The Schizosaccharomyces pombe PPR protein Ppr10 associates with a novel protein Mpa1 and acts as a mitochondrial translational activator

Abstract

The pentatricopeptide repeat (PPR) proteins characterized by tandem repeats of a degenerate 35-amino-acid motif function in all aspects of organellar RNA metabolism, many of which are essential for organellar gene expression. In this study, we report the characterization of a fission yeast Schizosaccharomyces pombe PPR protein, Ppr10 and a novel Ppr10-associated protein, designated Mpa1. The ppr10 deletion mutant exhibits growth defects in respiratory media, and is dramatically impaired for viability during the late-stationary phase. Deletion of ppr10 affects the accumulation of specific mitochondrial mRNAs. Furthermore, deletion of ppr10 severely impairs mitochondrial protein synthesis, suggesting that Ppr10 plays a general role in mitochondrial protein synthesis. Ppr10 interacts with Mpa1 in vivo and in vitro and the two proteins colocalize in the mitochondrial matrix. The ppr10 and mpa1 deletion mutants exhibit very similar phenotypes. One of Mpa1's functions is to maintain the normal protein level of Ppr10 protein by protecting it from degradation by the mitochondrial matrix protease Lon1. Our findings suggest that Ppr10 functions as a general mitochondrial translational activator, likely through interaction with mitochondrial mRNAs and mitochondrial translation initiation factor Mti2, and that Ppr10 requires Mpa1 association for stability and function.

Schizosaccharomyces pombe Mti2 and Mti3 act in conjunction during mitochondrial translation initiation

“…Therefore, we set out to determine whether deletion of mti2 or mti3 affected copy number of mtDNA. In order to determine quantitatively whether there was a change in the total levels of mtDNA in the Δ mti2 and Δ mti3 mutants, we measured mtDNA to nuclear DNA ratios by real‐time quantitative PCR (qPCR) and compared them with those in WT cells as previously reported . As a control, we also included the ptp1‐1 strain depleted of mtDNA .…”

“…Purification of mitochondria, protease protection, and isolation of soluble and membrane‐bound proteins in mitochondria by sodium carbonate extraction were performed as previously described .…”

“…Rabbit anti‐Cob1, anti‐Cox1, anti‐Cox2, anti‐Cox3, anti‐Atp8, anti‐Atp9, and anti‐HSP60 abs were prepared as described previously . Polyclonal abs against Mti2 (aa: 667–686: IVTGDKLQTFVEEYKPPDF), Mti3 (aa: 214–233: VNSHSATFLLQPLVSKSSEL), Mrp5 (aa: 368–387: IAMERGQKFVDVQREYYKQT), Mrpl40 (aa: 32–50: KIREHLNKSDPTVKDDKSA), and Mrpl1601 (aa: 37–54: GHQYAPRSIRNQKAQKGR) were raised in rabbits using synthetic peptides as antigens.…”

“…To analyze the steady‐state levels of mtDNA‐encoded proteins, mitochondrial extracts were prepared as described previously and total protein concentrations were determined with the BCA protein Assay Kit (Cwbiotech, Beijing, China). Mitochondrial proteins were fractionated by 12% SDS/PAGE.…”

“…However, the detailed mechanism remains to be determined. In S. pombe , two general mitochondrial translational activators, Ppr2 and Ppr10, and two mitochondrial mRNA‐specific translational activators, Ppr4 and Mtf2, have been found . However, their extract role in translation initiation remains to be determined.…”