Mtc6/Ehg2 is a novel endoplasmic reticulum-resident glycoprotein essential for high-pressure tolerance.

Hydrostatic pressure is a common mechanical stressor that modulates metabolism and reduces cell viability. Eukaryotic cells have genetic programs to cope with hydrostatic pressure stress and maintain intracellular homeostasis. However, the mechanism underlying hydrostatic pressure tolerance remains largely unknown. We have recently demonstrated that Maintenance of telomere capping protein 6 (Mtc6) plays a protective role in the survival of the budding yeast Saccharomyces cerevisiae under hydrostatic pressure stress by supporting the integrity of nutrient permeases. The current study demonstrate that Mtc6 acts as an endoplasmic reticulum (ER) membrane protein. Mtc6 comprises two transmembrane domains, a C-terminal cytoplasmic domain, and a luminal region with 12 Asn (N)-linked glycans attached to it. Serial mutational analyses showed that the cytoplasmic C-terminal amino acid residues GVPS are essential for Mtc6 activity. Multiple N-linked glycans in the luminal region are involved in the structural conformation of Mtc6. Moreover, deletion of MTC6 led to increased degradation of the leucine permease Bap2 under hydrostatic pressure, suggesting that Mtc6 facilitates proper folding of nutrient permeases in the ER under the stress condition. We propose a novel model of molecular function in which the glycosylated luminal domain and cytoplasmic GVPS sequences of Mtc6 cooperatively support the nutrient permease activity.

Activation of CWI pathway through high hydrostatic pressure, enhancing glycerol efflux via the aquaglyceroporin Fps1 in Saccharomyces cerevisiae.

The fungal cell wall is the initial barrier for the fungi against diverse external stresses, such as osmolarity changes, harmful drugs, and mechanical injuries. This study explores the roles of osmoregulation and the cell-wall integrity (CWI) pathway in response to high hydrostatic pressure in the yeast Saccharomyces cerevisiae. We demonstrate the roles of the transmembrane mechanosensor Wsc1 and aquaglyceroporin Fps1 in a general mechanism to maintain cell growth under high-pressure regimes. The promotion of water influx into cells at 25 MPa, as evident by an increase in cell volume and a loss of the plasma membrane eisosome structure, activates the CWI pathway through the function of Wsc1. Phosphorylation of Slt2, the downstream mitogen-activated protein kinase, was increased at 25 MPa. Glycerol efflux increases via Fps1 phosphorylation, which is initiated by downstream components of the CWI pathway, and contributes to the reduction in intracellular osmolarity under high pressure. The elucidation of the mechanisms underlying adaptation to high pressure through the well-established CWI pathway could potentially translate to mammalian cells and provide novel insights into cellular mechanosensation.

Substrate-induced differential degradation and partitioning of the two tryptophan permeases Tat1 and Tat2 into eisosomes in Saccharomyces cerevisiae.

Tryptophan is a relatively rare amino acid whose influx is strictly controlled to meet cellular demands. The yeast Saccharomyces cerevisiae has two tryptophan permeases, namely Tat1 (low-affinity type) and Tat2 (high-affinity type). These permeases are differentially regulated through ubiquitination based on inducible conditions and dependence on arrestin-related trafficking adaptors, although the physiological significance of their degradation remain unclear. Here, we demonstrated that Tat2 was rapidly degraded in an Rsp5-Bul1-dependent manner upon the addition of tryptophan, phenylalanine, or tyrosine, whereas Tat1 was unaffected. The expression of the ubiquitination-deficient variant Tat25K>R led to a reduction in cell yield at 4 µg/mL tryptophan, suggesting the occurrence of an uncontrolled, excessive consumption of tryptophan at low tryptophan concentrations. Eisosomes are membrane furrows that are thought to be storage compartments for some nutrient permeases. Tryptophan addition caused rapid Tat2 dissociation from eisosomes, whereas Tat1 distribution was unaffected. The 5 K > R mutation had no marked effect on Tat2 dissociation, suggesting that dissociation is independent of ubiquitination. Interestingly, the D74R mutation, which was created within the N-terminal acidic patch, stabilized Tat2 while reducing the degree of partitioning into eisosomes. Moreover, the hyperactive I285V mutation in Tat2, which increases Vmax/Km for tryptophan import by 2-fold, reduced the degree of segregation into eisosomes. Our findings illustrate the coordinated activity of Tat1 and Tat2 in the regulation of tryptophan transport at various tryptophan concentrations and suggest the positive role of substrates in inducing a conformational transition in Tat2, resulting in its dissociation from eisosomes and subsequent ubiquitination-dependent degradation.

