Mmi1, the Yeast Homologue of Mammalian TCTP,Associates with Stress Granules in Heat-Shocked Cells and Modulates Proteasome Activity

As we have shown previously, yeast Mmi1 protein translocates from the cytoplasm to the outer surface of mitochondria when vegetatively growing yeast cells are exposed to oxidative stress. Here we analyzed the effect of heat stress on Mmi1 distribution. We performed domain analyses and found that binding of Mmi1 to mitochondria is mediated by its central alpha-helical domain (V-domain) under all conditions tested. In contrast, the isolated N-terminal flexible loop domain of the protein always displays nuclear localization. Using immunoelectron microscopy we confirmed re-location of Mmi1 to the nucleus and showed association of Mmi1 with intact and heat shock-altered mitochondria. We also show here that mmi1Δ mutant strains are resistant to robust heat shock with respect to clonogenicity of the cells. To elucidate this phenotype we found that the cytosolic Mmi1 holoprotein re-localized to the nucleus even in cells heat-shocked at 40°C. Upon robust heat shock at 46°C, Mmi1 partly co-localized with the proteasome marker Rpn1 in the nuclear region as well as with the cytoplasmic stress granules defined by Rpg1 (eIF3a). We co-localized Mmi1 also with Bre5, Ubp3 and Cdc48 which are involved in the protein de-ubiquitination machinery, protecting protein substrates from proteasomal degradation. A comparison of proteolytic activities of wild type and mmi1Δ cells revealed that Mmi1 appears to be an inhibitor of the proteasome. We conclude that one of the physiological functions of the multifunctional protein module, Mmi1, is likely in regulating degradation and/or protection of proteins thereby indirectly regulating the pathways leading to cell death in stressed cells.     

CLASP regulates mitochondrial distribution in Schizosaccharomyces pombe

Movement of mitochondria in Schizosaccharomyces pombe depends on their association with the dynamic, or plus ends, of microtubules, yet the molecular basis for this interaction is poorly understood. We identified mmd4 in a screen of temperature-sensitive S. pombe strains for aberrant mitochondrial morphology and distribution. Cells with the mmd4 mutation display mitochondrial aggregation near the cell ends at elevated temperatures, a phenotype similar to mitochondrial defects observed in wild-type cells after microtubule depolymerization. However, microtubule morphology and function appear normal in the mmd4 mutant. The mmd4 lesion maps to peg1(+), which encodes a microtubule-associated protein with homology to cytoplasmic linker protein-associated proteins (mammalian microtubule plus end-binding proteins). Peg1p localizes to the plus end of microtubules and to mitochondria and is recovered with mitochondria during subcellular fractionation. This mitochondrial-associated fraction of Peg1p displays properties of a peripherally associated protein. Peg1p is the first identified microtubule plus end-binding protein required for mitochondrial distribution and likely functions as a molecular link between mitochondria and microtubules.     

Mmd1p, a novel, conserved protein essential for normal mitochondrial morphology and distribution in the fission yeast Schizosaccharomyces pombe

The mmd1 mutation causes temperature-sensitive growth and defects in mitochondrial morphology and distribution in the fission yeast Schizosaccharomyces pombe. In mutant cells, mitochondria aggregate at the two cell ends, with increased aggregation at elevated temperatures. Microtubules, which mediate mitochondrial positioning in fission yeast, seem normal in mmd1 cells at permissive temperature and after several hours at the nonpermissive temperature but display aberrant organization after prolonged periods at 37 degrees C. Additionally, cells harboring both mmd1 and ban5-4, a temperature-sensitive allele of alpha2-tubulin, display synthetic defects in growth and mitochondrial distribution. The mmd1 mutation maps to an open reading frame encoding a novel 35.7-kDa protein. The Mmd1p sequence features repeating EZ-HEAT motifs and displays high conservation with uncharacterized homologues found in a variety of organisms. Saccharomyces cerevisiae cells depleted for their MMD1 homologue show increased sensitivity to the antimicrotubule drug benomyl, and the S. cerevisiae gene complemented the S. pombe mutation. Mmd1p was localized to the cytosol. Mmd1p is the first identified component required for the alignment of mitochondria along microtubules in fission yeast.     

