Pressure-Induced Endocytic Degradation of the Saccharomyces cerevisiae Low-Affinity Tryptophan Permease Tat1 Is Mediated by Rsp5 Ubiquitin Ligase and Functionally Redundant PPxY Motif Proteins

Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1^K29R-K31R-GFP remained. The HPG1-1 (Rsp5^P514T) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.     

The N- and C-terminal mutations in tryptophan permease Tat2 confer cell growth in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> under high-pressure and low-temperature conditions

Tryptophan uptake appears to be the limiting factor in growth of tryptophan auxotrophic Saccharomyces cerevisiae strains under the conditions of high hydrostatic pressure and low temperature. When the cells are subjected to a pressure of 25 MPa, tryptophan permease Tat2 is degraded in a manner dependent on ubiquitination by Rsp5. One of the high-pressure growth-conferring genes, HPG2, was shown to be allelic to TAT2. The HPG2-1 (Tat2^E27F) mutation site is located within the ExKS motif in the N-terminus, and the HPG2-2 (Tat2^D563N) and HPG2-3 (Tat2^E570K) mutation sites are located at the KQEIAE sequence in the C-terminus. The HPG2 mutations enhance the stability of Tat2 during high-pressure or low-temperature incubation, leading to cell growth under these stressful conditions. These results suggest that the cytoplasmic tails are involved in Rsp5-mediated ubiquitination of Tat2 under high-pressure or low-temperature conditions.Communicated by K. Horikoshi     

Isolation of a highly copper-tolerant yeast, Cryptococcus sp., from the Japan Trench and the induction of superoxide dismutase activity by Cu2+

Thirteen yeast strains were isolated from deep-sea sediment samples collected at a depth of 4500 m to 6500 m in the Japan Trench. Amongst them, strain N6 possessed high tolerance against Cu²⁺ and could grow on yeast extract/peptone/dextrose/agar containing 50 mM CuSO₄. Analysis of the 18S rDNA sequence indicates strain N6 belongs to the genus Cryptococcus. In contrast, the type strain of C. albidus, a typical marine yeast Rhodotorula ingeniosa and Saccharomyces cerevisiae did not grow at high concentrations of CuSO₄. Superoxide dismutase (SOD) catalyzes the scavenging of superoxide radicals. The activity of SOD in cell extract of strain N6 was very weak (<1 mU g^–1 total protein) when the strain was grown in the absence of CuSO₄. However, the activity was stimulated (25.8 mU g^–1 total protein) when cells were grown with 1 mM CuSO₄ and further enhanced to 110 mU g^–1 total protein with 10 mM CuSO₄. Catalase activity was increased only 1.4 or 1.1-fold with 1 mM or 10 mM CuSO₄ in the growth medium, respectively. These results suggest that SOD may have a role in the defensive mechanisms against high concentrations of CuSO₄ in strain N6.     

T cells of atomic bomb survivors respond poorly to stimulation by Staphylococcus aureus toxins in vitro: does this stem from their peripheral lymphocyte populations having a diminished naïve CD4 T-cell content?

We found previously that the peripheral CD4 T-cell populations of heavily exposed A-bomb survivors contained fewer na?ve T cells than we detected in the corresponding unexposed controls. To determine whether this demonstrable impairment of the CD4 T-cell immunity of A-bomb survivors was likely to affect the responsiveness of their immune systems to infection by common pathogens, we tested the T cells of 723 survivors for their ability to proliferate in vitro after a challenge by each of the Staphylococcus aureus toxins SEB, SEC-2, SEC-3, SEE and TSST-1. The results presented here reveal that the proliferative responses of T cells of A-bomb survivors became progressively weaker as the radiation dose increased and did so in a manner that correlated well with the decreasing CD45RA-positive (na?ve) [but not CD45RA-negative (memory)] CD4 T-cell percentages that we found in their peripheral blood lymphocyte (PBL) populations. We also noted that the T cells of survivors with a history of myocardial infarction tended to respond poorly to several (or even all) of the S. aureus toxins, and that these same individuals had proportionally fewer CD45RA-positive (na?ve) CD4 T cells in their PBL populations than we detected in survivors with no myocardial infarction in their history. Taken together, these results clearly indicate that A-bomb irradiation led to an impairment of the ability of exposed individuals to maintain their na?ve T-cell pools. This may explain why A-bomb survivors tend to respond poorly to toxins encoded by the common pathogenic bacterium S. aureus.     

