scholarly journals The Rag4 Glucose Sensor Is Involved in the Hypoxic Induction ofKlPDC1Gene Expression in the Yeast Kluyveromyces lactis

2010 ◽  
Vol 10 (1) ◽  
pp. 146-148 ◽  
Author(s):  
C. Micolonghi ◽  
M. Wésolowski-Louvel ◽  
M. M. Bianchi
Keyword(s):  
Glucose Sensor ◽  
Oxygen Sensing ◽  
Kluyveromyces Lactis ◽  
Content Type ◽  
Hypoxic Induction ◽  
Regulation Of Metabolism

ABSTRACTKluyveromyces lactisis a yeast which cannot grow under strict anaerobiosis. To date, no factors responsible for oxygen sensing and oxygen-dependent regulation of metabolism have been identified. In this paper we present the identification of the glucose sensor Rag4 as a factor essential for oxygen-dependent regulation of the fermentative pathway.

scholarly journals Glycolysis Controls Plasma Membrane Glucose Sensors To Promote Glucose Signaling in Yeasts

2014 ◽  
Vol 35 (4) ◽  
pp. 747-757 ◽  
Author(s):  
Amélie Cairey-Remonnay ◽  
Julien Deffaud ◽  
Micheline Wésolowski-Louvel ◽  
Marc Lemaire ◽  
Alexandre Soulard
Keyword(s):  
Signaling Pathway ◽  
Glucose Sensor ◽  
Kluyveromyces Lactis ◽  
Glucose Sensors ◽  
Content Type ◽  
Glucose Signaling ◽  
Extracellular Glucose ◽  
Glucose Accumulation ◽  
The Stability ◽  
Intracellular Glucose

Sensing of extracellular glucose is necessary for cells to adapt to glucose variation in their environment. In the respiratory yeastKluyveromyces lactis, extracellular glucose controls the expression of major glucose permease geneRAG1through a cascade similar to theSaccharomyces cerevisiaeSnf3/Rgt2/Rgt1 glucose signaling pathway. This regulation depends also on intracellular glucose metabolism since we previously showed that glucose induction of theRAG1gene is abolished in glycolytic mutants. Here we show that glycolysis regulatesRAG1expression through theK. lactisRgt1 (KlRgt1) glucose signaling pathway by targeting the localization and probably the stability of Rag4, the single Snf3/Rgt2-type glucose sensor ofK. lactis. Additionally, the control exerted by glycolysis on glucose signaling seems to be conserved inS. cerevisiae. This retrocontrol might prevent yeasts from unnecessary glucose transport and intracellular glucose accumulation.

scholarly journals Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

2014 ◽  
Vol 80 (20) ◽  
pp. 6549-6559 ◽  
Author(s):  
Sabrina Wemhoff ◽  
Roland Klassen ◽  
Friedhelm Meinhardt
Keyword(s):  
Kluyveromyces Lactis ◽  
Killer Toxin ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Protein Toxin ◽  
Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

scholarly journals Recombination Can Cause Telomere Elongations as Well as Truncations Deep within Telomeres in Wild-Type Kluyveromyces lactis Cells

2010 ◽  
Vol 10 (2) ◽  
pp. 226-236 ◽  
Author(s):  
Laura H. Bechard ◽  
Nathan Jamieson ◽  
Michael J. McEachern
Keyword(s):  
Great Increase ◽  
Kluyveromyces Lactis ◽  
High Rate ◽  
Wild Type ◽  
Content Type ◽  
Normal Length ◽  
Highly Sensitive ◽  
Significant Process ◽  
Type Size

ABSTRACT In this study, we examined the role of recombination at the telomeres of the yeast Kluyveromyces lactis . We demonstrated that an abnormally long and mutationally tagged telomere was subject to high rates of telomere rapid deletion (TRD) that preferentially truncated the telomere to near-wild-type size. Unlike the case in Saccharomyces cerevisiae , however, there was not a great increase in TRD in meiosis. About half of mitotic TRD events were associated with deep turnover of telomeric repeats, suggesting that telomeres were often cleaved to well below normal length prior to being reextended by telomerase. Despite its high rate of TRD, the long telomere showed no increase in the rate of subtelomeric gene conversion, a highly sensitive test of telomere dysfunction. We also showed that the long telomere was subject to appreciable rates of becoming elongated substantially further through a recombinational mechanism that added additional tagged repeats. Finally, we showed that the deep turnover that occurs within normal-length telomeres was diminished in the absence of RAD52 . Taken together, our results suggest that homologous recombination is a significant process acting on both abnormally long and normally sized telomeres in K. lactis .