The dynamin-like protein DLP1 is essential for normal distribution and morphology of the endoplasmic reticulum and mitochondria in mammalian cells

The dynamin family of large GTPases has been implicated in vesicle formation from both the plasma membrane and various intracellular membrane compartments. The dynamin-like protein DLP1, recently identified in mammalian tissues, has been shown to be more closely related to the yeast dynamin proteins Vps1p and Dnm1p (42%) than to the mammalian dynamins (37%). Furthermore, DLP1 has been shown to associate with punctate vesicles that are in intimate contact with microtubules and the endoplasmic reticulum (ER) in mammalian cells. To define the function of DLP1, we have transiently expressed both wild-type and two mutant DLP1 proteins, tagged with green fluorescent protein, in cultured mammalian cells. Point mutations in the GTP-binding domain of DLP1 (K38A and D231N) dramatically changed its intracellular distribution from punctate vesicular structures to either an aggregated or a diffuse pattern. Strikingly, cells expressing DLP1 mutants or microinjected with DLP1 antibodies showed a marked reduction in ER fluorescence and a significant aggregation and tubulation of mitochondria by immunofluorescence microscopy. Consistent with these observations, electron microscopy of DLP1 mutant cells revealed a striking and quantitative change in the distribution and morphology of mitochondria and the ER. These data support very recent studies by other authors implicating DLP1 in the maintenance of mitochondrial morphology in both yeast and mammalian cells. Furthermore, this study provides the first evidence that a dynamin family member participates in the maintenance and distribution of the ER. How DLP1 might participate in the biogenesis of two presumably distinct organelle systems is discussed.     

Red1 promotes the elimination of meiosis-specific mRNAs in vegetatively growing fission yeast

Meiosis-specific mRNAs are transcribed in vegetative fission yeast, and these meiotic mRNAs are selectively removed from mitotic cells to suppress meiosis. This RNA elimination system requires degradation signal sequences called determinant of selective removal (DSR), an RNA-binding protein Mmi1, polyadenylation factors, and the nuclear exosome. However, the detailed mechanism by which meiotic mRNAs are selectively degraded in mitosis but not meiosis is not understood fully. Here we report that Red1, a novel protein, is essential for elimination of meiotic mRNAs from mitotic cells. A red1 deletion results in the accumulation of a large number of meiotic mRNAs in mitotic cells. Red1 interacts with Mmi1, Pla1, the canonical poly(A) polymerase, and Rrp6, a subunit of the nuclear exosome, and promotes the destabilization of DSR-containing mRNAs. Moreover, Red1 forms nuclear bodies in mitotic cells, and these foci are disassembled during meiosis. These results demonstrate that Red1 is involved in DSR-directed RNA decay to prevent ectopic expression of meiotic mRNAs in vegetative cells.     

Importance of polyadenylation in the selective elimination of meiotic mRNAs in growing S. pombe cells

A number of meiosis‐specific mRNAs are initially weakly transcribed, but then selectively removed during fission yeast mitotic growth. These mRNAs harbour a region termed DSR (determinant of selective removal), which is recognized by the YTH family RNA‐binding protein Mmi1p. Mmi1p directs the destruction of these mRNAs in collaboration with nuclear exosomes. However, detailed molecular mechanisms underlying this process of selective mRNA elimination have remained elusive. In this study, we demonstrate the critical role of polyadenylation in this process. Two‐hybrid and genetic screens revealed potential interactions between Mmi1p and proteins involved in polyadenylation. Additional investigations showed that destruction of DSR‐containing mRNAs by exosomes required polyadenylation by a canonical poly(A) polymerase. The recruitment of Pab2p, a poly(A)‐binding protein, to the poly(A) tail was also necessary for mRNA destruction. In cells undergoing vegetative growth, Mmi1p localized with exosomes, Pab2p, and components of the polyadenylation complex in several patchy structures in the nucleoplasm. These patches may represent the sites for degradation of meiosis‐specific mRNAs with untimely expression.     

