scholarly journals 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

2013 ◽  
Vol 12 (7) ◽  
pp. 990-997 ◽  
Author(s):  
Asaha Suzuki ◽  
Takahiro Mochizuki ◽  
Satoshi Uemura ◽  
Toshiki Hiraki ◽  
Fumiyoshi Abe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Pressure ◽  
Hydrostatic Pressure ◽  
Ubiquitin Ligase ◽  
High Hydrostatic Pressure ◽  
Intracellular Localization ◽  
Normal Growth ◽  
Growth Conditions ◽  
Content Type ◽  
Genes Encoding

ABSTRACT 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 bul1 Δ bul2 Δ 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.

scholarly journals Pressure-Induced Differential Regulation of the Two Tryptophan Permeases Tat1 and Tat2 by Ubiquitin Ligase Rsp5 and Its Binding Proteins, Bul1 and Bul2

2003 ◽  
Vol 23 (21) ◽  
pp. 7566-7584 ◽  
Author(s):  
Fumiyoshi Abe ◽  
Hidetoshi Iida
Keyword(s):  
High Pressure ◽  
Hydrostatic Pressure ◽  
Lipid Rafts ◽  
Ubiquitin Ligase ◽  
High Hydrostatic Pressure ◽  
State Level ◽  
Limiting Factor ◽  
Dependent Manner ◽  
Double Mutation ◽  
Tryptophan Uptake

ABSTRACT Tryptophan uptake appears to be the Achilles' heel in yeast physiology, since under a variety of seemingly diverse toxic conditions, it becomes the limiting factor for cell growth. When growing cells of Saccharomyces cerevisiae are subjected to high hydrostatic pressure, tryptophan uptake is down-regulated, leading to cell cycle arrest in the G1 phase. Here we present evidence that the two tryptophan permeases Tat1 and Tat2 are differentially regulated by Rsp5 ubiquitin ligase in response to high hydrostatic pressure. Analysis of high-pressure growth mutants revealed that the HPG1 gene was allelic to RSP5. The HPG1 mutation or the bul1Δ bul2Δ double mutation caused a marked increase in the steady-state level of Tat2 but not of Tat1, although both permeases were degraded at high pressure in an Rsp5-dependent manner. There were marked differences in subcellular localization. Tat1 localized predominantly in the plasma membrane, whereas Tat2 was abundant in the internal membranes. Moreover, Tat1 was associated with lipid rafts, whereas Tat2 localized in bulk lipids. Surprisingly, Tat2 became associated with lipid rafts upon the occurrence of a ubiquitination defect. These results suggest that ubiquitination is an important determinant of the localization and regulation of these tryptophan permeases. Determination of the activation volume (ΔV ≠) for Tat1- and Tat2-mediated tryptophan uptake (89.3 and 50.8 ml/mol, respectively) revealed that both permeases are highly sensitive to membrane perturbation and that Tat1 rather than Tat2 is likely to undergo a dramatic conformational change during tryptophan import. We suggest that hydrostatic pressure is a unique tool for elucidating the dynamics of integral membrane protein functions as well as for probing lipid microenvironments where they localize.

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

Biology ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1305
Author(s):  
Fumiyoshi Abe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Pressure ◽  
Hydrostatic Pressure ◽  
High Hydrostatic Pressure ◽  
High Pressures ◽  
Strong Association ◽  
Pressure Effects ◽  
Basic Knowledge ◽  
Challenger Deep ◽  
Human Joints

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.

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

2020 ◽  
Vol 133 (17) ◽  
pp. jcs245555
Author(s):  
Satoshi Uemura ◽  
Takahiro Mochizuki ◽  
Kengo Amemiya ◽  
Goyu Kurosaka ◽  
Miho Yazawa ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Pressure ◽  
Amino Acid ◽  
Hydrostatic Pressure ◽  
High Hydrostatic Pressure ◽  
Intracellular Accumulation ◽  
Intracellular Amino Acid ◽  
Regulatory Circuit ◽  
Nutrient Signaling ◽  
Glutamine Synthesis

ABSTRACTMechanical 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.

