Mitochondrial Biogenesis

Mitochondria are energy generating organelles of eukaryotic cells believed to have originated from a symbiotic association between an oxidative bacterium and a glycolytic proto-eukaryotic cell. The endosymbiotic origin of mitochondria is reflected in its bilayered membrane structure, about 86 kb circular genome, organelle-specific transcription and translation and protein assembly systems (Tzagoloff and Myers, 1986). However, during the stabilization of the symbiotic association, the majority of the mitochondrial genes were transferred to the nuclear genome. Recent analysis has revealed that around 477 proteins are required for mitochondrial function in yeast of which only 17 are coded by the mitochondrial genome; the remaining are nuclear encoded. This partial genetic autonomy of the mitochondria is borne out from the analysis of yeast mit- mutants that had point mutations or small deletions in the mtDNA. A second class of mutants was isolated that had a petite phenotype (pet mutants forming small colonies) when grown on a non-fermentable source of carbon such as glycerol, ethanol and lactate. Genetic analysis of these mutants revealed that unlike mit- mutants, pet genes were nuclear. Hundreds of pet genes were identified by exhaustive genetic screens and shown to regulate mitochondrial transcription, translation and assembly of the electron transport chain. More recently, yeast deletion mutants were used for the identification of new pet genes (Steinmetz et al., 2002). Briefly, 4706 homozygous deletion mutants were grown in nine different growth media and their growth characteristics in non-fermentable carbon sources analyzed to identify

1, Protein synthesis (B5)

3. Respiratory chain (21)

Unknown^ function (60)

1, Protein synthesis (B5)

3. Respiratory chain (21)

5. DNA and RNA metabolism (21) Assembly factors (17)

Figure 6.10 Participation of pet genes in biological processes. Genes were annotated using Gene Ontology (GO) resources (

pet mutants. Altogether, 341 pet genes belonging to different classes were identified. The function of 185 mitochondria-specific genes is shown in Figure 6.10. About half of the genes participate in protein synthesis, which is reasonable considering the fact that about 95% of mitochondrial proteins are coded by the nuclear genome.

That both nuclear and mitochondrial genes function together in mitochondrial biogenesis came from the work of Schatz's group (Schatz, 2001). They demonstrated that when yeast cells are grown under anaerobic conditions, their mitochondria are devoid of cytochromes and several other proteins and are difficult to detect by electron microscopy. These structures called proto-mitochondria are converted into functional mitochondria when cells are shifted to aerobic condition. This reversible process became a useful system to demonstrate that the assembly of the electron transport chain on the mitochondrial inner-membrane required the participation of both nuclear and mitochondrial genes. Especially noteworthy was the finding that in the absence of mitochondrial protein translation, cytochrome-c1 encoded by the nucleus became highly susceptible to proteolysis (Ross and Schatz, 1976). The increased susceptibility was not due to increased synthesis of proteases by the petite mutant but due to improper assembly and incorporation of cytochrome-c1 on the mitochon-drial membrane. This finding led to the discovery and characterization of an elaborate system by which proteins coded by the nuclear genes finally reach the mitochondrial matrix after traversing the double bilayered membrane of the organelle.

In the early 1990s, a breakthrough in the area of mitochondrial protein import was made by research groups of Gottfried Schatz and Walter Neupert, who established methods for the isolation of mitochondria from yeast cells and set up in vitro mitochondrial translocation assays using labeled proteins (Sollner et al., 1989). Briefly, mitochondria from yeast cells are mixed with proteins labeled with 35S-methionine by in vitro transcription-translation in rabbit reticulocyte lysates. Following incubation, mitochondria are isolated by density gradient centrifugation and the fate of the labeled proteins analyzed by biochemical and microscopic techniques. A common method involves the treatment of the incubation mixture with proteases and examination of the fate of the labeled proteins by gel electrophoresis. Complete translocation of the protein into the mitochondria renders them resistant to proteolytic digestion. Three significant conclusions were reached from these early studies. First, the mitochondrial outer membrane bears the import receptors that bind proteins destined to the mitochondria. Second, the proteins destined to the mitochondria carry import signals that are recognized by the import receptors. Third, mitochondrial protein import is an energy-driven process requiring ATP and a potential gradient across the mitochondrial membranes. In the last decade, the biochemical components of this complex process have been identified. Two specific receptor complexes have been characterized that reside on each of the two membranes. The "Tom" complex (translocase of outer membrane) and the "Tim" complex (translocase of inner membrane) form the translocation pore through which proteins are translocated from cytosol into mitochondria. About 30 proteins are present in these two complexes. Proteins destined to the mitochondria are unfolded before they can be recognized by the components of the "Tom" complex. The heat shock protein hsp70 and chaperone protein MSF mediate the process of unfolding and the stabilization of the unfolded protein by using the energy of ATP hydrolysis. Once the protein traverses the outer membrane, the "Tim" complex threads it in. Proteins residing in the mitochondrial lumen carry a mitochondrial import signal at their N-terminus that is processed by luminal peptidases (Wiedemann, et al. 2004). A simplified view of the mitochondrial protein import process is shown in Figure 6.11.

Like the eukaryotic cell cycle, mitochondrial protein import too is a conserved biochemical event. This concept is supported by functional complementation of yeast mitochondrial import mutants by mammalian genes and also from sequence similarities between yeast and mammalian mitochondrial import proteins. Significantly, function of several human disease genes has been revealed by the function of their yeast homologs (Steinmetz et al., 2002). As an example, when the gene associated with human deafness dystonia syndrome was cloned by positional cloning, no function could be assigned to the DDP1 peptide except that it had an N-terminal mitochondrial localization signal. Soon, however, a family of proteins from yeast bearing striking homology to DDP1 was characterized as components of the "Tim" complex. The human protein has been shown to function as a part of the yeast protein import machinery (Foury and Kucej, 2002). So far, about 102 human diseases have been attributed to defects in mitochondrial function. The yeast system offers a great tool to analyze the function of these human genes (Koutnikova et al., 1997).

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