Cox25 teams up with Mss51, Ssc1, and Cox14 to regulate mitochondrial cytochrome c oxidase subunit 1 expression and assembly in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, mitochondrial cytochrome c oxidase (COX) biogenesis is translationally regulated. Mss51, a specific COX1 mRNA translational activator and Cox1 chaperone, drives the regulatory mechanism. During translation and post-translationally, newly synthesized Cox1 physically interacts with a complex of proteins involving Ssc1, Mss51, and Cox14, which eventually hand over Cox1 to the assembly pathway. This step is probably catalyzed by assembly chaperones such as Shy1 in a process coupled to the release of Ssc1-Mss51 from the complex. Impaired COX assembly results in the trapping of Mss51 in the complex, thus limiting its availability for COX1 mRNA translation. An exception is a null mutation in COX14 that does not affect Cox1 synthesis because the Mss51 trapping complexes become unstable, and Mss51 is readily available for translation. Here we present evidence showing that Cox25 is a new essential COX assembly factor that plays some roles similar to Cox14. A null mutation in COX25 by itself or in combination with other COX mutations does not affect Cox1 synthesis. Cox25 is an inner mitochondrial membrane intrinsic protein with a hydrophilic C terminus protruding into the matrix. Cox25 is an essential component of the complexes containing newly synthesized Cox1, Ssc1, Mss51, and Cox14. In addition, Cox25 is also found to interact with Shy1 and Cox5 in a complex that does not contain Mss51. These results suggest that once Ssc1-Mss51 are released from the Cox1 stabilization complex, Cox25 continues to interact with Cox14 and Cox1 to facilitate the formation of multisubunit COX assembly intermediates.     

Coa1 links the Mss51 post-translational function to Cox1 cofactor insertion in cytochrome c oxidase assembly

The assembly of cytochrome c oxidase (CcO) in yeast mitochondria is shown to be dependent on a new assembly factor designated Coa1 that associates with the mitochondrial inner membrane. Translation of the mitochondrial-encoded subunits of CcO occurs normally in coa1Delta cells, but these subunits fail to accumulate. The respiratory defect in coa1Delta cells is suppressed by high-copy MSS51, MDJ1 and COX10. Mss51 functions in Cox1 translation and elongation, whereas Cox10 participates in the biosynthesis of heme a, a key cofactor of CcO. Respiration in coa1Delta and shy1Delta cells is enhanced when Mss51 and Cox10 are coexpressed. Shy1 has been implicated in formation of the heme a3-Cu(B) site in Cox1. The interaction between Coa1 and Cox1, and the physical and genetic interactions between Coa1 and Mss51, Shy1 and Cox14 suggest that Coa1 coordinates the transition of newly synthesized Cox1 from the Mss51:Cox14 complex to the heme a cofactor insertion involving Shy1. coa1Delta cells also display a mitochondrial copper defect suggesting that Coa1 may have a direct link to copper metallation of CcO.     

Two independent activities define Ccm1p as a moonlighting protein in Saccharomyces cerevisiae

Ccm1p is a nuclear-encoded PPR (pentatricopeptide repeat) protein that localizes into mitochondria of Saccharomyces cerevisiae. It was first defined as an essential factor to remove the bI4 [COB (cytochrome b) fourth intron)] and aI4 [COX1 (cytochrome c oxidase subunit 1) fourth intron] of pre-mRNAs, along with bI4 maturase, a protein encoded by part of bI4 and preceding exons that removes the intronic RNA sequence that codes for it. Later on, Ccm1p was described as key to maintain the steady-state levels of the mitoribosome small subunit RNA (15S rRNA). bI4 maturase is produced inside the mitochondria and therefore its activity depends on the functionality of mitochondrial translation. This report addresses the dilemma of whether Ccm1p supports bI4 maturase activity by keeping steady-state levels of 15S rRNA or separately and directly supports bI4 maturase activity per se. Experiments involving loss of Ccm1p, SMDC (sudden mitochondrial deprivation of Ccm1p) and mutations in one of the PPR (pentatricopeptide repeat) motifs revealed that the failure of bI4 maturase activity in CCM1 deletion mutants was not due to a malfunction of the translational machinery. Both functions were found to be independent, defining Ccm1p as a moonlighting protein. bI4 maturase activity was significantly more dependent on Ccm1p levels than the maintenance of 15S rRNA. The novel strategy of SMDC described here allowed the study of immediate short-term effects, before the mutant phenotype was definitively established. This approach can be also applied for further studies on 15S rRNA stability and mitoribosome assembly.     

