ABSTRACT
The Fanconi anemia DNA repair pathway is pivotal for the efficient repair of DNA interstrand cross-links. Here, we show that FA-defective (Fancc(-)) DT40 cells arrest in G2 phase following cross-link damage and trigger apoptosis. Strikingly, cell death was reduced in Fancc(-) cells by additional deletion of the BRCA1 tumor suppressor, resulting in elevated clonogenic survival. Increased resistance to cross-link damage was not due to loss of toxic BRCA1-mediated homologous recombination but rather through the loss of a G2 checkpoint. This proapoptotic role also required the BRCA1-A complex member ABRAXAS (FAM175A). Finally, we show that BRCA1 promotes G2 arrest and cell death by prolonging phosphorylation of Chk1 on serine 345 after DNA damage to sustain arrest. Our data imply that DNA-induced cross-link death in cells defective in the FA pathway is dependent on the ability of BRCA1 to prolong cell cycle arrest in G2 phase.
Subject(s)
Avian Proteins/metabolism , BRCA1 Protein/metabolism , DNA Repair , G2 Phase Cell Cycle Checkpoints , Protein Kinases/metabolism , Animals , Apoptosis , Avian Proteins/genetics , BRCA1 Protein/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line , Checkpoint Kinase 1 , Chickens , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Fanconi Anemia Complementation Group C Protein/genetics , Fanconi Anemia Complementation Group C Protein/metabolism , Gene Deletion , PhosphorylationABSTRACT
The vast majority of proteins trafficking across or into the bacterial cytoplasmic membrane occur via the translocon. The translocon consists of the SecYEG complex that forms an evolutionarily conserved heterotrimeric protein-conducting membrane channel that functions in conjunction with a variety of ancillary proteins. For posttranslational protein translocation, the translocon interacts with the cytosolic motor protein SecA that drives the ATP-dependent stepwise translocation of unfolded polypeptides across the membrane. For the cotranslational integration of membrane proteins, the translocon interacts with ribosome-nascent chain complexes and membrane insertion is coupled to polypeptide chain elongation at the ribosome. These processes are assisted by the YidC and SecDF(yajC) complex that transiently interacts with the translocon. This review summarizes our current understanding of the structure-function relationship of the translocon and its interactions with ancillary components during protein translocation and membrane protein insertion. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Protein Transport , SEC Translocation Channels , SecA Proteins , Structure-Activity RelationshipABSTRACT
The SecYEG translocon of Escherichia coli mediates the translocation of preproteins across the cytoplasmic membrane. Here, we have examined the role of the proposed lateral gate of the translocon in translocation. A dual cysteine cross-linking approach allowed the introduction of cross-linker arms of various lengths between adjoining aminoacyl positions of transmembrane segments 2b and 7 of the lateral gate. Oxidation and short spacer linkers that fix the gate in the closed state abolished preprotein translocation, whereas long spacer linkers support translocation. The cross-linking data further suggests that SecYEG lateral gate opening and activation of the SecA ATPase are coupled processes. It is concluded that lateral gate opening is a critical step during SecA-dependent protein translocation.
Subject(s)
Bacterial Proteins/genetics , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Bacterial Proteins/metabolism , Cysteine/metabolism , Cytoplasm/metabolism , Disulfides/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Euryarchaeota/genetics , Euryarchaeota/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Peptide Hydrolases/metabolism , Porins/metabolism , Protein Conformation , Protein Precursors/genetics , Protein Precursors/metabolism , Protein Transport/genetics , SEC Translocation ChannelsABSTRACT
The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that facilitate the insertion of membrane proteins. Depletion of YidC in E. coli leads to a specific defect in the functional assembly of major energy transducing complexes such as the F1F0 ATPase and cytochrome bo3 oxidase. Here we report on the in vitro reconstitution of the membrane insertion of the CyoA subunit of cytochrome bo3 oxidase. Efficient insertion of in vitro synthesized pre-CyoA into proteoliposomes requires YidC, SecYEG, and SecA and occurs independently of the proton motive force. These data demonstrate that pre-CyoA is a substrate of a novel pathway that involves both SecYEG and YidC.