An alternative translation initiation start site within the TPI mRNA.

<div><p> <b>A)</b> The first 17 codons of the human TPI sequence and the netstart scores are presented. The introduced mutations are indicated in bold letters.</p> <p> <b>B)</b> MR100 <i>Δtpi1</i> yeast cells were transformed with the respective plasmids encoding TPI, TPI_{Met1_AAG}, TPI_{Ser3_TER} or the variant TPI_2ndATG.</p> <p>Ethanol lysates were prepared from logarithmically growing yeast cultures and the expression level of the different TPI variants was analyzed by immunoblotting using polyclonal α-TPI serum.</p> <p>Please note that no sample was loaded in case of the lane marked with a dash.</p> <p> <b>C)</b> COS1 cells were transfected with the plasmids pEGFP-N1, pEGFP-N1-TPI, pEGFP-N1-TPI_{Met1_AAG} or pEGFP-N1-TPI_{Ser3_TER}.</p> <p>Cell lysates were prepared and the expression level of the different fusion proteins was analyzed by immunoblot using an α-GFP antibody.</p> <p> <b>D)</b> MR100 <i>Δtpi1</i> yeast was transformed with expression plasmids encoding wild-type TPI, TPI_{Met1_AAG} or TPI_2ndATG.</p> <p>Afterwards, single yeast clones were selected and grown on the respective SC^-leu-ura plates for 3 days at 30°C as indicated.</p></div

Consequences of mutations within human TPI.

<div><p>Homozygous mutations within TPI affecting enzyme dimerization cause TPI deficiency, whereas homozygous mutations resulting in an inactive <i>TPI</i> allele are lethal.</p> <p>Compound heterozygous individuals having inherited one inactive and one allele defective in dimerization properties will develop TPI deficiency, whereas heterozygote individuals having inherited a heterozygote null allele have an evolutionary advantage. (+: wild-type TPI; −: TPI with aberrant dimerization property; 0: allele encoding no or a catalytically inactive TPI).</p></div

Human wild-type and pathogenic TPI variants can substitute for yeast TPI1.

<div><p> <b>A)</b> MR100 yeast cells (<i>Δtpi1</i>) were transformed with the various p416GPD-based expression plasmids encoding wild-type human TPI as well as the pathogenic variants Met1_AAG, Cys41Tyr, Glu104Asp, Gly122Arg, Ile170Val or Phe240Leu, respectively, and plated on minimal SC ^-leu-ura medium supplemented with 3% ethanol/0.1% glucose.</p> <p>Afterwards, single yeast clones were selected and grown as represented by the schemes on SC ^-leu-ura medium plates supplemented either with 2% glucose or with 3% ethanol/0.1% glucose at 30°C. <b>B)</b> MR100 <i>Δtpi1</i> yeast cells expressing wild-type TPI or the different pathogenic TPI variants were grown until logarithmic phase.</p> <p>Then, the same cell number of each culture was spotted as 5-fold serial dilutions onto glucose media or onto glucose media supplemented with different concentrations of lithium chloride. </p> <p>Plates were incubated for 3 days at 30°C and growth of the different yeast strains was analyzed.</p></div

MR100 Δ<i>tpi1</i> yeast cells expressing the Ile170Val TPI variant are hyperresistant to diamide.

<div><p>MR100 <i>Δtpi1</i> yeast cells expressing wild-type as well as the pathogenic TPI variants Cys41Tyr, Glu104Asp, Gly122Arg, Ile170Val or Phe240Leu were grown to stationary phase, serially diluted to OD₆₀₀ values of 3.0, 1.0, 0.3, 0.1 and spotted onto SC medium plates containing different concentrations of diamide.</p> <p>Sensitivity/resistance was determined by comparing the growth between the different yeast strains after incubating the plates for 3 days at 28°C.</p></div

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, 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

Primers used for cloning.

<p>Primers used for cloning.</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p