In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 49 ( 2012-12-04)
Abstract:
In conclusion, our investigation demonstrated that comparative analysis of S. cerevisiae and C. albicans is of great utility in exploring how and why protein import pathways function, how they change through evolution, and how mitochondrial biogenesis is regulated with respect to the cell-division cycle and metabolic control. Finally, our study addressed whether a given protein uses the same route for entry into mitochondria in all organisms. We would have answered “yes” but instead found evidence of evolutionary rewiring of import routes. The cytochromes b 2 and c 1 , required by all yeasts for a comprehensive oxidative phosphorylation capability to generate ATP from diverse environmental carbon sources ( Fig. P1 ), have different targeting sequences to follow distinct pathways for entry into mitochondria in C. albicans and S. cerevisiae . This difference suggests further examples of rewiring, wherein specific targeting sequences have been adapted through evolution distinctly to drive the most effective localization of a given protein, thus ensuring its efficient import into mitochondria. Differences between S. cerevisiae and C. albicans related to the properties of Sam35 revealed that Sam35 is a receptor that binds the targeting sequences in newly imported β-barrel proteins and thereby engages them into the SAM complex for assembly into the outer membrane ( Fig. P1 ). This receptor function must occur on the inner face of the outer membrane, although Sam35 is exposed on the outer face of the outer membrane. We found that in C. albicans Sam35 is an integral membrane protein, a topology consistent with its being largely exposed on the outer face of the outer membrane but also being at least partially in the intermembrane space, thus explaining how it can bind substrates for SAM. We further showed that assembly of the β-barrel protein Tom40 into TOM, a process catalyzed by SAM, is extremely rapid in C. albicans and that Sam50, Sam51, Sam35, and Sam37 all play roles in this assembly. Assays of protein import into mitochondria isolated from C. albicans revealed that the voltage-dependent anion channel (VDAC) is located in the outer mitochondrial membrane where it allows small molecules such as ATP to be distributed from the mitochondria to the rest of the cell. In S. cerevisiae , individual VDAC monomers can form a molecular sieve for rapid exchange of small molecules. In C. albicans , a more static picture is apparent with a single, stable oligomeric form of VDAC predominating, so that C. albicans is less able to increase rapidly the mobilization of ATP from out of its mitochondria. We first searched the genome of C. albicans for factors similar to the protein import apparatus of S. cerevisiae and found common elements but with several differences, particularly in two key molecular machines in the mitochondrial outer membrane, the translocase of the outer membrane (TOM) and mitochondrial sorting and assembly machinery (SAM) complexes. TOM forms a channel in the outer mitochondrial membrane that serves as the gateway for protein import into mitochondria, and SAM assembles integral membrane proteins with a β-barrel architecture into the mitochondrial outer membrane. C. albicans lacks Tom70, which is replaced by a related protein, Tom71; the sequences of Tom5, Tom6, and Tom7 differ in their cytosolic domains, which serve as phosphorylation sites in S. cerevisiae ( 3 ). We found Sam51 in C. albicans , and genomics analyses revealed that Sam50 and Sam51 are related in ancestry and that Sam51 and Sam50 are present in a wide variety of yeasts, but not S. cerevisiae . The pathways for protein import into mitochondria are mediated by a series of molecular machines in the mitochondrial membranes ( 1 , 2 ). Studies of the yeast Saccharomyces cerevisiae showed that the activity of protein import in response to metabolic demand is modified by protein phosphorylation ( 3 ). When S. cerevisiae grows optimally, it ferments sugars anaerobically regardless of oxygen availability and produces ethanol when mitochondrial function is inhibited. Moreover, S. cerevisiae responds to glucose through metabolic cycling, with mitochondrial activity and mitochondrial biogenesis cordoned into specific metabolic phases ( 4 , 5 ). The human pathogen Candida albicans , which is separated from S. cerevisiae by 300 million years of evolution, uses oxygen and maintains mitochondrial function when growing on glucose. We showed here that C. albicans does not display metabolic cycling, so the regulation of mitochondrial activity in C. albicans is different from that than in S. cerevisiae . All eukaryotic cells have mitochondria derived through evolution from an intracellular bacterium. Only remnants of the ancestral bacterial genome remain in these organelles. Thus, in each round of the cell division cycle, the building of new mitochondria requires the import and assembly of proteins that are encoded by genes in the nucleus and synthesized in the cytosol ( Fig. P1 ). Here, we present insights into the function, evolution, and regulation of mitochondrial biogenesis through the study of the human fungal pathogen Candida albicans . Analysis of targeting sequences and assays of mitochondrial protein import showed that components of the electron transport chain are imported by evolutionary rewiring of mitochondrial import pathways.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1206345109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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