scholarly journals Overproduction of Human Zip (SLC39) Zinc Transporters in Saccharomyces cerevisiae for Biophysical Characterization

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 213
Author(s):  
Eva Ramos Becares ◽  
Per Amstrup Pedersen ◽  
Pontus Gourdon ◽  
Kamil Gotfryd
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biochemical Analysis ◽  
Toxic Metal ◽  
Zinc Homeostasis ◽  
Animal Kingdom ◽  
Biophysical Characterization ◽  
Functional Studies ◽  
Cellular Processes ◽  
Dna And Protein Synthesis ◽  
Solute Carrier

Zinc constitutes the second most abundant transition metal in the human body, and it is implicated in numerous cellular processes, including cell division, DNA and protein synthesis as well as for the catalytic activity of many enzymes. Two major membrane protein families facilitate zinc homeostasis in the animal kingdom, i.e., Zrt/Irt-like proteins (ZIPs aka solute carrier 39, SLC39, family) and Zn transporters (ZnTs), essentially conducting zinc flux in the opposite directions. Human ZIPs (hZIPs) regulate import of extracellular zinc to the cytosol, being critical in preventing overaccumulation of this potentially toxic metal, and crucial for diverse physiological and pathological processes, including development of neurodegenerative disorders and several cancers. To date, our understanding of structure–function relationships governing hZIP-mediated zinc transport mechanism is scarce, mainly due to the notorious difficulty in overproduction of these proteins for biophysical characterization. Here we describe employment of a Saccharomyces cerevisiae-based platform for heterologous expression of hZIPs. We demonstrate that yeast is able to produce four full-length hZIP members belonging to three different subfamilies. One target (hZIP1) is purified in the high quantity and homogeneity required for the downstream biochemical analysis. Our work demonstrates the potential of the described production system for future structural and functional studies of hZIP transporters.

Saccharomyces cerevisiae SSD1-VConfers Longevity by a Sir2p-Independent Mechanism

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1661-1672 ◽  
Author(s):  
Matt Kaeberlein ◽  
Alex A Andalis ◽  
Gregory B Liszt ◽  
Gerald R Fink ◽  
Leonard Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Life Span ◽  
Cell Cycle Progression ◽  
Polymorphic Locus ◽  
Cell Integrity ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Maximal Life Span ◽  
Independent Mechanism ◽  
Yeast Aging

AbstractThe SSD1 gene of Saccharomyces cerevisiae is a polymorphic locus that affects diverse cellular processes including cell integrity, cell cycle progression, and growth at high temperature. We show here that the SSD1-V allele is necessary for cells to achieve extremely long life span. Furthermore, addition of SSD1-V to cells can increase longevity independently of SIR2, although SIR2 is necessary for SSD1-V cells to attain maximal life span. Past studies of yeast aging have been performed in short-lived ssd1-d strain backgrounds. We propose that SSD1-V defines a previously undescribed pathway affecting cellular longevity and suggest that future studies on longevity-promoting genes should be carried out in long-lived SSD1-V strains.

scholarly journals Phylogenetic comparison and splice site conservation of eukaryotic U1 snRNP-specific U1-70K gene family

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tao Fan ◽  
Yu-Zhen Zhao ◽  
Jing-Fang Yang ◽  
Qin-Lai Liu ◽  
Yuan Tian ◽  
...  
Keyword(s):  
Gene Family ◽  
Splice Site ◽  
Messenger Rna ◽  
Expression Patterns ◽  
Protein Structures ◽  
Single Copy ◽  
Central Component ◽  
Animal Kingdom ◽  
Functional Studies ◽  
U1 Snrnp

