Nuclear Import of UBL-Domain Protein Mdy2 Is Required for Heat-Induced Stress Response in Saccharomyces cerevisiae

Institute for Microbiology, Heinrich Heine University, Düsseldorf, Germany.
PLoS ONE (Impact Factor: 3.23). 12/2012; 7(12):e52956. DOI: 10.1371/journal.pone.0052956
Source: PubMed


Ubiquitin (Ub) and ubiquitin-like (UBL) proteins regulate a diverse array of cellular pathways through covalent as well as non-covalent interactions with target proteins. Yeast protein Mdy2 (Get5) and its human homolog GdX (Ubl4a) belong to the class of UBL proteins which do not form conjugates with other proteins. Mdy2 is required for cell survival under heat stress and for efficient mating. As part of a complex with Sgt2 and Get4 it has been implicated in the biogenesis of tail-anchored proteins. Interestingly, in response to heat stress, Mdy2 protein that is predominantly localized in the nucleus co-localized with poly(A)-binding protein Pab1 to cytoplasmic stress granules suggesting that nucleocytoplasmic shuttling is of functional importance. Here we investigate the nuclear import of Mdy2, a process that is independent of the Get4/Sgt2 complex but required for stress response. Nuclear import is mediated by an N-terminal nuclear localization signal (NLS) and this process is essential for the heat stress response. In contrast, cells expressing Mdy2 lacking a nuclear export signal (NES) behave like wild type. Importantly, both Mdy2 and Mdy2-ΔNES, but not Mdy2-ΔNLS, physically interact with Pab1 and this interaction correlates with the accumulation in cytoplasmic stress granules. Thus, the nuclear history of the UBL Mdy2 appears to be essential for its function in cytoplasmic stress granules during the rapid cellular response to heat stress.

  • Source
    • "Until now, we have shown that CERKL is present in SGs, polysomes, PB and other mRNP compact particles. All these structures share mRNAs and proteins involved in mRNA metabolism, most of which also shift from the nucleus to the cytoplasm [18], [19]. To study if CERKL interacted with some of them, we performed immunoprecipitation experiments followed by liquid chromatography-tandem mass spectrometry. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The function of CERKL (CERamide Kinase Like), a causative gene of retinitis pigmentosa and cone-rod dystrophy, still awaits characterization. To approach its cellular role we have investigated the subcellular localization and interaction partners of the full length CERKL isoform, CERKLa of 532 amino acids, in different cell lines, including a photoreceptor-derived cell line. We demonstrate that CERKLa is a main component of compact and untranslated mRNPs and that associates with other RNP complexes such as stress granules, P-bodies and polysomes. CERKLa is a protein that binds through its N-terminus to mRNAs and interacts with other mRNA-binding proteins like eIF3B, PABP, HSP70 and RPS3. Except for eIF3B, these interactions depend on the integrity of mRNAs but not of ribosomes. Interestingly, the C125W CERKLa pathological mutant does not interact with eIF3B and is absent from these complexes. Compact mRNPs containing CERKLa also associate with microtubules and are found in neurites of neural differentiated cells. These localizations had not been reported previously for any member of the retinal disorders gene family and should be considered when investigating the pathogenic mechanisms and therapeutical approaches in these diseases.
    PLoS ONE 02/2014; 9(2):e87898. DOI:10.1371/journal.pone.0087898 · 3.23 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The insertion of tail-anchored membrane (TA) proteins into the appropriate membrane is a post-translational event that requires stabilization of the transmembrane domain and targeting to the proper destination. Sgt2, a small glutamine-rich tetratricopeptide-repeat protein, is a heat-shock protein cognate (HSC) co-chaperone that preferentially binds endoplasmic reticulum-destined TA proteins and directs them to the GET pathway via Get4 and Get5. The N-terminal domain of Sgt2 seems to exert dual functions. It mediates Get5 interaction and allows substrate delivery to Get3. Following the N-terminus of Get5 is a ubiquitin-like (Ubl) domain that interacts with the N-terminus of Sgt2. Here, the crystal structure of the Sgt2 dimerization domain complexed with the Get5 Ubl domain (Sgt2N–Get5Ubl) is reported. This complex reveals an intimate interaction between one Sgt2 dimer and one Get5 monomer. This research further demonstrates that hydrophobic residues from both Sgt2 and Get5 play an important role in cell survival under heat stress. This study provides detailed molecular insights into the specific binding of this GET-pathway complex.
    Acta Crystallographica Section D Biological Crystallography 10/2013; 69(Pt 10):2081-2090. DOI:10.1107/S0907444913019379 · 2.67 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To reduce expression of gene products not required under stress conditions, eukaryotic cells form large and complex cytoplasmic aggregates of RNA and proteins (stress granules; SGs), where transcripts are kept translationally inert. The overall composition of SGs, as well as their assembly requirements and regulation through stress-activated signaling pathways remain largely unknown. We have performed a genome-wide screen of S. cerevisiae gene deletion mutants for defects in SG formation upon glucose starvation stress. The screen revealed numerous genes not previously implicated in SG formation. Most mutants with strong phenotypes are equally SG defective when challenged with other stresses, but a considerable fraction is stress-specific. Proteins associated with SG defects are enriched in low-complexity regions, indicating that multiple weak macromolecule interactions are responsible for the structural integrity of SGs. Certain SG-defective mutants, but not all, display an enhanced heat-induced mutation rate. We found several mutations affecting the Ran GTPase, regulating nucleocytoplasmic transport of RNA and proteins, to confer SG defects. Unexpectedly, we found stress-regulated transcripts to reach more extreme levels in mutants unable to form SGs: stress-induced mRNAs accumulate to higher levels than in the wild-type, whereas stress-repressed mRNAs are reduced further in such mutants. Our findings are consistent with the view that, not only are SGs being regulated by stress signaling pathways, but SGs also modulate the extent of stress responses. We speculate that nucleocytoplasmic shuttling of RNA-binding proteins is required for gene expression regulation during stress, and that SGs modulate this traffic. The absence of SGs thus leads the cell to excessive, and potentially deleterious, reactions to stress.
    PLoS Genetics 11/2014; 10(11):e1004763. DOI:10.1371/journal.pgen.1004763 · 7.53 Impact Factor