A mutant of Neurospora crassa deficient in cytochrome c heme lyase activity cannot import cytochrome c into mitochondria

The nuclear cyt-2-1 mutant of Neurospora crassa is characterized by a gross deficiency of cytochrome c (Bertrand, H., and Collins, R. A. (1978) Mol. Gen. Genet. 166, 1-13). The mutant produces mRNA that can be translated into apocytochrome c in vitro. Apocytochrome c is also synthesized in vivo in cyt-2-1, but it is rapidly degraded and thus does not accumulate in the cytosol. Mitochondria from wild-type cells bind apocytochrome c made in vitro from either wild-type or cyt-2-1 mRNA and convert it to holocytochrome c. This conversion depends on the addition of heme by cytochrome c heme lyase and is coupled to translocation of cytochrome c into the intermembrane space. Mitochondria from the cyt-2-1 strain are deficient in the ability to bind apocytochrome c. They are also completely devoid of cytochrome c heme lyase activity. These defects explain the inability of the cyt-2-1 mutant to convert apocytochrome c to the holo form and to import it into mitochondria

A crucial role of the mitochondrial protein import receptor MOM19 for the biogenesis of mitochondria

The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but the cells remained viable. The number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activity. Protein import into isolated mutant mitochondria was decreased by factors of 6 to 30 for most proteins from all suborganellar compartments. Proteins like the ADP/ATP carrier, MOM19, and cytochrome c, whose import into wild-type mitochondria occurs independently of MOM19 became imported normally showing that the reduced import activities are solely caused by a lack of MOM19. Depletion of MOM19 reveals a close functional relationship between MOM19 and MOM22, since loss of MOM19 led to decreased levels of MOM22 and reduced protein import through MOM22. Furthermore, MOM72 does not function as a general backup receptor for MOM19 suggesting that these two proteins have distinct precursor specificities. These findings demonstrate that the import receptor MOM19 fulfills an important role in the biogenesis of mitochondria and that it is essential for the formation of mitochondria competent in respiration and phosphorylation

Import of cytochrome c heme lyase into mitochondria

Cytochrome c heme lyase (CCHL) catalyses the covalent attachment of the heme group to apocytochrome c during its import into mitochondria. The enzyme is membrane-associated and is located within the intermembrane space. The precursor of CCHL synthesized in vitro was efficiently translocated into isolated mitochondria from Neurospora crassa. The imported CCHL, like the native protein, was correctly localized to the intermembrane space, where it was membrane-bound. As with the majority of mitochondrial precursor proteins, CCHL uses the MOM19-GIP receptor complex in the outer membrane for import. In contrast to proteins taking the general import route, CCHL was imported independently of both ATP-hydrolysis and an electrochemical potential as external energy sources. CCHL which lacks a cleavable signal sequence apparently does not traverse the inner membrane to reach the intermembrane space; rather, it translocates through the outer membrane only. Thus, CCHL represents an example of a novel, 'non-conservative' import pathway into the intermembrane space, thereby also showing that the import apparatus in the outer membrane acts separately from the import machinery in the inner membrane

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

Apocytochrome c

The cytochrome c import pathway differs markedly from the general route taken by the majority of other imported proteins, which is characterized by the import involvement of namely, surface receptors, the general insertion protein (GIP), contact sites and by the requirement of a membrane potential (Δψ). Unique features of both the cytochrome c precursor (apocytochrome c) and of the mechanism that transports it into mitochondria, have contributed to the evolution of a distinct import pathway that is not shared by any other mitochondrial protein analysed thus far. The cytochrome c pathway is particularly unique because i) apocytochrome c appears to have spontaneous membrane insertion-activity; ii) cytochrome c heme lyase seems to act as a specific binding site in lieu of a surface receptor and; iii) covalent heme addition and the associated refolding of the polypeptide appears to provide the free energy for the translocation of the cytochrome c polypeptide across the outer mitochondrial membrane

Strain-Minimizing Hyperbolic Network Embeddings with Landmarks

We introduce L-hydra (landmarked hyperbolic distance recovery and approximation), a method for embedding network- or distance-based data into hyperbolic space, which requires only the distance measurements to a few 'landmark nodes'. This landmark heuristic makes L-hydra applicable to large-scale graphs and improves upon previously introduced methods. As a mathematical justification, we show that a point configuration in d-dimensional hyperbolic space can be perfectly recovered (up to isometry) from distance measurements to just d+1 landmarks. We also show that L-hydra solves a two-stage strain-minimization problem, similar to our previous (unlandmarked) method 'hydra'. Testing on real network data, we show that L-hydra is an order of magnitude faster than existing hyperbolic embedding methods and scales linearly in the number of nodes. While the embedding error of L-hydra is higher than the error of existing methods, we introduce an extension, L-hydra+, which outperforms existing methods in both runtime and embedding quality