Dynamic light scattering and circular dichroism studies on heat-induced gelation of hard-keratin protein aqueous solutions

Animal hairs consist of aggregates of dead cells filled with keratin protein gel. We succeeded in preparing water-soluble hard-keratin proteins and reconstructing the keratin gels by heat-induced disulfide linkages in vitro. Here, the roles of intermolecular hydrophobic interaction and disulfide bonding between the proteins in the gel were discussed. Water-soluble keratin proteins consisting of mixtures of type I ( approximately 48 kDa) and type II ( approximately 61 kDa) were prepared from wool fibers as S-carboxymethyl alanyl disulfide keratin (CMADK). The gelation was achieved by heating an aqueous solution containing at least 0.8 wt % CMADK at 100 degrees C. CMADK solutions with different urea or N-ethylmaleimide concentrations or pH were exposed to dynamic light scattering (DLS) and circular dichroism (CD). DLS clarified the gelation point of CMADK solutions and provided information on the changes in keratin cluster size. DLS suggested two types of gelation mechanism. One was the regenerated chemical disulfide bonding between keratins from CMAD parts of chains. After the gel formed, this bond became important to maintain the gel structure. The other was the physical assembly due to hydrophobic interaction between alpha-helix parts of keratin chains. This hydrophobic assembly also played an important role during gelation. CD confirmed a conformational change in the keratin protein, resulting heat-induced gelation. CD clarified the relationship between keratin protein conformation and gelation, i.e., a rodlike conformation with many alpha-helix structures was necessary to associate keratin chains and form a gel network.     

Display of both N- and C-terminal target fusion proteins on the Aspergillus oryzae cell surface using a chitin-binding module

A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.     

Cloning and characterization of the gene encoding endo-beta-1,3-glucanase from Arthrobacter sp. NHB-10

The gluA gene, encoding an endo-beta-1,3-glucanase from Arthrobacter sp. (strain NHB-10), was cloned and analyzed. The deduced endo-beta-1,3-glucanase amino acid sequence was 750 amino acids long and contained a 42 amino acid signal peptide with a mature protein of 708 amino acids. There was no similarity to known endo-beta-1,3-glucanases, but GluA was partially similar to two fungal exo-beta-1,3-glucanases in glycoside hydrolase (GH) family 55. Of five possible residues for catalysis and two motifs in two beta-helix heads of GH family 55, three residues and one motif were conserved in GluA, suggesting that GluA is the first bacterial endo-beta-1,3-glucanase in GH family 55. Significant similarity was also found to two proteins of unknown function from Streptomyces coelicolor A3(2) and S. avermitilis.     

Exploration of the effects of high hydrostatic pressure on microbial growth, physiology and survival: perspectives from piezophysiology

The discovery of piezophiles (previously referred to as barophiles) prompted researchers to investigate the survival strategies they employ in high-pressure environments. There have been innovative high-pressure studies on biological processes applying modern techniques of genetics and molecular biology in bacteria and yeasts as model organisms. Recent advanced studies in this field have shown unexpected outcomes in microbial growth, physiology and survival when living cells are subjected to high hydrostatic pressure. The effects are conceptually dependent on the sign and magnitude of volume changes associated with any chemical reaction in the cells. Nevertheless, it is difficult to explain the pressure effects on complex metabolic networks based on a simple volume law. The challenges in piezophysiology are to discover whether the physiological responses of living cells to high pressure are relevant to their growth and to identify the critical factors in cell viability and lethality under high pressure from the general and organism-specific viewpoints.