Effect of High Hydrostatic Pressure on Salmonella Inoculated into Creamy Peanut Butter with Modified Composition

2014 ◽  
Vol 77 (10) ◽  
pp. 1664-1668 ◽  
Author(s):  
TANYA D'SOUZA ◽  
MUKUND KARWE ◽  
DONALD W. SCHAFFNER
Keyword(s):  
High Pressure ◽  
Hydrostatic Pressure ◽  
Water Activity ◽  
High Hydrostatic Pressure ◽  
Peanut Butter ◽  
High Pressure Processing ◽  
Peanut Oil ◽  
Peanut Flour ◽  
Log Reduction ◽  
High Hydrostatic Pressure Processing

Peanut butter has been associated with several large foodborne salmonellosis outbreaks. This research investigates the potential of high hydrostatic pressure processing (HPP) for inactivation of Salmonella in peanut butter of modified composition, both by modifying its water activity as well by the addition of various amounts of nisin. A cocktail of six Salmonella strains associated with peanut butter and nut-related outbreaks was used for all experiments. Different volumes of sterile distilled water were added to peanut butter to increase water activity, and different volumes of peanut oil were added to decrease water activity. Inactivation in 12% fat, light roast, partially defatted peanut flour, and peanut oil was also quantified. Nisaplin was incorporated into peanut butter at four concentrations corresponding to 2.5, 5.0, 12.5, and 25.0 ppm of pure nisin. All samples were subjected to 600 MPa for 18 min. A steady and statistically significant increase in log reduction was seen as added moisture was increased from 50 to 90%. The color of all peanut butter samples containing added moisture contents darkened after high pressure processing. The addition of peanut oil to further lower the water activity of peanut butter further reduced the effectiveness of HPP. Just over a 1-log reduction was obtained in peanut flour, while inactivation to below detection limits (2 log CFU/g) was observed in peanut oil. Nisin alone without HPP had no effect. Recovery of Salmonella after a combined nisin and HPP treatment did show increased log reduction with longer storage times. The maximum log reduction of Salmonella achieved was 1.7 log CFU/g, which was comparable to that achieved by noncycling pressure treatment alone. High pressure processing alone or with other formulation modification, including added nisin, is not a suitable technology to manage the microbiological safety of Salmonella-contaminated peanut butter.

scholarly journals High-Pressure Inactivation of Hepatitis A Virus within Oysters

2005 ◽  
Vol 71 (1) ◽  
pp. 339-343 ◽  
Author(s):  
Kevin R. Calci ◽  
Gloria K. Meade ◽  
Robert C. Tezloff ◽  
David H. Kingsley
Keyword(s):  
High Pressure ◽  
Hydrostatic Pressure ◽  
High Hydrostatic Pressure ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Natural Conditions ◽  
Direct Evaluation ◽  
Temperature Range

ABSTRACT Previous results demonstrated that hepatitis A virus (HAV) could be inactivated by high hydrostatic pressure (HHP) (D. H. Kingsley, D. Hoover, E. Papafragkou, and G. P. Richards, J. Food Prot. 65:1605-1609, 2002); however, direct evaluation of HAV inactivation within contaminated oysters was not performed. In this study, we report confirmation that HAV within contaminated shellfish is inactivated by HHP. Shellfish were initially contaminated with HAV by using a flowthrough system. PFU reductions of >1, >2, and >3 log10 were observed for 1-min treatments at 350, 375, and 400 megapascals, respectively, within a temperature range of 8.7 to 10.3�C. Bioconcentration of nearly 6 log10 PFU of HAV per oyster was achieved under simulated natural conditions. These results suggest that HHP treatment of raw shellfish will be a viable strategy for the reduction of infectious HAV.

scholarly journals Selective Microautophagy of Proteasomes is Initiated by ESCRT-0 and Is Promoted by Proteasome Ubiquitylation

2021 ◽  
Author(s):  
Jianhui Li ◽  
Mark Hochstrasser
Keyword(s):  
Ubiquitin Ligase ◽  
Normal Growth ◽  
Ubiquitin Proteasome System ◽  
Growth Conditions ◽  
Endosomal Sorting ◽  
Glucose Depletion ◽  
Ubiquitin Binding ◽  
Ubiquitin Proteasome ◽  
Low Glucose ◽  
Amp Activated Protein Kinase