Ty1 transposition induced by carcinogens in Saccharomyces cerevisiae yeast depends on mitochondrial function

The transposition of the Ty mobile genetic element of Saccharomyces cerevisiae is induced by carcinogens. While the molecular background of spontaneous Ty1 transposition is well understood, the detailed mechanism of carcinogen induced Ty1 transposition is not clear. We found that mitochondrial functions participate in the Ty induced transposition induced by carcinogens. Contrary to the parental rho(+) cells rho(-) mutants (spontaneous or induced by ethidium bromide) do not increase the rate of Ty1 transposition upon treatment with carcinogens. Preliminary results strongly suggest that the absence of oxidative phosphorylation in rho(-) mutants is the reason for the inhibited Ty transposition. The lack of carcinogen induced Ty1 transposition in rho(-) cells is not specific for a particular carcinogen and represents a general feature of different carcinogenic substances inducing rho(-). It is concluded that carcinogen induced Ty1 transposition depends on the functional state of mitochondria and cannot take place in cells with compromised mitochondrial function (rho(-)).     

Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity

We previously cloned RRP14/YKL082c, whose product exhibits two-hybrid interaction with Ebp2p, a regulatory factor of assembly of 60S ribosomal subunits. Depletion of Rrp14p results in shortage of 60S ribosomal subunits and retardation of processing from 27S pre-rRNA to 25S rRNA. Furthermore, 35S pre-rRNA synthesis appears to decline in Rrp14p-depleted cells. Rrp14p interacts with regulatory factors of 60S subunit assembly and also with Utp11p and Faf1p, which are regulatory factors required for assembly of 40S ribosomal subunits. We propose that Rrp14p is involved in ribosome synthesis from the beginning of 35S pre-rRNA synthesis to assembly of the 60S ribosomal subunit. Disruption of RRP14 causes an extremely slow growth rate of the cell, a severe defect in ribosome synthesis, and a depolarized localization of cortical actin patches throughout the cell cycle. These results suggest that Rrp14p has dual functions in ribosome synthesis and polarized cell growth.     

Saccharomyces cerevisiae Pra1p/Yip3p interacts with Yip1p and Rab proteins.

The regulation of membrane traffic involves the Rab family of Ras-related GTPases, of which there are a total of 11 members in the yeast Saccharomyces cerevisiae. Previous work has identified PRA1 as a dual prenylated Rab GTPase and VAMP2 interacting protein [Martinic et al. (1999) J. Biol. Chem. 272, 26991-26998]. In this study we demonstrate that the yeast counterpart of PRA1 interacts with Rab proteins and with Yip1p, a membrane protein of unknown function that has been reported to interact specifically with the Rab proteins Ypt1p and Ypt31p. Yeast Pra1p/Yip3p is a factor capable of biochemical interaction with a panel of different Rab proteins and does not show in vitro specificity for any particular Rab. The interactions between Pra1p/Yip3p and Rab proteins are dependent on the presence of the Rab protein C-terminal cysteines and require C-terminal prenylation.     

Mmm1p, a mitochondrial outer membrane protein, is connected to mitochondrial DNA (mtDNA) nucleoids and required for mtDNA stability

In the yeast Saccharomyces cerevisiae, mitochondria form a branched, tubular reticulum in the periphery of the cell. Mmm1p is required to maintain normal mitochondrial shape and in mmm1 mutants mitochondria form large, spherical organelles. To further explore Mmm1p function, we examined the localization of a Mmm1p-green fluorescent protein (GFP) fusion in living cells. We found that Mmm1p-GFP is located in small, punctate structures on the mitochondrial outer membrane, adjacent to a subset of matrix-localized mitochondrial DNA nucleoids. We also found that the temperature-sensitive mmm1-1 mutant was defective in transmission of mitochondrial DNA to daughter cells immediately after the shift to restrictive temperature. Normal mitochondrial nucleoid structure also collapsed at the nonpermissive temperature with similar kinetics. Moreover, we found that mitochondrial inner membrane structure is dramatically disorganized in mmm1 disruption strains. We propose that Mmm1p is part of a connection between the mitochondrial outer and inner membranes, anchoring mitochondrial DNA nucleoids in the matrix.     