SOD1 mutations cause hypersensitivity to high-pressure-induced oxidative stress in Saccharomyces cerevisiae.

Living organisms are subject to various mechanical stressors, such as high hydrostatic pressure. Empirical evidence shows that under high pressure, the oxidative stress response is activated in Saccharomyces cerevisiae. However, the mechanisms involved in its antioxidant systems are unclear. Here, we demonstrate that superoxide dismutase 1 (Sod1) plays a role in resisting high pressure for cell growth. Mutants lacking Sod1 or Ccs1, the copper chaperone for Sod1, displayed growth defects under 25 MPa. Of the various SOD1 mutations associated with familial amyotrophic lateral sclerosis, H46Q and S134N substitutions diminished SOD activity to levels comparable to those of catalytically deficient H63A and null mutants. When these mutant cells were cultured under 25 MPa, their intracellular O2•- levels increased while sod1∆ mutant genome stability was unaffected. The high-pressure sensitive sod1 mutants were also susceptible to sublethal levels of the O2•- generator paraquat. The sod1∆ mutant is known to exhibit methionine and lysine auxotrophy. However, excess methionine addition or overexpression of the lysine permease gene LYP1 did not counteract high-pressure sensitivity in the sod1 mutants, suggesting that their amino acid availability might be intact under 25 MPa. Interestingly, an exclusive localization of Sco2-Sod1 to the intermembrane space (IMS) of mitochondria appeared to partially restore the high-pressure growth ability in the sod1 mutants. Taken these results together, we suggest that high pressure enhances O2•- production and Sod1 within the IMS plays a role in scavenging O2•- allowing the cells to grow under high pressure. BACKGROUND: Empirical evidence shows that under high hydrostatic pressure, the oxidative stress response is activated in Saccharomyces cerevisiae. However, the mechanisms involved in its antioxidant systems are unclear. In the current study, we aimed to explore the role of superoxide dismutase 1 (Sod1) in yeast able to grow under high pressure. METHODS: Wild type and sod1 mutant cells were cultured in high-pressure chambers under 25 MPa (~250 kg/cm2). The SOD activity in whole cell extracts and 6His-tagged Sod1 recombinant proteins was analyzed using an SOD assay kit. The O2•- generation in cells was estimated by fluorescence staining. RESULTS: Mutants lacking Sod1 or Ccs1, the copper chaperone for Sod1, displayed growth defects under 25 MPa. Of the various SOD1 mutations associated with familial amyotrophic lateral sclerosis, H46Q and S134N substitutions diminished SOD activity to levels comparable to those of catalytically deficient H63A and null mutants. The high-pressure sensitive sod1 mutants were also susceptible to sublethal levels of the O2•- generator paraquat. Exclusive localization of Sco2-Sod1 to the intermembrane space (IMS) of mitochondria partially restored the high-pressure growth ability in the sod1 mutants. CONCLUSIONS: High pressure enhances O2•- production and Sod1 within the IMS plays a role in scavenging O2•- allowing the cells to grow under high pressure. GENERAL SIGNIFICANCE: Unlike external free radical-generating compounds, high-pressure treatment appeared to increase endogenous O2•- levels in yeast cells. Our experimental system offers a unique approach to investigating the physiological responses to mechanical and oxidative stresses in human body.