scholarly journals Intracellular NADPH Levels Affect the Oligomeric State of the Glucose 6-Phosphate Dehydrogenase

2012 ◽  
Vol 11 (12) ◽  
pp. 1503-1511 ◽  
Author(s):  
Michele Saliola ◽  
Angela Tramonti ◽  
Claudio Lanini ◽  
Samantha Cialfi ◽  
Daniela De Biase ◽  
...  
Keyword(s):  
Specific Activity ◽  
Kluyveromyces Lactis ◽  
Growth Conditions ◽  
G6pdh Activity ◽  
Oligomeric State ◽  
Content Type ◽  
Metabolic Role ◽  
Activity Band ◽  
Glucose 6 Phosphate Dehydrogenase

ABSTRACTIn the yeastKluyveromyces lactis, glucose 6-phosphate dehydrogenase (G6PDH) is detected as two differently migrating forms on native polyacrylamide gels. The pivotal metabolic role of G6PDH inK. lactisled us to investigate the mechanism controlling the two activities in respiratory and fermentative mutant strains. An extensive analysis of these mutants showed that the NAD+(H)/NADP+(H)-dependent cytosolic alcohol (ADH) and aldehyde (ALD) dehydrogenase balance affects the expression of the G6PDH activity pattern. Under fermentative/ethanol growth conditions, the concomitant activation of ADH and ALD activities led to cytosolic accumulation of NADPH, triggering an alteration in the oligomeric state of the G6PDH caused by displacement/release of the structural NADP+bound to each subunit of the enzyme. The new oligomeric G6PDH form with faster-migrating properties increases as a consequence of intracellular redox unbalance/NADPH accumulation, which inhibits G6PDH activityin vivo. The appearance of a new G6PDH-specific activity band, following incubation ofSaccharomyces cerevisiaeand human cellular extracts with NADP+, also suggests that a regulatory mechanism of this activity through NADPH accumulation is highly conserved among eukaryotes.

scholarly journals Interaction with Enzyme IIBMpo(EIIBMpo) and Phosphorylation by Phosphorylated EIIBMpoExert Antagonistic Effects on the Transcriptional Activator ManR of Listeria monocytogenes

2015 ◽  
Vol 197 (9) ◽  
pp. 1559-1572 ◽  
Author(s):  
Arthur Constant Zébré ◽  
Francine Moussan Aké ◽  
Magali Ventroux ◽  
Rose Koffi-Nevry ◽  
Marie-Françoise Noirot-Gros ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Glucose Sensor ◽  
Dual Role ◽  
Transcription Activator ◽  
Phosphotransferase System ◽  
Content Type ◽  
Antagonistic Effects ◽  
Virulence Gene Expression ◽  
Low Efficiency ◽  
Terminal Domains

ABSTRACTListeriae take up glucose and mannose predominantly through a mannose class phosphoenolpyruvate:carbohydrate phosphotransferase system (PTSMan), whose three components are encoded by themanLMNgenes. The expression of these genes is controlled by ManR, a LevR-type transcription activator containing two PTS regulation domains (PRDs) and two PTS-like domains (enzyme IIAMan[EIIAMan]- and EIIBGat-like). We demonstrate here that inListeria monocytogenes, ManR is activated via the phosphorylation of His585 in the EIIAMan-like domain by the general PTS components enzyme I and HPr. We also show that ManR is regulated by the PTSMpoand that EIIBMpoplays a dual role in ManR regulation. First, yeast two-hybrid experiments revealed that unphosphorylated EIIBMpointeracts with the two C-terminal domains of ManR (EIIBGat-like and PRD2) and that this interaction is required for ManR activity. Second, in the absence of glucose/mannose, phosphorylated EIIBMpo(P∼EIIBMpo) inhibits ManR activity by phosphorylating His871 in PRD2. The presence of glucose/mannose causes the dephosphorylation of P∼EIIBMpoand P∼PRD2 of ManR, which together lead to the induction of themanLMNoperon. Complementation of a ΔmanRmutant with variousmanRalleles confirmed the antagonistic effects of PTS-catalyzed phosphorylation at the two different histidine residues of ManR. Deletion ofmanRprevented not only the expression of themanLMNoperon but also glucose-mediated repression of virulence gene expression; however, repression by other carbohydrates was unaffected. Interestingly, the expression ofmanLMNinListeria innocuawas reported to require not only ManR but also the Crp-like transcription activator Lin0142. Unlike Lin0142, theL. monocytogeneshomologue, Lmo0095, is not required formanLMNexpression; its absence rather stimulatesmanexpression.IMPORTANCEListeria monocytogenesis a human pathogen causing the foodborne disease listeriosis. The expression of most virulence genes is controlled by the transcription activator PrfA. Its activity is strongly repressed by carbohydrates, including glucose, which is transported intoL. monocytogenesmainly via a mannose/glucose-specific phosphotransferase system (PTSMan). Expression of themanoperon is regulated by the transcription activator ManR, the activity of which is controlled by a second, low-efficiency PTS of the mannose family, which functions as glucose sensor. Here we demonstrate that the EIIBMpocomponent plays a dual role in ManR regulation: it inactivates ManR by phosphorylating its His871 residue and stimulates ManR by interacting with its two C-terminal domains.