Uth1p: a yeast mitochondrial protein at the crossroads of stress, degradation and cell death

UTH1 is a yeast aging gene that has been identified on the basis of stress resistance and longer life span of mutants. It was also shown to participate in mitochondrial biogenesis. The absence of Uth1p was found to trigger resistance to autophagy induced by rapamycin. Uth1p is therefore the first mitochondrial protein proven to be required for the autophagic degradation of mitochondria. Since this protein is also involved in yeast cell death induced by heterologous expression of the pro-apoptotic protein Bax, the results are discussed in the light of evidence suggesting a co-regulation of apoptosis and autophagy in mammalian cells.     

Sec61β facilitates the maintenance of endoplasmic reticulum homeostasis by associating microtubules

Sec61β, a subunit of the Sec61 translocon complex, is not essential in yeast and commonly used as a marker of endoplasmic reticulum (ER). In higher eukaryotes, such as Drosophila, deletion of Sec61β causes lethality, but its physiological role is unclear. Here, we show that Sec61β interacts directly with microtubules. Overexpression of Sec61β containing small epitope tags, but not a RFP tag, induces dramatic bundling of the ER and microtubule. A basic region in the cytosolic domain of Sec61β is critical for microtubule association. Depletion of Sec61β induces ER stress in both mammalian cells and Caenorhabditis elegans, and subsequent restoration of ER homeostasis correlates with the microtubule binding ability of Sec61β. Loss of Sec61β causes increased mobility of translocon complexes and reduced level of membrane-bound ribosomes. These results suggest that Sec61β may stabilize protein translocation by linking translocon complex to microtubule and provide insight into the physiological function of ER-microtubule interaction.     

Role of Tom5 in maintaining the structural stability of the TOM complex of mitochondria

Transport of nuclear encoded proteins into mitochondria is mediated by multisubunit translocation machineries in the outer and inner membranes of mitochondria. The TOM complex contains receptor and pore components that facilitate the recognition of preproteins and their transfer through the outer membrane. In addition, the complex contains a set of small proteins. Tom7 and Tom6 have been found in Neurospora and yeast, Tom5 has been found so far only in the latter organism. In the present study, we identified Neurospora Tom5 and analyzed its function in comparison to yeast Tom5, which has been proposed to play a role as a receptor-like component. Neurospora Tom5 crosses the outer membrane with its carboxyl terminus facing the intermembrane space like the other small Tom components. The temperature-sensitive growth phenotype of the yeast TOM5 deletion was rescued by overexpression of Neurospora Tom5. On the other hand, Neurospora cells deficient in tom5 did not exhibit any defect in growth. The structural stability of TOM complexes from cells devoid of Tom5 was significantly altered in yeast but not in Neurospora. The efficiency of protein import in Neurospora mitochondria was not affected by deletion of tom5, whereas in yeast it was reduced as compared with wild type. We conclude that the main role of Tom5, rather than being a receptor, is maintaining the structural integrity of the TOM complex.     

Mitochondrial import of human and yeast fumarase in live mammalian cells: retrograde translocation of the yeast enzyme is mainly caused by its poor targeting sequence

Studies on yeast fumarase provide the main evidence for dual localization of a protein in mitochondria and cytosol by means of retrograde translocation. We have examined the subcellular targeting of yeast and human fumarase in live cells to identify factors responsible for this. The cDNAs for mature yeast or human fumarase were fused to the gene for enhanced green fluorescent protein (eGFP) and they contained, at their N-terminus, a mitochondrial targeting sequence (MTS) derived from either yeast fumarase, human fumarase, or cytochrome c oxidase subunit VIII (COX) protein. Two nuclear localization sequences (2x NLS) were also added to these constructs to facilitate detection of any cytosolic protein by its targeting to nucleus. In Cos-1 cells transfected with these constructs, human fumarase with either the native or COX MTSs was detected exclusively in mitochondria in >98% of the cells, while the remainder 1-2% of the cells showed varying amounts of nuclear labeling. In contrast, when human fumarase was fused to the yeast MTS, >50% of the cells showed nuclear labeling. Similar studies with yeast fumarase showed that with its native MTS, nuclear labeling was seen in 80-85% of the cells, but upon fusion to either human or COX MTS, nuclear labeling was observed in only 10-15% of the cells. These results provide evidence that extramitochondrial presence of yeast fumarase is mainly caused by the poor mitochondrial targeting characteristics of its MTS (but also affected by its primary sequence), and that the retrograde translocation mechanism does not play a significant role in the extramitochondrial presence of mammalian fumarase.     

