ABSTRACT
Centromere function requires the presence of the histone H3 variant CENP-A in most eukaryotes. The precise localization and protein amount of CENP-A are crucial for correct chromosome segregation, and misregulation can lead to aneuploidy. To characterize the loading of CENP-A to non-centromeric chromatin, we utilized different truncation- and localization-deficient CENP-A mutant constructs in Drosophila melanogaster cultured cells, and show that the N-terminus of Drosophila melanogaster CENP-A is required for nuclear localization and protein stability, and that CENP-A associated proteins, rather than CENP-A itself, determine its localization. Co-expression of mutant CENP-A with its loading factor CAL1 leads to exclusive centromere loading of CENP-A whereas co-expression with the histone-binding protein RbAp48 leads to exclusive non-centromeric CENP-A incorporation. Mass spectrometry analysis of non-centromeric CENP-A interacting partners identified the RbAp48-containing NuRD chromatin remodeling complex. Further analysis confirmed that NuRD is required for ectopic CENP-A incorporation, and RbAp48 and MTA1-like subunits of NuRD together with the N-terminal tail of CENP-A mediate the interaction. In summary, our data show that Drosophila CENP-A has no intrinsic specificity for centromeric chromatin and utilizes separate loading mechanisms for its incorporation into centromeric and ectopic sites. This suggests that the specific association and availability of CENP-A interacting factors are the major determinants of CENP-A loading specificity.
Subject(s)
Centromere Protein A/metabolism , Centromere/metabolism , Chromatin Assembly and Disassembly/physiology , Drosophila Proteins/metabolism , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Animals , Cells, Cultured , Drosophila Proteins/genetics , Drosophila melanogaster , Protein Domains , Retinoblastoma-Binding Protein 4/genetics , Retinoblastoma-Binding Protein 4/metabolism , Trans-Activators/metabolismABSTRACT
Aggregation processes can cause severe perturbations of cellular homeostasis and are frequently associated with diseases. We performed a comprehensive analysis of mitochondrial quality and function in the presence of aggregation-prone polypeptides. Despite a significant aggregate formation inside mitochondria, we observed only a minor impairment of mitochondrial function. Detoxification of aggregated reporter polypeptides as well as misfolded endogenous proteins inside mitochondria takes place via their sequestration into a specific organellar deposit site we termed intramitochondrial protein quality control compartment (IMiQ). Only minor amounts of endogenous proteins coaggregated with IMiQ deposits and neither resolubilization nor degradation by the mitochondrial protein quality control system were observed. The single IMiQ aggregate deposit was not transferred to daughter cells during cell division. Detoxification of aggregates via IMiQ formation was highly dependent on a functional mitochondrial fission machinery. We conclude that the formation of an aggregate deposit is an important mechanism to maintain full functionality of mitochondria under proteotoxic stress conditions.