Molecular Responses to High Hydrostatic Pressure in Eukaryotes: Genetic Insights from Studies on Saccharomyces cerevisiae.

High hydrostatic pressure is common mechanical stress in nature and is also experienced by the human body. Organisms in the Challenger Deep of the Mariana Trench are habitually exposed to pressures up to 110 MPa. Human joints are intermittently exposed to hydrostatic pressures of 3-10 MPa. Pressures less than 50 MPa do not deform or kill the cells. However, high pressure can have various effects on the cell's biological processes. Although Saccharomyces cerevisiae is not a deep-sea piezophile, it can be used to elucidate the molecular mechanism underlying the cell's responses to high pressures by applying basic knowledge of the effects of pressure on industrial processes involving microorganisms. We have explored the genes associated with the growth of S. cerevisiae under high pressure by employing functional genomic strategies and transcriptomics analysis and indicated a strong association between high-pressure signaling and the cell's response to nutrient availability. This review summarizes the occurrence and significance of high-pressure effects on complex metabolic and genetic networks in eukaryotic cells and how the cell responds to increasing pressure by particularly focusing on the physiology of S. cerevisiae at the molecular level. Mechanosensation in humans has also been discussed.

Cellular NADH and NADPH Conformation as a Real-Time Fluorescence-Based Metabolic Indicator under Pressurized Conditions.

Cellular conformation of reduced pyridine nucleotides NADH and NADPH sensed using autofluorescence spectroscopy is presented as a real-time metabolic indicator under pressurized conditions. The approach provides information on the role of pressure in energy metabolism and antioxidant defense with applications in agriculture and food technologies. Here, we use spectral phasor analysis on UV-excited autofluorescence from Saccharomyces cerevisiae (baker's yeast) to assess the involvement of one or multiple NADH- or NADPH-linked pathways based on the presence of two-component spectral behavior during a metabolic response. To demonstrate metabolic monitoring under pressure, we first present the autofluorescence response to cyanide (a respiratory inhibitor) at 32 MPa. Although ambient and high-pressure responses remain similar, pressure itself also induces a response that is consistent with a change in cellular redox state and ROS production. Next, as an example of an autofluorescence response altered by pressurization, we investigate the response to ethanol at ambient, 12 MPa, and 30 MPa pressure. Ethanol (another respiratory inhibitor) and cyanide induce similar responses at ambient pressure. The onset of non-two-component spectral behavior upon pressurization suggests a change in the mechanism of ethanol action. Overall, results point to new avenues of investigation in piezophysiology by providing a way of visualizing metabolism and mitochondrial function under pressurized conditions.

Draft Genome Sequence of Naganishia liquefaciens Strain N6, Isolated from the Japan Trench.

The draft genome sequence of the deep-sea yeast Naganishia liquefaciens strain N6, isolated from the Japan Trench, is reported here. This strain was previously classified into a Cryptococcus clade. Phylogenetic analysis using the presented sequence suggests that strain N6 is in the clade of the genus Naganishia.

Amino acid homeostatic control by TORC1 in Saccharomyces cerevisiae under high hydrostatic pressure.