Microtubules, mitochondria, and molecular chaperones: a new hypothesis for in vivo assembly of microtubules

In Chinese hamster ovary cells, a number of independent mutants selected for resistance to antimitotic drugs have been found to be specifically altered in two major cellular proteins, designated P1 (relative mass (Mr) approximately 60-63 kilodaltons (kDa] and P2 (Mr approximately 69-70 kDa), which appeared microtubule related by a number of genetic and biochemical criteria. Antibodies to P1 have been found to bind specifically to mitochondria that showed specific association with microtubules in interphase cells. Biochemical and cDNA sequence studies on P1 showed that this protein, which is localized in the matrix compartment, is the mammalian homolog of the highly conserved chaperonin family of proteins (other members include the GroEL protein of Escherichia coli, the 60-kDa heat-shock protein of yeast, and the rubisco subunit binding protein of plant chloroplasts). The chaperonin proteins in various systems play a transient but essential molecular chaperone role in the proper folding of polypeptide chains and their assembly into oligomeric protein complexes. Our studies on P2 protein established that it corresponds to the constitutive form of the major 70-kDa heat-shock protein of mammalian cells (i.e., hsc70), which also acts as a molecular chaperone in the intracellular transport of nascent proteins to organelles such as mitochondria and endoplasmic reticulum. To account for the above, as well as a number of other observations (e.g., binding of fluorescent-labeled antimitotic drugs to mitochondria, association of tubulin with mitochondria as well as other membranes, and high affinity binding of antimitotic drugs to free tubulin but not to assembled microtubules), a new model for the in vivo assembly of interphase microtubules is proposed. The model ascribes a central role to the mitochondrially localized chaperonin (i.e., P1) protein in the intracellular formation of tubulin dimers and in their addition to the growth sites in microtubules. The proposed model also explains a number of other observations related to microtubule assembly in the literature.     

Moe1 and spInt6, the fission yeast homologues of mammalian translation initiation factor 3 subunits p66 (eIF3d) and p48 (eIF3e), respectively, are required for stable association of eIF3 subunits

The protein encoded by the fission yeast gene, moe1(+) is the homologue of the p66/eIF3d subunit of mammalian translation initiation factor eIF3. In this study, we show that in fission yeast, Moe1 physically associates with eIF3 core subunits as well as with 40 S ribosomal particles as a constituent of the eIF3 protein complex that is similar in size to multisubunit mammalian eIF3. However, strains lacking moe1(+) (Deltamoe1) are viable and show no gross defects in translation initiation, although the rate of translation in the Deltamoe1 cells is about 30-40% slower than wild-type cells. Mutant Deltamoe1 cells are hypersensitive to caffeine and defective in spore formation. These phenotypes of Deltamoe1 cells are similar to those reported previously for deletion of the fission yeast int6(+) gene that encodes the fission yeast homologue of the p48/Int6/eIF3e subunit of mammalian eIF3. Further analysis of eIF3 subunits in Deltamoe1 or Deltaint6 cells shows that in these deletion strains, while all the eIF3 subunits are bound to 40 S particles, dissociation of ribosome-bound eIF3 results in the loss of stable association between the eIF3 subunits. In contrast, eIF3 isolated from ribosomes of wild-type cells are associated with one another in a protein complex. These observations suggest that Moe1 and spInt6 are each required for stable association of eIF3 subunits in fission yeast.     

