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To maintain cellular homeostasis, subcellular organelles communicate with each other and form physical and functional networks through membrane contact sites coupled by protein tethers. In particular, endoplasmic reticulum (ER)-mitochondrial contacts (EMC) regulate diverse cellular activities such as metabolite exchange (Ca2+ and lipids), intracellular signaling, apoptosis, and autophagy. The significance of EMCs has been highlighted by reports indicating that EMC dysregulation is linked to neurodegenerative diseases. Therefore, obtaining a better understanding of the physical and functional components of EMCs should provide new insights into the pathogenesis of several neurodegenerative diseases. Here, we applied engineered ascorbate peroxidase (APEX) to map the proteome at EMCs in live HEK293 cells. APEX was targeted to the outer mitochondrial membrane, and proximity-labeled proteins were analyzed by stable isotope labeling with amino acids in culture (SILAC)-LC/MS-MS. We further refined the specificity of the proteins identified by combining biochemical subcellular fractionation to the protein isolation method. We identified 405 proteins with a 2.0-fold cutoff ratio (log base 2) in SILAC quantification from replicate experiments. We performed validation screening with a Split-Rluc8 complementation assay that identified reticulon 1A (RTN1A), an ER-shaping protein localized to EMCs as an EMC promoter. Proximity mapping augmented with biochemical fractionation and additional validation methods reported here could be useful to discover other components of EMCs, identify mitochondrial contacts with other organelles, and further unravel their communication. [ABSTRACT FROM AUTHOR]

Flap endonuclease 1 (FEN1) is the enzyme responsible for specifically removing the flap structure produced during DNA replication, repair, and recombination. Here we report that the human replication factor C (RFC) complex stimulates the nuclease activity of human FEN1 in an ATP-independent manner. Although proliferating cell nuclear antigen is also known to stimulate FEN1, less RFC was required for comparable FEN1 stimulation. Kinetic analyses indicate that the mechanism by which RFC stimulates FEN1 is distinct from that by proliferating cell nuclear antigen. Heat-denatured RFC or its subunit retained, fully or partially, the ability to stimulate FEN1. Via systematic deletion analyses, we have defined three specific regions of RFC4 capable of stimulating FEN1. The region of RFC4 with the highest activity spans amino acids 170-194 and contains RFC box VII. Four amino acid residues (i.e. Tyr-182, Glu-188, Pro-189, and Ser-192) are especially important for FEN1 stimulatory activity. Thus, RFC, via several stimulatory motifs per molecule, potently activates FEN1. This function makes RFC a critical partner with FEN1 for the processing of eukaryotic Okazaki fragments. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae MPH1 was first identified as a gene encoding a 3′ to 5′p DNA helicase, which when deleted leads to a mutator phenotype. In this study, we isolated MPH1 as a multicopy suppressor of the dna2K1080E helicase-negative lethal mutant. Purified Mph1 stimulated the endonuclease activities of both Fen1 and Dna2, which act faithfully in the processing of Okazaki fragments. This stimulation required neither ATP hydrolysis nor the helicase activity of Mph1. Multicopy expression of MPH1 also suppressed the temperature-sensitive growth defects in cells expressing dna2Δ405N, which lacks the N-terminal 405 amino acids of Dna2. However, Mph1 did not stimulate the endonuclease activity of the Dna2Δ405N mutant protein. The stimulation of Fen1 by Mph1 was limited to flap-structured substrates; Mph1 hardly stimulated the 5′ to 3′ exonuclease activity of Fen1, Mph1 binds to flap- structured substrate more efficiently than to nicked duplex structures, suggesting that the stimulatory effect of Mph1 is exerted through its binding to DNA substrates. In addition, we found that Mph1 reversed the inhibitory effects of replication protein A on Fen1 activity. Our biochemical and genetic data indicate that the in vivo suppression of Dna2 defects observed with both dna2K1080E and dna2Δ405N mutants occur via stimulation of Fen1 activity. These findings suggest that Mph1 plays an important, although not essential, role in processing of Okazaki fragments by facilitating the formation of ligatable nicks. [ABSTRACT FROM AUTHOR]