A quantitative fluorescence-based approach to study mitochondrial protein import.

Mitochondria play central roles in cellular energy production and metabolism. Most proteins required to carry out these functions are synthesized in the cytosol and imported into mitochondria. A growing number of metabolic disorders arising from mitochondrial dysfunction can be traced to errors in mitochondrial protein import. The mechanisms underlying the import of precursor proteins are commonly studied using radioactively labeled precursor proteins imported into purified mitochondria. Here, we establish a fluorescence-based import assay to analyze protein import into mitochondria. We show that fluorescently labeled precursors enable import analysis with similar sensitivity to those using radioactive precursors, yet they provide the advantage of quantifying import with picomole resolution. We adapted the import assay to a 96-well plate format allowing for fast analysis in a screening-compatible format. Moreover, we show that fluorescently labeled precursors can be used to monitor the assembly of the F1 F0 ATP synthase in purified mitochondria. Thus, we provide a sensitive fluorescence-based import assay that enables quantitative and fast import analysis.

Defective Mitochondrial Cardiolipin Remodeling Dampens HIF-1α Expression in Hypoxia.

Mitochondria fulfill vital metabolic functions and act as crucial cellular signaling hubs, integrating their metabolic status into the cellular context. Here, we show that defective cardiolipin remodeling, upon loss of the cardiolipin acyl transferase tafazzin, decreases HIF-1α signaling in hypoxia. Tafazzin deficiency does not affect posttranslational HIF-1α regulation but rather HIF-1α gene expression, a dysfunction recapitulated in iPSC-derived cardiomyocytes from Barth syndrome patients with tafazzin deficiency. RNA-seq analyses confirmed drastically altered signaling in tafazzin mutant cells. In hypoxia, tafazzin-deficient cells display reduced production of reactive oxygen species (ROS) perturbing NF-κB activation and concomitantly HIF-1α gene expression. Tafazzin-deficient mice hearts display reduced HIF-1α levels and undergo maladaptive hypertrophy with heart failure in response to pressure overload challenge. We conclude that defective mitochondrial cardiolipin remodeling dampens HIF-1α signaling due to a lack of NF-κB activation through reduced mitochondrial ROS production, decreasing HIF-1α transcription.

Recognition and ER Quality Control of Misfolded Formylglycine-Generating Enzyme by Protein Disulfide Isomerase.

Multiple sulfatase deficiency (MSD) is a fatal, inherited lysosomal storage disorder characterized by reduced activities of all sulfatases in patients. Sulfatases require a unique post-translational modification of an active-site cysteine to formylglycine that is catalyzed by the formylglycine-generating enzyme (FGE). FGE mutations that affect intracellular protein stability determine residual enzyme activity and disease severity in MSD patients. Here, we show that protein disulfide isomerase (PDI) plays a pivotal role in the recognition and quality control of MSD-causing FGE variants. Overexpression of PDI reduces the residual activity of unstable FGE variants, whereas inhibition of PDI function rescues the residual activity of sulfatases in MSD fibroblasts. Mass spectrometric analysis of a PDI+FGE variant covalent complex allowed determination of the molecular signature for FGE recognition by PDI. Our findings highlight the role of PDI as a disease modifier in MSD, which may also be relevant for other ER-associated protein folding pathologies.

Presequence recognition by the tom40 channel contributes to precursor translocation into the mitochondrial matrix.

More than 70% of mitochondrial proteins utilize N-terminal presequences as targeting signals. Presequence interactions with redundant cytosolic receptor domains of the translocase of the outer mitochondrial membrane (TOM) are well established. However, after the presequence enters the protein-conducting Tom40 channel, the recognition events that occur at the trans side leading up to the engagement of the presequence with inner membrane-bound receptors are less well defined. Using a photoaffinity-labeling approach with modified presequence peptides, we identified Tom40 as a presequence interactor of the TOM complex. Utilizing mass spectrometry, we mapped Tom40's presequence-interacting regions to both sides of the ß-barrel. Analysis of a phosphorylation site within one of the presequence-interacting regions revealed altered translocation kinetics along the presequence pathway. Our analyses assess the relation between the identified presequence-binding region of Tom40 and the intermembrane space domain of Tom22. The identified presequence-interacting region of Tom40 is capable of functioning independently of the established trans-acting TOM presequence-binding domain during matrix import.

Occlusal contact patterns--population-based data.

The reconstruction of functionally appropriate contacts between antagonistic teeth substantially determines the quality of prosthetic-restorative work. In a population-based sample comprising 2597 subjects, static occlusal contacts were registered and analysed using the GEDAS (Greifswald Digital Analyzing System), which digitally represents the contact point situation by means of silicone bite impressions. The number of contacting teeth is approximately equal on the Left- and right-hand side amounting to 8.3 on the left and 8.4 on the right. Furthermore, it was shown that 39% of the maxillary bridge pontics and 33% of the mandibular bridge units are not in contact. Antagonistic contacts are missing in 41% of the maxillary and 39% of the mandibular removable denture teeth. These results show that the fabrication of fixed dentures, particularly in bridge pontics, and the inspection of removable dentures needs to be done with more care to this detail.

Partial- or full-mouth examination assessing the dental and prosthetic status among elderly individuals.

The aim of this study was to determine whether half-mouth examinations accurately reflect the dental and prosthetic status of the entire mouth. Samples of 1,830 adults aged 55- 79 years were examined. The rate of agreement between half- and full-mouth examinations was estimated using weighted and unweighted Kappa values comparing findings of each tooth bilaterally. A power analysis was performed to estimate the number of subjects representing complete dental recordings within a certain power. Subjects showed unweighted Kappa values from 0.34-0.96. Weighted Kappa values ranged from 0. 74-0.99. A power analysis for unweighted Kappa scores computed that.findings from 122-335 individuals were necessary to equal the results obtained using complete dental recordings.

AP-1A and AP-3A lysosomal sorting functions.

Heterotetrameric adaptor-protein complexes AP-1A and AP-3A mediate protein sorting in post-Golgi vesicular transport. AP-1A and AP-3A have been localized to the trans-Golgi network, indicating a function in protein sorting at this compartment. AP-3A appears to mediate trans-Golgi network-to-lysosome and also endosome-to-lysosome protein sorting. AP-1A is thought to be required for both trans-Golgi network-to-endosome transport and endosome-to-trans-Golgi network transport. However, the recent discovery of a role for monomeric GGA (Golgi localized gamma-ear containing, ARF binding protein) adaptor proteins in trans-Golgi network to endosome protein transport has brought into question the long-discussed trans-Golgi network-to-endosome sorting function of AP-1A. Murine cytomegalovirus gp48 contains an unusual di-leucine-based lysosome sorting signal motif and mediates lysosomal sorting of gp48/major histocompatibility complex class I receptor complexes, preventing exposure of major histocompatibility complex class I at the plasma membrane. We analyzed lysosomal sorting of gp48/major histocompatibility complex class I receptor complexes in cell lines deficient for AP-1A, AP-3A and both, to determine their sorting functions. We find that AP1-A and AP3-A mediate distinct and sequential steps in the lysosomal sorting. Both sorting functions are required to prevent MHC class I exposure at the plasma membrane at steady-state.