Upstream open reading frames regulate the cell cycle-dependent expression of the RNA helicase Rok1 in Saccharomyces cerevisiae

The RNA helicase Rok1 plays a role in rRNA processing and in control of cell cycle progression in Saccharomyces cerevisiae. We identified two upstream open reading frames (uORFs) within the ROK1 5′ untranslated region, which inhibited Rok1 translation. Mutating uATG to uAAG or generation of a premature stop codon in the uORFs resulted in increased Rok1p levels. Rok1 protein levels oscillated during the cell cycle, declining at G1/S and increasing at G2. The uAAG1 mutation caused a constitutive level of Rok1 proteins throughout the cell cycle, resulting in significant delays in mitotic bud emergence and recovery from pheromone arrest. Our study reveals that the Rok1 protein level is regulated by uORFs, which is critical in cell cycle progression.     

Site-1 protease inhibits mitochondrial respiration by controlling the TGF-β target gene Mss51

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Lack of GTP-bound Rho1p in secretory vesicles of Saccharomyces cerevisiae

Rho1p, an essential Rho-type GTPase in Saccharomyces cerevisiae, activates its effectors in the GTP-bound form. Here, we show that Rho1p in secretory vesicles cannot activate 1,3-beta-glucan synthase, a cell wall synthesizing enzyme, during vesicular transport to the plasma membrane. Analyses with an antibody preferentially reacting with the GTP-bound form of Rho1p revealed that Rho1p remains in the inactive form in secretory vesicles. Rom2p, the GDP/GTP exchange factor of Rho1p, is preferentially localized on the plasma membrane even when vesicular transport is blocked. Overexpression of Rom2p results in delocalization of Rom2p and accumulation of 1,3-beta-glucan in secretory vesicles. Based on these results, we propose that Rho1p is kept inactive in intracellular secretory organelles, resulting in repression of the activity of the cell wall-synthesizing enzyme within cells.     

Population structure of mitochondrial genomes in Saccharomyces cerevisiae

Background

Rigorous study of mitochondrial functions and cell biology in the budding yeast, Saccharomyces cerevisiae has advanced our understanding of mitochondrial genetics. This yeast is now a powerful model for population genetics, owing to large genetic diversity and highly structured populations among wild isolates. Comparative mitochondrial genomic analyses between yeast species have revealed broad evolutionary changes in genome organization and architecture. A fine-scale view of recent evolutionary changes within S. cerevisiae has not been possible due to low numbers of complete mitochondrial sequences.

Results

To address challenges of sequencing AT-rich and repetitive mitochondrial DNAs (mtDNAs), we sequenced two divergent S. cerevisiae mtDNAs using a single-molecule sequencing platform (PacBio RS). Using de novo assemblies, we generated highly accurate complete mtDNA sequences. These mtDNA sequences were compared with 98 additional mtDNA sequences gathered from various published collections. Phylogenies based on mitochondrial coding sequences and intron profiles revealed that intraspecific diversity in mitochondrial genomes generally recapitulated the population structure of nuclear genomes. Analysis of intergenic sequence indicated a recent expansion of mobile elements in certain populations. Additionally, our analyses revealed that certain populations lacked introns previously believed conserved throughout the species, as well as the presence of introns never before reported in S. cerevisiae.

Conclusions

Our results revealed that the extensive variation in S. cerevisiae mtDNAs is often population specific, thus offering a window into the recent evolutionary processes shaping these genomes. In addition, we offer an effective strategy for sequencing these challenging AT-rich mitochondrial genomes for small scale projects.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1664-4) contains supplementary material, which is available to authorized users.     

