Replication-Independent Histone Variant H3.3 Controls Animal Lifespan through the Regulation of Pro-longevity Transcriptional Programs.

Chromatin structure orchestrates the accessibility to the genetic material. Replication-independent histone variants control transcriptional plasticity in postmitotic cells. The life-long accumulation of these histones has been described, yet the implications on organismal aging remain elusive. Here, we study the importance of the histone variant H3.3 in Caenorhabditis elegans longevity pathways. We show that H3.3-deficient nematodes have negligible lifespan differences compared to wild-type animals. However, H3.3 is essential for the lifespan extension of C. elegans mutants in which pronounced transcriptional changes control longevity programs. Notably, H3.3 loss critically affects the expression of a very large number of genes in long-lived nematodes, resulting in transcriptional profiles similar to wild-type animals. We conclude that H3.3 positively contributes to diverse lifespan-extending signaling pathways, with potential implications on age-related processes in multicellular organisms.

The role of Djp1 in import of the mitochondrial protein Mim1 demonstrates specificity between a cochaperone and its substrate protein.

A special group of mitochondrial outer membrane proteins spans the membrane once, exposing soluble domains to both sides of the membrane. These proteins are synthesized in the cytosol and then inserted into the membrane by an unknown mechanism. To identify proteins that are involved in the biogenesis of the single-span model protein Mim1, we performed a high-throughput screen in yeast. Two interesting candidates were the cytosolic cochaperone Djp1 and the mitochondrial import receptor Tom70. Our results indeed demonstrate a direct interaction of newly synthesized Mim1 molecules with Tom70. We further observed lower steady-state levels of Mim1 in mitochondria from djp1Δ and tom70 tom71Δ cells and massive mislocalization of overexpressed GFP-Mim1 to the endoplasmic reticulum in the absence of Djp1. Importantly, these phenotypes were observed specifically for the deletion of DJP1 and were not detected in mutant cells lacking any of the other cytosolic cochaperones of the Hsp40 family. Furthermore, the djp1Δ tom70Δ tom71Δ triple deletion resulted in a severe synthetic sick/lethal growth phenotype. Taking our results together, we identified Tom70 and Djp1 as crucial players in the biogenesis of Mim1. Moreover, the involvement of Djp1 provides a unique case of specificity between a cochaperone and its substrate protein.

A crucial role for Mim2 in the biogenesis of mitochondrial outer membrane proteins.

Most of the mitochondrial outer membrane (MOM) proteins contain helical transmembrane domains. Some of the single-span proteins and all known multiple-span proteins are inserted into the membrane in a pathway that depends on the MOM protein Mitochondrial Import 1 (Mim1). So far it has been unknown whether additional proteins are required for this process. Here, we describe the identification and characterization of Mim2, a novel protein of the MOM that has a crucial role in the biogenesis of MOM helical proteins. Mim2 physically and genetically interacts with Mim1, and both proteins form the MIM complex. Cells lacking Mim2 exhibit a severely reduced growth rate and lower steady-state levels of helical MOM proteins. In addition, absence of Mim2 leads to compromised assembly of the translocase of the outer mitochondrial membrane (TOM complex), hampered mitochondrial protein import, and defects in mitochondrial morphology. In summary, the current study demonstrates that Mim2 is a novel central player in the biogenesis of MOM proteins.

Multispan mitochondrial outer membrane protein Ugo1 follows a unique Mim1-dependent import pathway.

The mitochondrial outer membrane (MOM) harbors several multispan proteins that execute various functions. Despite their importance, the mechanisms by which these proteins are recognized and inserted into the outer membrane remain largely unclear. In this paper, we address this issue using yeast mitochondria and the multispan protein Ugo1. Using a specific insertion assay and analysis by native gel electrophoresis, we show that the import receptor Tom70, but not its partner Tom20, is involved in the initial recognition of the Ugo1 precursor. Surprisingly, the import pore formed by the translocase of the outer membrane complex appears not to be required for the insertion process. Conversely, the multifunctional outer membrane protein mitochondrial import 1 (Mim1) plays a central role in mediating the insertion of Ugo1. Collectively, these results suggest that Ugo1 is inserted into the MOM by a novel pathway in which Tom70 and Mim1 contribute to the efficiency and selectivity of the process.

Mitochondria can recognize and assemble fragments of a beta-barrel structure.

ß-barrel proteins are found in the outer membranes of eukaryotic organelles of endosymbiotic origin as well as in the outer membrane of Gram-negative bacteria. Precursors of mitochondrial ß-barrel proteins are synthesized in the cytosol and have to be targeted to the organelle. Currently, the signal that assures their specific targeting to mitochondria is poorly defined. To characterize the structural features needed for specific mitochondrial targeting and to test whether a full ß-barrel structure is required, we expressed in yeast cells the ß-barrel domain of the trimeric autotransporter Yersinia adhesin A (YadA). Trimeric autotransporters are found only in prokaryotes, where they are anchored to the outer membrane by a single 12-stranded ß-barrel structure to which each monomer is contributing four ß-strands. Importantly, we found that YadA is solely localized to the mitochondrial outer membrane, where it exists in a native trimeric conformation. These findings demonstrate that, rather than a linear sequence or a complete ß-barrel structure, four ß-strands are sufficient for the mitochondria to recognize and assemble a ß-barrel protein. Remarkably, the evolutionary origin of mitochondria from bacteria enables them to import and assemble even proteins belonging to a class that is absent in eukaryotes.

Signals in bacterial beta-barrel proteins are functional in eukaryotic cells for targeting to and assembly in mitochondria.

The outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts harbor beta-barrel proteins. The signals that allow precursors of such proteins to be targeted to mitochondria were not characterized so far. To better understand the mechanism by which beta-barrel precursor proteins are recognized and sorted within eukaryotic cells, we expressed the bacterial beta-barrel proteins PhoE, OmpA, Omp85, and OmpC in Saccharomyces cerevisiae and demonstrated that they were imported into mitochondria. A detailed investigation of the import pathway of PhoE revealed that it is shared with mitochondrial beta-barrel proteins. PhoE interacts initially with surface import receptors, and its further sorting depends on components of the TOB/SAM complex. The bacterial Omp85 and PhoE integrated into the mitochondrial outer membrane as native-like oligomers. For the latter protein this assembly depended on the C-terminal Phe residue, which is important also for the correct assembly of PhoE into the bacterial outer membrane. Collectively, it appears that mitochondrial beta-barrel proteins have not evolved eukaryotic-specific signals to ensure their import into mitochondria. Furthermore, the signal for assembly of beta-barrel proteins into the bacterial outer membrane is functional in mitochondria.