A 22kDa protein specific for yeast mitochondrial nucleoids is an unidentified putative ribosomal protein encoded in open reading frame YGL068W

Abstract

Mitochondrial-nucleoid (mt-nucleoid) proteins of the yeast Saccharomyces cerevisiae were separated by two-dimensional gel electrophoresis. Analysis of the N-terminal amino acid sequence showed that a 22 kDa protein which is unique in the mt-nucleoid fraction is an unidentified protein encoded in the open reading frame YGL068W and shows a homology with the ribosomal protein L7/L12 of bacteria. We named this protein Mnp1p (for the mitochondrial-nucleoid protein 1). Immunoblotting of each fraction with an anti-Mnp1p antibody during the mt-nucleoid isolation showed that Mnp1p is highly concentrated in the mt-nucleoid fraction. Immunofluorescence microscopy suggested that Mnp1p is localized to mitochondria in vivo, and a significant amount of Mnp1p is associated with the mt-nucleoids. On the other hand, Northern blotting showed that a large amount of large and small mitochondrial ribosomal RNAs was not associated with the mt-nucleoids and remained in the supernatant after the isolation of mt-nucleoids. The null mutation of MNP1 led to a respiratory-deficient phenotype, but the morphology of the mt-nucleoids in the transformants carrying the null mutation was normal. These results suggest that a significant amount of Mnp1p plays a role as a major component of the mt-nucleoids.

The Novel ATP-Binding Cassette Protein ARB1 Is a Shuttling Factor That Stimulates 40S and 60S Ribosome Biogenesis

“…The proteins copurifying with TAP-ARB1 were identified by multidimensional microcapillary liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (43). We identified 43 proteins with a PAF of 3 or greater for the tagged sample and a PAF of 0 for the untagged control sample (see Table S2 in the supplemental material) (3,5,9,13,18,23,27,28,36,41,54,56,60,64,75,77,78). The PAF expresses the number of nonredundant peptides identified by mass spectrometry normalized for the molecular weight of the cognate protein.…”

Dynamic Changes in Mitochondrial Nucleoids during the Transition from Anaerobic to Aerobic Culture in the Yeast Saccharomyces cerevisiae

“…Subsequently, we have measured the exact DNA content of individual mt-nucleoids by a combination of DAPI staining and a VIMPCS (Miyakawa et al 2004). These studies together demonstrated that giant mt-nucleoids in anaerobically grown cells are extraordinarily large mtDNA-protein complexes that are each composed of about 20 mtDNA molecules and a number of protein components.…”

“…Therefore, Abf2p may be of primary importance in the organization of mtDNA into the mt-nucleoid structure under both aerobic and anaerobic conditions. On the other hand, the identification of novel mt-nucleoid proteins other than Abf2p is in progress, and unexpected proteins including citric acid cycle enzymes and a mitochondrial ribosomal protein have been detected as components of mt-nucleoids (Kaufman et al 2000, Sato et al 2002, Sato and Miyakawa 2004, Chen et al 2005. It is possible that both the 67-and 52-kDa proteins have some roles in regulating aggregation in mt-nucleoids.…”

“…That was the first report showing that the protein composition of yeast mt-nucleoids changes depending on the culture conditions of yeast cells. The DNA contents of individual mt-nucleoids in cells and in isolated mtnucleoids were quantitatively measured by a combination of DAPI staining and a video-intensified microscope photon counting system (VIMPCS) (Miyakawa et al 2004). We have shown that each giant mt-nucleoid in anaerobically grown cells contains about 20 mtDNA molecules, while each small mt-nucleoid in aerobic cells cultured in the same culture medium contains only 1-2 mtDNA molecules.…”

Morphology and protein composition of the mitochondrial nucleoids in yeast cells lacking Abf2p, a high mobility group protein

“…In addition, the mtnucleoids contain many other proteins Nosek et al, 2006), which include three components of α-ketoglutarate dehydrogenase (Kaufman et al, 2000;Sato et al, 2002) and mitochondrial ribosomal protein Mnp1p (Sato and Miyakawa, 2004). A high-resolution atomic force microscopy indicated that the interaction between Abf2p and mtDNA is rather weak, compared to that between nuclear DNA and histones, and that Abf2p compacts DNA by simply introducing a number of sharp bends into the DNA backbone (Brewer et al, 2003;Friddle et al, 2004).…”