Mechanical stresses, including high hydrostatic pressure, elicit diverse physiological effects on organisms. Gtr1, Gtr2, Ego1 (also known as Meh1) and Ego3 (also known as Slm4), central regulators of the TOR complex 1 (TORC1) nutrient signaling pathway, are required for the growth of Saccharomyces cerevisiae cells under high pressure. Here, we showed that a pressure of 25 MPa (∼250 kg/cm2) stimulates TORC1 to promote phosphorylation of Sch9, which depends on the EGO complex (EGOC) and Pib2. Incubation of cells at this pressure aberrantly increased glutamine and alanine levels in the ego1Δ, gtr1Δ, tor1Δ and pib2Δ mutants, whereas the polysome profiles were unaffected. Moreover, we found that glutamine levels were reduced by combined deletions of EGO1, GTR1, TOR1 and PIB2 with GLN3 These results suggest that high pressure leads to the intracellular accumulation of amino acids. Subsequently, Pib2 loaded with glutamine stimulates the EGOC-TORC1 complex to inactivate Gln3, downregulating glutamine synthesis. Our findings illustrate the regulatory circuit that maintains intracellular amino acid homeostasis and suggest critical roles for the EGOC-TORC1 and Pib2-TORC1 complexes in the growth of yeast under high hydrostatic pressure.

A novel ER membrane protein Ehg1/May24 plays a critical role in maintaining multiple nutrient permeases in yeast under high-pressure perturbation.

Previously, we isolated 84 deletion mutants in Saccharomyces cerevisiae auxotrophic background that exhibited hypersensitive growth under high hydrostatic pressure and/or low temperature. Here, we observed that 24 deletion mutants were rescued by the introduction of four plasmids (LEU2, HIS3, LYS2, and URA3) together to grow at 25 MPa, thereby suggesting close links between the genes and nutrient uptake. Most of the highly ranked genes were poorly characterized, including MAY24/YPR153W. May24 appeared to be localized in the endoplasmic reticulum (ER) membrane. Therefore, we designated this gene as EHG (ER-associated high-pressure growth gene) 1. Deletion of EHG1 led to reduced nutrient transport rates and decreases in the nutrient permease levels at 25 MPa. These results suggest that Ehg1 is required for the stability and functionality of the permeases under high pressure. Ehg1 physically interacted with nutrient permeases Hip1, Bap2, and Fur4; however, alanine substitutions for Pro17, Phe19, and Pro20, which were highly conserved among Ehg1 homologues in various yeast species, eliminated interactions with the permeases as well as the high-pressure growth ability. By functioning as a novel chaperone that facilitated coping with high-pressure-induced perturbations, Ehg1 could exert a stabilizing effect on nutrient permeases when they are present in the ER.

Hyperactive mutation occurs adjacent to the essential glutamate 286 for transport in the yeast tryptophan permease Tat2.

In Saccharomyces cerevisiae, high-affinity tryptophan import is mediated by the plasma membrane permease Tat2. Herein, we identified hyperactive Tat2 mutations, I285V and I285T, which allowed the cells to grow at very low tryptophan concentrations (<4 µg/mL). The Km value of wild-type Tat2 for tryptophan appeared to be 24 µg/mL, whereas that of Tat2I285V and Tat2I285T was 17 and 11 µg/mL, respectively. Normalized values of Vmax/Km for Tat2I285V- and Tat2I285T-mediated tryptophan import were 2-fold higher than that for Tat2, suggesting that these mutations increase the affinity for tryptophan, and mediate transport at very low tryptophan concentrations. I285 resides adjacent to E286, a fully conserved residue among amino acid pemreases. According to a pKa prediction for E208 (pKa ∼8.3-11.7) of Escherichia coli AdiC antiporter, a structural homologue of Tat2, the E286 carboxyl chain of Tat2 could get loaded with a proton during tryptophan/H+ symport. Hence, I285V and I285T mutations might affect the buried residue environment of Tat2, thereby facilitating tryptophan import. Additionally, Tat2I285V and Tat2I285T levels increased rapidly, and were efficiently localized to the cell surface after transferring the cells to low tryptophan medium (0.5 µg/mL). Our findings provide a clue to gain insights into the property of high-affinity transport mechanisms, and offer a unique approach to improve the functionality of broad types of amino acid permeases.

Simulated microgravity triggers characteristic morphology and stress response in Saccharomyces cerevisiae.