AbstractEukaryotic cells can expand their coding ability by using their splicing machinery, spliceosome, to process precursor mRNA (pre-mRNA) into mature messenger RNA. The mega-macromolecular spliceosome contains multiple subcomplexes, referred to as small nuclear ribonucleoproteins (snRNPs). Among these, U1 snRNP and its central component, U1-70K, are crucial for splice site recognition during early spliceosome assembly. The human U1-70K has been linked to several types of human autoimmune and neurodegenerative diseases. However, its phylogenetic relationship has been seldom reported. To this end, we carried out a systemic analysis of 95 animal U1-70K genes and compare these proteins to their yeast and plant counterparts. Analysis of their gene and protein structures, expression patterns and splicing conservation suggest that animal U1-70Ks are conserved in their molecular function, and may play essential role in cancers and juvenile development. In particular, animal U1-70Ks display unique characteristics of single copy number and a splicing isoform with truncated C-terminal, suggesting the specific role of these U1-70Ks in animal kingdom. In summary, our results provide phylogenetic overview of U1-70K gene family in vertebrates. In silico analyses conducted in this work will act as a reference for future functional studies of this crucial U1 splicing factor in animal kingdom.

scholarly journals Global mapping of protein–metabolite interactions in Saccharomyces cerevisiae reveals that Ser-Leu dipeptide regulates phosphoglycerate kinase activity

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Marcin Luzarowski ◽  
Rubén Vicente ◽  
Andrei Kiselev ◽  
Mateusz Wagner ◽  
Dennis Schlossarek ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Small Molecules ◽  
Phosphoglycerate Kinase ◽  
Glycolytic Enzyme ◽  
Diauxic Shift ◽  
Cellular Processes ◽  
Targeted Analysis ◽  
Global Mapping ◽  
Biochemical Approach ◽  
User Friendly

AbstractProtein–metabolite interactions are of crucial importance for all cellular processes but remain understudied. Here, we applied a biochemical approach named PROMIS, to address the complexity of the protein–small molecule interactome in the model yeast Saccharomyces cerevisiae. By doing so, we provide a unique dataset, which can be queried for interactions between 74 small molecules and 3982 proteins using a user-friendly interface available at https://promis.mpimp-golm.mpg.de/yeastpmi/. By interpolating PROMIS with the list of predicted protein–metabolite interactions, we provided experimental validation for 225 binding events. Remarkably, of the 74 small molecules co-eluting with proteins, 36 were proteogenic dipeptides. Targeted analysis of a representative dipeptide, Ser-Leu, revealed numerous protein interactors comprising chaperones, proteasomal subunits, and metabolic enzymes. We could further demonstrate that Ser-Leu binding increases activity of a glycolytic enzyme phosphoglycerate kinase (Pgk1). Consistent with the binding analysis, Ser-Leu supplementation leads to the acute metabolic changes and delays timing of a diauxic shift. Supported by the dipeptide accumulation analysis our work attests to the role of Ser-Leu as a metabolic regulator at the interface of protein degradation and central metabolism.

scholarly journals Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria

2017 ◽  
Vol 114 (35) ◽  
pp. E7226-E7235 ◽  
Author(s):  
Philip H. Choi ◽  
Thu Minh Ngoc Vu ◽  
Huong Thi Pham ◽  
Joshua J. Woodward ◽  
Mark S. Turner ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Pyruvate Carboxylase ◽  
Adenosine Monophosphate ◽  
Binding Mode ◽  
Functional Studies ◽  
Cellular Processes ◽  
Wide Range ◽  
A Site ◽  
Amp Inhibition

Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism inListeria monocytogenesby inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition ofLactococcus lactisPC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. InL. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool inL. lactisis negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP–insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

scholarly journals Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Blake W Tye ◽  
Nicoletta Commins ◽  
Lillia V Ryazanova ◽  
Martin Wühr ◽  
Michael Springer ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosome Assembly ◽  
Maximal Growth ◽  
Proliferating Cells ◽  
Cellular Processes ◽  
Direct Contribution ◽  
The Face ◽  
Cellular Phenotypes

