13 research outputs found
Mmi1, the Yeast Homologue of Mammalian TCTP, Associates with Stress Granules in Heat-Shocked Cells and Modulates Proteasome Activity
<div><p>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 <i>mmi1</i>Δ 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 <i>mmi1</i>Δ 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.</p></div
Mmi1 co-localizes with stress granules.
<p>(A) Distribution of Mmi1-RFP and the stress granule marker Rpg1-GFP co-expressed from the chromosome sites (strain CRY1309) was analyzed in cells before and after heat shock at 46°C for 10 min where the two proteins were co-localized to a high degree in cytoplasmic granules (B) During recovery from heat shock both proteins returned to their uniform “unstressed” cytoplasmic location. (C) Cells expressing Mmi1-GFP from the chromosomal locus (strain CRY1226) were heat-shocked at 46°C for 10 min in the absence (Control) or in the presence of cycloheximide (CYH; 50 µg/ml). The nuclear DNA was stained with Hoechst 33342. Cycloheximide affected formation of large Mmi1 cytoplasmic accumulations but did not prevent the translocation of Mmi1 to the nucleus. Scale bar 4 µm.</p
Electron microscopy of Mmi1-GFP.
<p>Exponentially growing cells expressing Mmi1-GFP from the chromosomal locus (strain CRY1103) were processed for immunogold labeling (see Materials and Methods for details) either prior to (A, B, full bars in E) or immediately following the 10 min long heat shock at 46°C (C, D, empty bars in E). Representative examples of whole cell sections (A, C) and detailed views in negative contrast allowing for identification of individual gold particles (B, D) are presented. Mitochondria (m), the cytosol (c), the nucleus (n) and vacuoles (v) are marked. Cytoplasmic clusters of gold particles frequently observed in heat-shocked cells are highlighted (arrows). Scale bar 1 µm. Density of the immunogold labeling (number of the gold particles per the area of the corresponding cellular compartment) was counted by analyzing 237 cells. Relative errors of both the measured quantities were determined as SDs from random repetitions of measurements on identical images. Relative error of the ratio was calculated as a sum of these relative errors. Gold particle densities are plotted relative to the average density (gold particles per cell), equal to 1. In total, 105 untreated and 132 heat-shocked cells (4868 and 7565 gold particles, respectively) were analyzed. A significant enrichment of Mmi1 in the nucleus after heat shock was found.</p
Mmi1 localization studies.
<p>(A) Structure prediction of Mmi1 with Swissmodel (<a href="http://swissmodel.expasy.org/" target="_blank">http://swissmodel.expasy.org/</a>). (B) Amino acid sequence of the open reading frame of MMI1. Three domains which are obvious in structure (A) are color-marked here. Blue is the N-terminal domain, (Mmi1(N)); green is the middle V-domain (Mmi1(V)) which is characterized by two large alpha helices; red is the C-terminal domain, which contains two short beta sheets and which is not shown in the fluorescence pictures. The three domains were cloned separately including the respective overlapping sequences (yellow) adding a start methionine where necessary and fused C-terminally with GFP as described in Materials and Methods. (C) Mmi1-RFP expressed from its chromosomal locus compared with the mitochondrial marker Aco1-GFP expressed from plasmid pUG35 in strain CRY1844 and the nuclear stain Hoechst 33342. In control cells, Mmi1-RFP was excluded from the nucleus (arrow points to nucleus). After heat shock, Mmi1 accumulates in the nucleus and partially overlaps with mitochondria (double arrow). (D) Mmi1(N)-GFP compared with MITO-RFP and Hoechst 33342 in strain CRY1924. The N-terminal domain is nuclear at all temperatures. It is shown here at 30°C. (E) Mmi1(V)-GFP co-expressed with MITO-RFP and Hoechst 33342 staining in strain CRY1839. The V-domain is mitochondrial at all temperatures. It is shown here at 30°C. (F) Mmi1(N+V) is cytoplasmic at 30°C, and nuclear after heat shock with a part of the proteins forming cytoplasmic granules (strain CRY1842). It appears that the combination of the two domains has re-gained heat shock-dependent regulation of Mmi1 subcellular localization. Scale bar 4 µm.</p
Mmi1 in times of stress.
<p>(A) Growth curves of BY4742 wild type (WT) cells as well as <i>mmi1</i>Δ cells in the BY4742 strain (strain CRY1981) with and without the addition of 1 mM and 3 mM hydrogen peroxide. (B) Survival of WT and <i>mmi1</i>Δ cells of both mating types (strains BY4741, BY4742, CRY1107, CRY1981) after a temperature shift from 30°C to 46°C for time periods of up to 100 min. Note the very marked increase of heat shock resistance of the deletion mutants. Error bars denote standard deviations of the mean obtained form 3 independent repeats of the experiment. (C) Changes of Mmi1-GFP distribution after a temperature shift from 30°C to 37°C, 40°C, 42°C and 46°C for 10 min each (strain CRY1838). Scale bar 4 µm. The figures show transfer of Mmi1 to part of the nuclear compartment at intermediate temperatures and transfer to both the nucleus and cytoplasmic granules upon 10 min heat shock at 46°C.</p
Deletion of <i>MMI1</i> gene does not affect SGs assembly and dissolution.
<p>We compared the distribution of stress granules markers Pab1-GFP and Rpg1-RFP fusion proteins produced from sites on chromosomes in wild type (strain CRY527) and <i>mmi1</i>Δ (strain CRY1060) cells. (A) Stress granule formation after robust heat shock was not influenced at all by the absence of Mmi1 in the <i>mmi1</i>Δ deletion strain. (B) In the same strains that were used in (A) recovery from heat shock was observed for 60 min (shown) and 120 min. As shown, absence of Mmi1 had no influence on the dissolution of stress granules during recovery from the heat shock. Scale bar 4 µm.</p
Primers used for chromosomal integration of RFP-tags.
<p>Primers used for chromosomal integration of RFP-tags.</p
Mmi1 and the de-ubiquitination complex.
<p>Mmi1-RFP was tested for co-localization with either Bre5-GFP, or Ubp3-GFP (A – C) after a heat shock in strains CRY1698 and CRY1696, co-expressing particular fusion proteins from chromosomal sites. (A) Unstressed cells displayed uniformly cytosolic distribution of the fusion proteins. (B) In cells heat-shocked at 41°C for 10 min, co-localization of Mmi1-RFP with Bre5-GFP was observed in the nuclear region (arrows). In these cells, Ubp3-GFP was already accumulated in cytoplasmic granules whereas Mmi1-RFP remained uniformly cytosolic (double arrows). (C) Robust heat shock induced the accumulation of Bre5-GFP and Ubp3-GFP fusion proteins together with Mmi1-RFP in cytoplasmic granules but not with Mmi1-RFP accumulations in the nuclear region. (D) After a robust heat shock at 46°C, extensive co-localization of Mmi1 with Cdc48 in cytoplasmic granules was observed (strain CRY1308). Scale bars 4 µm.</p