Reduction of gravity results in changes in gene expression and morphology in the budding yeast Saccharomyces cerevisiae. We studied the genes responsible for the morphological changes induced by simulated microgravity (SMG) using the yeast morphology data. We comprehensively captured the features of the morphological changes in yeast cells cultured in SMG with CalMorph, a high-throughput image-processing system. Statistical analysis revealed that 95 of 501 morphological traits were significantly affected, which included changes in bud direction, the ratio of daughter to mother cell size, the random daughter cell shape, the large mother cell size, bright nuclei in the M phase, and the decrease in angle between two nuclei. We identified downregulated genes that impacted the morphological changes in conditions of SMG by focusing on each of the morphological features individually. Gene Ontology (GO)-enrichment analysis indicated that morphological changes under conditions of SMG were caused by cooperative downregulation of 103 genes annotated to six GO terms, which included cytoplasmic ribonucleoprotein granule, RNA elongation, mitotic cell cycle phase transition, nucleocytoplasmic transport, protein-DNA complex subunit organization, and RNA localization. P-body formation was also promoted under conditions of SMG. These results suggest that cooperative downregulation of multiple genes occurs in conditions of SMG.

Functional analysis of human aromatic amino acid transporter MCT10/TAT1 using the yeast Saccharomyces cerevisiae.

Tryptophan is an essential amino acid in humans and an important serotonin and melatonin precursor. Monocarboxylate transporter MCT10 is a member of the SLC16A family proteins that mediates low-affinity tryptophan transport across basolateral membranes of kidney, small intestine, and liver epithelial cells, although the precise transport mechanism remains unclear. Here we developed a simple functional assay to analyze tryptophan transport by human MCT10 using a deletion mutant for the high-affinity tryptophan permease Tat2 in Saccharomyces cerevisiae. tat2Δtrp1 cells are defective in growth in YPD medium because tyrosine present in the medium competes for the low-affinity tryptophan permease Tat1 with tryptophan. MCT10 appeared to allow growth of tat2Δtrp1 cells in YPD medium, and accumulate in cells deficient for Rsp5 ubiquitin ligase. These results suggest that MCT10 is functional in yeast, and is subject to ubiquitin-dependent quality control. Whereas growth of Tat2-expressing cells was significantly impaired by neutral pH, that of MCT10-expressing cells was nearly unaffected. This property is consistent with the transport mechanism of MCT10 via facilitated diffusion without a need for pH gradient across the plasma membrane. Single-nucleotide polymorphisms (SNPs) are known to occur in the human MCT10 coding region. Among eight SNP amino acid changes in MCT10, the N81K mutation completely abrogated tryptophan import without any abnormalities in the expression or localization. In the MCT10 modeled structure, N81 appeared to protrude into the putative trajectory of tryptophan. Plasma membrane localization of MCT10 and the variant proteins was also verified in human embryonic kidney 293T cells.

Critical role of the proton-dependent oligopeptide transporter (POT) in the cellular uptake of the peptidyl nucleoside antibiotic, blasticidin S.

Blasticidin S (BlaS) interferes in the cell growth of both eukaryotes and prokaryotes. Its mode of action as a protein synthesis inhibitor has been investigated extensively. However, the mechanism of BlaS transport into the target cells is not understood well. Here, we show that Ptr2, a member of the proton-dependent oligopeptide transporter (POT) family, is responsible for the uptake of BlaS in yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Notably, some mutants of Ptr2 that are dysfunctional in dipeptide uptake were still competent to transport BlaS. Mouse-derived oligopeptide transporter PepT1 conferred BlaS sensitivity in the S. cerevisiae ptr2∆ mutant. Furthermore, bacterial POT family proteins also potentiated the BlaS sensitivity of E. coli. The role of the POT family oligopeptide transporters in the uptake of BlaS is conserved across species from bacteria to mammals.

Involvement of flocculin in negative potential-applied ITO electrode adhesion of yeast cells.

The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between -0.2 and -0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications.