scholarly journals Induction by Hypoxia of Heterologous-Protein Production with the KlPDC1 Promoter in Yeasts

2006 ◽  
Vol 73 (3) ◽  
pp. 922-929 ◽  
Author(s):  
Andrea Camattari ◽  
Michele M. Bianchi ◽  
Paola Branduardi ◽  
Danilo Porro ◽  
Luca Brambilla
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Heterologous Gene Expression ◽  
Kluyveromyces Lactis ◽  
Expression System ◽  
Promoter Sequence ◽  
Heterologous Protein Production ◽  
Heterologous Proteins ◽  
Hypoxic Induction ◽  
Production Ratio

ABSTRACT The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the KlPDC1 promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in Kluyveromyces lactis under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1β was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The lacZ gene from Escherichia coli was cloned downstream of the KlPDC1 promoter in order to get β-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in Saccharomyces cerevisiae and in Zygosaccharomyces bailii, and effects of hypoxic induction were measured and compared to those already observed in K. lactis cells. Interestingly, we found that the induction still worked in Z. bailii; thus, this promotor constitutes a possible inducible system for this new nonconventional host.

scholarly journals Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F 1 -ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

2013 ◽  
Vol 12 (11) ◽  
pp. 1451-1461 ◽  
Author(s):  
Thuy La ◽  
George Desmond Clark-Walker ◽  
Xiaowen Wang ◽  
Stephan Wilkens ◽  
Xin Jie Chen
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Kluyveromyces Lactis ◽  
Β Subunit ◽  
Α Subunit ◽  
Content Type ◽  
Subunit Stoichiometry ◽  
Catalytic Sites ◽  
A Subunit

ABSTRACT F 1 -ATPase is a rotary molecular machine with a subunit stoichiometry of α 3 β 3 γ 1 δ 1 ε 1 . It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α 3 β 3 core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F 1 -ATPase have suggested that the α 3 β 3 core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F 1 -ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ o ) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F 1 complexes retained 21.7 to 44.6% of the native F 1 -ATPase activity. The γ-less F 1 subcomplex was assembled but was structurally and functionally labile in vitro . Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ o conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α 3 β 3 subcomplex.

scholarly journals Ineffective Phosphorylation of Mitogen-Activated Protein Kinase Hog1p in Response to High Osmotic Stress in the Yeast Kluyveromyces lactis

2015 ◽  
Vol 14 (9) ◽  
pp. 922-930 ◽  
Author(s):  
Nancy Velázquez-Zavala ◽  
Miriam Rodríguez-González ◽  
Rocío Navarro-Olmos ◽  
Laura Ongay-Larios ◽  
Laura Kawasaki ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Osmotic Stress ◽  
Kluyveromyces Lactis ◽  
Chimeric Protein ◽  
High Sensitivity ◽  
Hyperosmotic Stress ◽  
Mitogen Activated Protein Kinase ◽  
Hog Pathway ◽  
Content Type ◽  
Mitogen Activated Protein

ABSTRACT When treated with a hyperosmotic stimulus, Kluyveromyces lactis cells respond by activating the mitogen-activated protein kinase (MAPK) K. lactis Hog1 (KlHog1) protein via two conserved branches, SLN1 and SHO1. Mutants affected in only one branch can cope with external hyperosmolarity by activating KlHog1p by phosphorylation, except for single Δ Klste11 and Δ Klste50 mutants, which showed high sensitivity to osmotic stress, even though the other branch (SLN1) was intact. Inactivation of both branches by deletion of KlSHO1 and KlSSK2 also produced sensitivity to high salt. Interestingly, we have observed that in Δ Klste11 and Δ Klsho1 Δ Klssk2 mutants, which exhibit sensitivity to hyperosmotic stress, and contrary to what would be expected, KlHog1p becomes phosphorylated. Additionally, in mutants lacking both MAPK kinase kinases (MAPKKKs) present in K. lactis (KlSte11p and KlSsk2p), the hyperosmotic stress induced the phosphorylation and nuclear internalization of KlHog1p, but it failed to induce the transcriptional expression of KlSTL1 and the cell was unable to grow in high-osmolarity medium. KlHog1p phosphorylation via the canonical HOG pathway or in mutants where the SHO1 and SLN1 branches have been inactivated requires not only the presence of KlPbs2p but also its kinase activity. This indicates that when the SHO1 and SLN1 branches are inactivated, high-osmotic-stress conditions activate an independent input that yields active KlPbs2p, which, in turn, renders KlHog1p phosphorylation ineffective. Finally, we found that KlSte11p can alleviate the sensitivity to hyperosmotic stress displayed by a Δ Klsho1 Δ Klssk2 mutant when it is anchored to the plasma membrane by adding the KlSho1p transmembrane segments, indicating that this chimeric protein can substitute for KlSho1p and KlSsk2p.