Prohibitins and Ras2 protein cooperate in the maintenance of mitochondrial function during yeast aging

The yeast Saccharomyces cerevisiae has a finite replicative life span. Yeasts possess two prohibitins, Phb1p and Phb2p, in similarity to mammalian cells. These proteins are located in the inner mitochondrial membrane, where they are involved in the processing of newly-synthesized membrane proteins. We demonstrate that the elimination of one or both of the prohibitin genes in yeast markedly diminished the replicative life span of cells that lack fully-functional mitochondria, while having no effect on cells with functioning mitochondria. This deleterious effect was suppressed by the deletion of the RAS2 gene. The expression of PHB1 and PHB2 declined gradually up to 5-fold during the life span. Cells in which PHB1 was deleted in conjunction with the absence of a mitochondrial genome displayed remarkable changes in mitochondrial morphology, distribution, and inheritance. This loss of mitochondrial integrity was not seen in cells devoid of PHB1 but possessing an intact mitochondrial genome. In a subset of the cells, the changes in mitochondrial integrity were associated with increased production of reactive oxygen species, which co-localized with the altered mitochondria. The mitochondrial deficits described above were all suppressed by deletion of RAS2. Our data, together with published information, are interpreted to provide a unified view of the role of the prohibitins in yeast aging. This model posits that the key initiating event is a decline in mitochondrial function, which leads to progressive oxidative damage that is exacerbated in the absence of the prohibitins. This aggravation of the initial damage is ameliorated by the suppression of the production of mitochondrial proteins in the absence of Ras2p signaling of mitochondrial biogenesis.     

An interaction between the SRP receptor and the translocon is critical during cotranslational protein translocation

The signal recognition particle (SRP)-dependent targeting pathway facilitates rapid, efficient delivery of the ribosome-nascent chain complex (RNC) to the protein translocation channel. We test whether the SRP receptor (SR) locates a vacant protein translocation channel by interacting with the yeast Sec61 and Ssh1 translocons. Surprisingly, the slow growth and cotranslational translocation defects caused by deletion of the transmembrane (TM) span of yeast SRbeta (SRbeta-DeltaTM) are exaggerated when the SSH1 gene is disrupted. Disruption of the SBH2 gene, which encodes the beta subunit of the Ssh1p complex, likewise causes a growth defect when combined with SRbeta-DeltaTM. Cotranslational translocation defects in the ssh1DeltaSRbeta-DeltaTM mutant are explained by slow and inefficient in vivo gating of translocons by RNCs. A critical function for translocation channel beta subunits in the SR-channel interaction is supported by the observation that simultaneous deletion of Sbh1p and Sbh2p causes a defect in the cotranslational targeting pathway that is similar to the translocation defect caused by deletion of either subunit of the SR.     

Highly divergent amino termini of the homologous human ALR and yeast scERV1 gene products define species specific differences in cellular localization.

The yeast scERV1 gene product is involved in the biogenesis of mitochondria and is indispensable for viability and regulation of the cell cycle. Recently the general importance of this gene for the eukaryotic cell was shown by the identification of a structural and functional human homologue. The homologous mammalian ALR (Augmenter of Liver Regeneration) genes from man, mouse and rat are involved in the phenomenon of liver regeneration. A low expression rate of the genes is found in all investigated cells and mammalian tissues but it is specifically induced after damage of liver organs and is especially high during spermatogenesis. The alignment of the different proteins identifies a highly conserved carboxy terminus with more than 40% identical amino acids between yeast and mammals. The conserved carboxy terminus is functionally interchangeable between distantly related species like yeast and man. In contrast, the amino terminal parts of the proteins display a high degree of variability and significant differences even among closely related species. This finding leads to the problem whether the amino termini have comparable or divergent functions in different species. In this study we demonstrate by heterologous complementation experiments in yeast that the complete human ALR protein with its own amino terminus is not able to substitute for the yeast scERV1 protein. Fusion proteins of Alrp and scErv1p with the green fluorescence protein were created to investigate the respective subcellular localizations of these homologous proteins in yeast and human cells. In yeast cells human Alrp accumulates in the cytoplasm in contrast to yeast scErv1p that is preferentially associated with yeast mitochondria. Comparable studies with human cells clearly show that the homologous human Alrp is located in the cytosol of these cells. Fractionation experiments and antibody tests with yeast and human mitochondria and cellular extracts verify these findings.