Effects of High Hydrostatic Pressure on Microbial Cell Membranes: Structural and Functional Perspectives.

Biological processes associated with dynamic structural features of membranes are highly sensitive to changes in hydrostatic pressure and temperature. Marine organisms potentially experience a broad range of pressure and temperature fluctuations. Hence, they have specialized cell membranes to perform membrane protein functions under various environmental conditions. Although the effects of high pressure on artificial lipid bilayers have been investigated in detail, little is known about how high pressure affects the structure of natural cell membranes and how organisms cope with pressure alterations. This review focused on the recent advances in research on the effects of high pressure on microbial membranes, particularly on the use of time-resolved fluorescence anisotropy measurement to determine membrane dynamics in deep-sea piezophiles.

Retention of chimeric Tat2-Gap1 permease in the endoplasmic reticulum induces unfolded protein response in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, high-affinity tryptophan import is performed by subtle mechanisms involving tryptophan permease Tat2. We have shown that Tat2 requires 15 amino acid residues in the transmembrane domains (TMDs) for its import activity, whereas leucine permease Bap2 requires only seven corresponding residues for its leucine import. For this reason, the structure of Tat2 is elaborately designed to transport the hydrophobic and bulky tryptophan. Newly synthesized cell surface proteins first undergo endoplasmic reticulum (ER)-associated quality check before entering the secretory pathway. In this study, we used domain replacement with general amino acid permease Gap1 to show that Tat2 chimeric proteins were dysfunctional when TMD10 or TMD11 was replaced. These chimeras formed large 270-800-kDa protein complexes and were stably retained in the ER membrane without efficient degradation. In contrast, Tat2 chimeras of TMD9 or TMD12 retained some of their tryptophan import activity and underwent vacuolar degradation as observed with wild-type Tat2. Thus, ours results suggest that TMD10 and TMD11 are essential for the correct folding of Tat2, probably because of their interdomain interactions. Notably, overexpression of Tat2-Gap1 chimera of TMD10 activated the unfolded protein response (UPR) element-lacZ reporter, suggesting that ER retention of the protein aggregates induces the UPR.

Glucosylceramide Contained in Koji Mold-Cultured Cereal Confers Membrane and Flavor Modification and Stress Tolerance to Saccharomyces cerevisiae during Coculture Fermentation.

In nature, different microorganisms create communities through their physiochemical and metabolic interactions. Many fermenting microbes, such as yeasts, lactic acid bacteria, and acetic acid bacteria, secrete acidic substances and grow faster at acidic pH values. However, on the surface of cereals, the pH is neutral to alkaline. Therefore, in order to grow on cereals, microbes must adapt to the alkaline environment at the initial stage of colonization; such adaptations are also crucial for industrial fermentation. Here, we show that the yeast Saccharomyces cerevisiae, which is incapable of synthesizing glucosylceramide (GlcCer), adapted to alkaline conditions after exposure to GlcCer from koji cereal cultured with Aspergillus kawachii. We also show that various species of GlcCer derived from different plants and fungi similarly conferred alkali tolerance to yeast. Although exogenous ceramide also enhanced the alkali tolerance of yeast, no discernible degradation of GlcCer to ceramide was observed in the yeast culture, suggesting that exogenous GlcCer itself exerted the activity. Exogenous GlcCer also increased ethanol tolerance and modified the flavor profile of the yeast cells by altering the membrane properties. These results indicate that GlcCer from A. kawachii modifies the physiology of the yeast S. cerevisiae and demonstrate a new mechanism for cooperation between microbes in food fermentation.

Carbohydrate-active enzymes identified by metagenomic analysis of deep-sea sediment bacteria.

Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psychrophilic aerobic bacteria on cellulose, xylan, chitin, and starch. A variety of species belonging to Alpha- and Gammaproteobacteria and to Flavobacteria were isolated from sediment depths between 12 and 42 mbsf. Metagenomic DNA purified from the pooled enrichments was sequenced and analyzed for phylogenetic composition and presence of genes encoding carbohydrate-active enzymes. More than 200 open reading frames coding for glycoside hydrolases were identified, and more than 60 of them relevant for enzymatic degradation of lignocellulose. Four genes encoding ß-glucosidases with less than 52% identities to characterized enzymes were chosen for recombinant expression in Escherichia coli. In addition one endomannanase, two endoxylanases, and three ß-xylosidases were produced recombinantly. All genes could be actively expressed. Functional analysis revealed discrepancies and additional variability for the recombinant enzymes as compared to the sequence-based predictions.

Functional implications and ubiquitin-dependent degradation of the peptide transporter Ptr2 in Saccharomyces cerevisiae.

The peptide transporter Ptr2 plays a central role in di- or tripeptide import in Saccharomyces cerevisiae. Although PTR2 transcription has been extensively analyzed in terms of upregulation by the Ubr1-Cup9 circuit, the structural and functional information for this transporter is limited. Here we identified 14 amino acid residues required for peptide import through Ptr2 based on the crystallographic information of Streptococcus thermophilus peptide transporter PepTst and based on the conservation of primary sequences among the proton-dependent oligopeptide transporters (POTs). Expression of Ptr2 carrying one of the 14 mutations of which the corresponding residues of PepTst are involved in peptide recognition, salt bridge interaction, or peptide translocation failed to enable ptr2Δtrp1 cell growth in alanyl-tryptophan (Ala-Trp) medium. We observed that Ptr2 underwent rapid degradation after cycloheximide treatment (half-life, approximately 1 h), and this degradation depended on Rsp5 ubiquitin ligase. The ubiquitination of Ptr2 most likely occurs at the N-terminal lysines 16, 27, and 34. Simultaneous substitution of arginine for the three lysines fully prevented Ptr2 degradation. Ptr2 mutants of the presumed peptide-binding site (E92Q, R93K, K205R, W362L, and E480D) exhibited severe defects in peptide import and were subjected to Rsp5-dependent degradation when cells were moved to Ala-Trp medium, whereas, similar to what occurs in the wild-type Ptr2, mutant proteins of the intracellular gate were upregulated. These results suggest that Ptr2 undergoes quality control and the defects in peptide binding and the concomitant conformational change render Ptr2 subject to efficient ubiquitination and subsequent degradation.

Loss of hydroxyl groups from the ceramide moiety can modify the lateral diffusion of membrane proteins in S. cerevisiae.

In the yeast Saccharomyces cerevisiae, structural diversities of complex sphingolipids [inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide] are often observed in the presence or absence of hydroxyl groups on the C-4 position of long-chain base (C4-OH) and the C-2 position of very long-chain fatty acids (C2-OH), but the biological significance of these groups remains unclear. Here, we evaluated cellular membrane fluidity in hydroxyl group-defective yeast mutants by fluorescence recovery after photobleaching. The lateral diffusion of enhanced green fluorescent protein-tagged hexose transporter 1 (Hxt1-EGFP) was influenced by the absence of C4-OH and/or C2-OH. Notably, the fluorescence recovery of Hxt1-EGFP was dramatically decreased in the sur2Δ mutant (absence of C4-OH) under the csg1Δcsh1Δ background, in which mannosylation of IPC is blocked leading to IPC accumulation, while the recovery in the scs7Δ mutant (absence of C2-OH) under the same background was modestly decreased. In addition, the amount of low affinity tryptophan transporter 1 (Tat1)-EGFP was markedly decreased in the sur2Δcsg1Δcsh1Δ mutant and accumulated in intracellular membranes in the scs7Δcsg1Δcsh1Δ mutant without altering its protein expression. These results suggest that C4-OH and C2-OH are most probably critical factors for maintaining membrane fluidity and proper turnover of membrane molecules in yeast containing complex sphingolipids with only one hydrophilic head group.