitochondrial protein import may preferentially take place at or near the presumably stable contact sites

In vitro characterization of mitochondrial function and structure in rat and human cells with a deficiency of the NADH:ubiquinone oxidoreductase Ndufc2 subunit

Ndufc2, a subunit of the NADH:ubiquinone oxidoreductase, plays a key role in the assembly and activity of complex I within the mitochondrial OXPHOS chain. Its deficiency has been shown to be involved in diabetes, cancer and stroke. To improve our knowledge on the mechanisms underlying the increased disease risk due to Ndufc2 reduction, we performed the present in vitro study aimed at the fine characterization of the derangements in mitochondrial structure and function consequent to Ndufc2 deficiency. We found that both fibroblasts obtained from skin of heterozygous Ndufc2 knock-out rat model showed marked mitochondrial dysfunction and PBMC obtained from subjects homozygous for the TT genotype of the rs11237379/NDUFC2 variant, previously shown to associate with reduced gene expression, demonstrated increased generation of reactive oxygen species and mitochondrial damage. The latter was associated with increased oxidative stress and significant ultrastructural impairment of mitochondrial morphology with a loss of internal cristae. In both models the exposure to stress stimuli, such as high-NaCl concentration or LPS, exacerbated the mitochondrial damage and dysfunction. Resveratrol significantly counteracted the ROS generation. These findings provide additional insights on the role of an altered pattern of mitochondrial structure-function as a cause of human diseases. In particular, they contribute to underscore a potential genetic risk factor for cardiovascular diseases, including stroke

A dynamic model of the mitochondrial protein import machinery

Many proteins are translocated into or across two mem-branes in order to reach their functional destination; these include many nuclear-encoded mitochondrial and chloro-plast proteins, as well as proteins transported into or across the outer membrane of gram-negative bacteria. In eukaryotes, mechanistic insights have been obtained mainly with the mitochondrial two-membrane transport system. By generating translocation intermediates that span both mitochondrial membranes at the same time, it has been demonstrated that the outer and inner mem-brane translocation machineries cooperate in the import of preproteins (Hart1 and Neupert, 1990; Baker and Schatz, 1991). Translocation contact sites were defined as mito-chondrial import sites where the outer and inner mem-branes are so close together that they can be spanned b

Contact sites between inner and outer membranes

Contact sites between both mitochondrial membranes play a predominant role in the transport of nuclear-coded precursor proteins into mitochondria. The characterization of contact sites was greatly advanced by the reversible accumulation of precursor proteins in transit (translocation intermediates). It was found that the sites are saturable, apparently contain proteinaceous components and mediate extensive unfolding of the polypeptide chain in translocation. Some components of mitochondrial contact sites are currently being identified

Purification and Characterisation of a Pore Protein of the Outer Mitochondrial Membrane from Neurospora crassa

The major protein of the outer mitochondrial membrane of Neurospora was purified. On dodecylsulfate-containing gels it displayed a single bend with an apparent molecular weight of 31000. reconstitution experiments with artifical lipid bilayers showed that this protein forms pores. Pore conductance was dependent on the voltage across the membrane. The protein inserted into the membrane in an oriented fashion, the membrane current being dependent on the sign of the voltage. Single pore conductance was 5nS, suggesting a diameter of 2nm of the open pore. This mitochondrial protein shows a number of similarities to the outer membrane porins of gram-negative bacteria

Pink1 and Parkin regulate Drosophila intestinal stem cell proliferation during stress and aging.

Intestinal stem cells (ISCs) maintain the midgut epithelium in Drosophila melanogaster Proper cellular turnover and tissue function rely on tightly regulated rates of ISC division and appropriate differentiation of daughter cells. However, aging and epithelial injury cause elevated ISC proliferation and decreased capacity for terminal differentiation of daughter enteroblasts (EBs). The mechanisms causing functional decline of stem cells with age remain elusive; however, recent findings suggest that stem cell metabolism plays an important role in the regulation of stem cell activity. Here, we investigate how alterations in mitochondrial homeostasis modulate stem cell behavior in vivo via RNA interference-mediated knockdown of factors involved in mitochondrial dynamics. ISC/EB-specific knockdown of the mitophagy-related genes Pink1 or Parkin suppresses the age-related loss of tissue homeostasis, despite dramatic changes in mitochondrial ultrastructure and mitochondrial damage in ISCs/EBs. Maintenance of tissue homeostasis upon reduction of Pink1 or Parkin appears to result from reduction of age- and stress-induced ISC proliferation, in part, through induction of ISC senescence. Our results indicate an uncoupling of cellular, tissue, and organismal aging through inhibition of ISC proliferation and provide insight into strategies used by stem cells to maintain tissue homeostasis despite severe damage to organelles