The proteasome is central to proteolysis by the ubiquitin-proteasome system under normal growth conditions but is itself degraded through macroautophagy under nutrient stress. A recently described AMPK (AMP-activated protein kinase)-regulated ESCRT (endosomal sorting complex required for transport)-dependent microautophagy pathway also regulates proteasome trafficking and degradation in low glucose conditions in yeast. Aberrant proteasomes are more prone to microautophagy, suggesting the ESCRT system fine-tunes proteasome quality control under low glucose stress. Here we uncover additional features of the selective microautophagy of proteasomes. Genetic or pharmacological induction of aberrant proteasomes is associated with increased mono- or oligo-ubiquitylation of proteasome components, which appear to be recognized by ESCRT-0. AMPK controls this pathway in part by regulating the trafficking of ESCRT-0 to the vacuole surface, which also leads to degradation of the Vps27 subunit of ESCRT-0. The Rsp5 ubiquitin ligase contributes to proteasome subunit ubiquitylation, and multiple ubiquitin-binding elements in Vps27 are involved in their recognition. We propose that ESCRT-0 at the vacuole surface recognizes ubiquitylated proteasomes and initiates their microautophagic elimination during glucose depletion.

Constitutive and inducible Saccharomyces cerevisiae promoters: evidence for two distinct molecular mechanisms

1986 ◽  
Vol 6 (11) ◽  
pp. 3847-3853
Author(s):  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mechanisms ◽  
Normal Growth ◽  
Regulatory Elements ◽  
Growth Conditions ◽  
Physical Structure ◽  
Base Pairs ◽  
Inducible Promoters ◽  
Upstream Promoter ◽  
Selection Of

his3 and pet56 are adjacent Saccharomyces cerevisiae genes that are transcribed in opposite directions from initiation sites that are separated by 200 base pairs. Under normal growth conditions, in which his3 and pet56 are transcribed at similar basal levels, a poly(dA-dT) sequence located between the genes serves as the upstream promoter element for both. In contrast, his3 but not pet56 transcription is induced during conditions of amino acid starvation, even though the critical regulatory site is located upstream of both respective TATA regions. Moreover, only one of the two normal his3 initiation sites is subject to induction. From genetic and biochemical evidence, I suggest that the his3-pet56 intergenic region contains constitutive and inducible promoters with different properties. In particular, two classes of TATA elements, constitutive (Tc) and regulatory (Tr), can be distinguished by their ability to respond to upstream regulatory elements, by their effects on the selection of initiation sites, and by their physical structure in nuclear chromatin. Constitutive and inducible his3 transcription is mediated by distinct promoters representing each class, whereas pet56 transcription is mediated by a constitutive promoter. Molecular mechanisms for these different kinds of S. cerevisiae promoters are proposed.

scholarly journals Overexpression of WAX INDUCER1/SHINE1 Gene Enhances Wax Accumulation under Osmotic Stress and Oil Synthesis in Brassica napus

2019 ◽  
Vol 20 (18) ◽  
pp. 4435 ◽  
Author(s):  
Ning Liu ◽  
Jie Chen ◽  
Tiehu Wang ◽  
Qing Li ◽  
Pengpeng Cui ◽  
...  
Keyword(s):  
Salt Stress ◽  
Brassica Napus ◽  
De Novo ◽  
Normal Growth ◽  
Acid Synthesis ◽  
Growth Conditions ◽  
Lipid Profiling ◽  
Intracellular Lipids ◽  
Oil Synthesis ◽  
Genes Encoding

WAX INDUCER1/SHINE1 (WIN1) belongs to the AP2/EREBP transcription factor family and plays an important role in wax and cutin accumulation in plants. Here we show that BnWIN1 from Brassica napus (Bn) has dual functions in wax accumulation and oil synthesis. Overexpression (OE) of BnWIN1 led to enhanced wax accumulation and promoted growth without adverse effects on oil synthesis under salt stress conditions. Lipid profiling revealed that BnWIN1-OE plants accumulated more waxes with elevated C29-alkanes, C31-alkanes, C28-alcohol, and C29-alcohol relative to wild type (WT) under salt stress. Moreover, overexpression of BnWIN1 also increased seed oil content under normal growth conditions. BnWIN1 directly bound to the promoter region of genes encoding biotin carboxyl carrier protein 1 (BCCP1), glycerol-3-phosphate acyltransferase 9 (GPAT9), lysophosphatidic acid acyltransferase 5 (LPAT5), and diacylglycerol acyltransferase 2 (DGAT2) involved in the lipid anabolic process. Overexpression of BnWIN1 resulted in upregulated expression of numerous genes involved in de novo fatty acid synthesis, wax accumulation, and oil production. The results suggest that BnWIN1 is a transcriptional activator to regulate the biosynthesis of both extracellular and intracellular lipids.

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