The Paf1 Complex Subunit Rtf1 Buffers Cells Against the Toxic Effects of [PSI+] and Defects in Rkr1-Dependent Protein Quality Control in Saccharomyces cerevisiae

The Rtf1 subunit of the Paf1 complex is required for specific histone modifications, including histone H2B lysine 123 monoubiquitylation. In Saccharomyces cerevisiae, deletion of RTF1 is lethal in the absence of Rkr1, a ubiquitin-protein ligase involved in the destruction of nonstop proteins, which arise from mRNAs lacking stop codons or translational readthrough into the poly(A) tail. We performed a transposon-based mutagenesis screen to identify suppressors of rtf1Δ rkr1Δ lethality and found that a mutation in the gene encoding the protein chaperone Hsp104 rescued viability. Hsp104 plays a role in prion propagation, including the maintenance of [PSI(+)], which contributes to the synthesis of nonstop proteins. We demonstrate that rtf1Δ and rkr1Δ are synthetically lethal only in the presence of [PSI(+)]. The deletion, inactivation, and overexpression of HSP104 or the overexpression of prion-encoding genes URE2 and LSM4 clear [PSI(+)] and rescue rtf1Δ rkr1Δ lethality. In addition, the presence of [PSI(+)] decreases the fitness of rkr1Δ strains. We investigated whether the loss of RTF1 exacerbates an overload in nonstop proteins in rkr1Δ [PSI(+)] strains but, using reporter plasmids, found that rtf1Δ decreases nonstop protein levels, indicating that excess nonstop proteins may not be the cause of synthetic lethality. Instead, our data suggest that the loss of Rtf1-dependent histone modifications increases the burden on quality control pathways in cells lacking Rkr1 and containing [PSI(+)].     

Novel Ree1 regulates the expression of ENO1 via the Snf1 complex pathway in Saccharomyces cerevisiae

Using cDNA microarray analysis, we found that the mRNA of YJL217W and several other genes related to cell wall organization and biogenesis were up-regulated by galactose in Saccharomyces cerevisiae early during the induction process. YJL217W is also known as REE1 (Regulation of Enolase I). Both the Gal4 regulatory region and the Mac1 binding domain were found on the upstream region of REE1, and the expression of REE1 was up-regulated by galactose but not by glucose. The up-regulation of REE1 by galactose was not observed in the Δgal4 strain. From the two-hybrid analysis, we found that Ree1 physically interacted with Gal83. Furthermore, from 2-D gel electrophoresis we found that the deletion of REE1 resulted in the up-regulation of Eno1. From Western blotting, we learned that the expression of Eno1 in the Δree1 strain was different from that in wild-type strains and that Eno1 expression was not changed by glucose stimulation. Taken together, these results suggest that Ree1p functions in the galactose metabolic pathway via the Gal83 protein and that it may control the level of Eno1p, which is also affected by the Snf1 complex, in S. cerevisiae.     

Role of the putative structural protein Sed1p in mitochondrial genome maintenance

The nuclear gene MIP1 encodes the mitochondrial DNA polymerase responsible for replicating the mitochondrial genome in Saccharomyces cerevisiae. A number of other factors involved in replicating and segregating the mitochondrial genome are yet to be identified. Here, we report that a bacterial two-hybrid screen using the mitochondrial polymerase, Mip1p, as bait identified the yeast protein Sed1p. Sed1p is a cell surface protein highly expressed in the stationary phase. We find that several modified forms of Sed1p are expressed and the largest of these forms interacts with the mitochondrial polymerase in vitro. Deletion of SED1 causes a 3.5-fold increase in the rate of mitochondrial DNA point mutations as well as a 4.3-fold increase in the rate of loss of respiration. In contrast, we see no change in the rate of nuclear point mutations indicating the specific role of Sed1p function in mitochondrial genome stability. Indirect immunofluorescence analysis of Sed1p localization shows that Sed1p is targeted to the mitochondria. Moreover, Sed1p is detected in purified mitochondrial fractions and the localization to the mitochondria of the largest modified form is insensitive to the action of proteinase K. Deletion of the sed1 gene results in a reduction in the quantity of Mip1p and also affects the levels of a mitochondrially-expressed protein, Cox3p. Our results point towards a role for Sed1p in mitochondrial genome maintenance.