scholarly journals The hypoxic expression of the glucose transporter RAG1 reveals the role of the bHLH transcription factor Sck1 as a novel hypoxic modulator in Kluyveromyces lactis

2019 ◽  
Vol 19 (4) ◽  
Author(s):  
Rosa Santomartino ◽  
Daniela Ottaviano ◽  
Ilaria Camponeschi ◽  
Tracy Ann Alcarpio Landicho ◽  
Luca Falato ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Intracellular Signaling ◽  
Molecular Mechanisms ◽  
Glucose Transporter ◽  
Kluyveromyces Lactis ◽  
Transcriptional Activator ◽  
Bhlh Transcription Factor ◽  
Cellular Processes ◽  
Hypoxic Induction ◽  
Rag1 Gene

ABSTRACTGlucose is the preferred nutrient for most living cells and is also a signaling molecule that modulates several cellular processes. Glucose regulates the expression of glucose permease genes in yeasts through signaling pathways dependent on plasma membrane glucose sensors. In the yeast Kluyveromyces lactis, sufficient levels of glucose induction of the low-affinity glucose transporter RAG1 gene also depends on a functional glycolysis, suggesting additional intracellular signaling. We have found that the expression of RAG1 gene is also induced by hypoxia in the presence of glucose, indicating that glucose and oxygen signaling pathways are interconnected. In this study we investigated the molecular mechanisms underlying this crosstalk. By analyzing RAG1 expression in various K. lactis mutants, we found that the bHLH transcriptional activator Sck1 is required for the hypoxic induction of RAG1 gene. The RAG1 promoter region essential for its hypoxic induction was identified by promoter deletion experiments. Taken together, these results show that the RAG1 glucose permease gene is synergistically induced by hypoxia and glucose and highlighted a novel role for the transcriptional activator Sck1 as a key mediator in this mechanism.

scholarly journals Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function inCandida albicans

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Swati Bijlani ◽  
Mathuravani A. Thevandavakkam ◽  
Hung-Ji Tsai ◽  
Judith Berman
Keyword(s):  
Candida Albicans ◽  
Colony Size ◽  
Transformation Efficiency ◽  
Kluyveromyces Lactis ◽  
Baker’S Yeast ◽  
Dependent Manner ◽  
Baker's Yeast ◽  
Linear Plasmids ◽  
Content Type ◽  
Bona Fide

ABSTRACTThe ability to generate autonomously replicating plasmids has been elusive inCandida albicans, a prevalent human fungal commensal and pathogen. Instead, plasmids generally integrate into the genome. Here, we assessed plasmid and transformant properties, including plasmid geometry, transformant colony size, four selectable markers, and potential origins of replication, for their ability to drive autonomous plasmid maintenance. Importantly, linear plasmids with terminal telomere repeats yielded many more autonomous transformants than circular plasmids with the identical sequences. Furthermore, we could distinguish (by colony size) transient, autonomously replicating, and chromosomally integrated transformants (tiny, medium, and large, respectively).Candida albicansURA3and a heterologous marker,ARG4,yielded many transient transformants indicative of weak origin activity; the replication of the plasmid carrying the heterologousLEU2marker was highly dependent upon the addition of abona fideorigin sequence. Severalbona fidechromosomal origins, with an origin fragment of ∼100 bp as well as a heterologous origin,panARS, fromKluyveromyces lactis, drove autonomous replication, yielding moderate transformation efficiency and plasmid stability. Thus,C. albicansmaintains linear plasmids that yield high transformation efficiency and are maintained autonomously in an origin-dependent manner.IMPORTANCECircular plasmids are important tools for molecular manipulation in model fungi such as baker’s yeast, yet, inCandida albicans, an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate inC. albicans. We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that an ∼100-bp fragment of aC. albicansorigin and an origin sequence from a distantly related yeast can both function as origins inC. albicans. Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ betweenC. albicansand baker’s yeast.

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