To achieve maximal growth, cells must manage a massive economy of ribosomal proteins (r-proteins) and RNAs (rRNAs) to produce thousands of ribosomes every minute. Although ribosomes are essential in all cells, natural disruptions to ribosome biogenesis lead to heterogeneous phenotypes. Here, we model these perturbations in Saccharomyces cerevisiae and show that challenges to ribosome biogenesis result in acute loss of proteostasis. Imbalances in the synthesis of r-proteins and rRNAs lead to the rapid aggregation of newly synthesized orphan r-proteins and compromise essential cellular processes, which cells alleviate by activating proteostasis genes. Exogenously bolstering the proteostasis network increases cellular fitness in the face of challenges to ribosome assembly, demonstrating the direct contribution of orphan r-proteins to cellular phenotypes. We propose that ribosome assembly is a key vulnerability of proteostasis maintenance in proliferating cells that may be compromised by diverse genetic, environmental, and xenobiotic perturbations that generate orphan r-proteins.

scholarly journals The GIS2 Gene Is Repressed by a Zinc-Regulated Bicistronic RNA in Saccharomyces cerevisiae

Genes ◽  
2018 ◽  
Vol 9 (9) ◽  
pp. 462 ◽  
Author(s):  
Janet Taggart ◽  
Yirong Wang ◽  
Erin Weisenhorn ◽  
Colin W. MacDiarmid ◽  
Jason Russell ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Zinc Deficiency ◽  
Transcriptional Activation ◽  
Open Reading Frames ◽  
Zinc Homeostasis ◽  
Protein Accumulation ◽  
Coding Region ◽  
Yeast Saccharomyces Cerevisiae ◽  
Short Transcript

Zinc homeostasis is essential for all organisms. The Zap1 transcriptional activator regulates these processes in the yeast Saccharomyces cerevisiae. During zinc deficiency, Zap1 increases expression of zinc transporters and proteins involved in adapting to the stress of zinc deficiency. Transcriptional activation by Zap1 can also repress expression of some genes, e.g., RTC4. In zinc-replete cells, RTC4 mRNA is produced with a short transcript leader that is efficiently translated. During deficiency, Zap1-dependent expression of an RNA with a longer transcript leader represses the RTC4 promoter. This long leader transcript (LLT) is not translated due to the presence of small open reading frames upstream of the RTC4 coding region. In this study, we show that the RTC4 LLT RNA also plays a second function, i.e., repression of the adjacent GIS2 gene. In generating the LLT transcript, RNA polymerase II transcribes RTC4 through the GIS2 promoter. Production of the LLT RNA correlates with the decreased expression of GIS2 mRNA and mutations that prevent synthesis of the LLT RNA or terminate it before the GIS2 promoter renders GIS2 mRNA expression and Gis2 protein accumulation constitutive. Thus, we have discovered an unusual regulatory mechanism that uses a bicistronic RNA to control two genes simultaneously.

scholarly journals Pathway swapping: Toward modular engineering of essential cellular processes

2016 ◽  
Vol 113 (52) ◽  
pp. 15060-15065 ◽  
Author(s):  
Niels G. A. Kuijpers ◽  
Daniel Solis-Escalante ◽  
Marijke A. H. Luttik ◽  
Markus M. M. Bisschops ◽  
Francine J. Boonekamp ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Pathways ◽  
Applied Research ◽  
Central Metabolism ◽  
Microbial Genomes ◽  
Cellular Processes ◽  
Recent Developments ◽  
One Step ◽  
Saccharomyces Kudriavzevii

Recent developments in synthetic biology enable one-step implementation of entire metabolic pathways in industrial microorganisms. A similarly radical remodelling of central metabolism could greatly accelerate fundamental and applied research, but is impeded by the mosaic organization of microbial genomes. To eliminate this limitation, we propose and explore the concept of “pathway swapping,” using yeast glycolysis as the experimental model. Construction of a “single-locus glycolysis” Saccharomyces cerevisiae platform enabled quick and easy replacement of this yeast’s entire complement of 26 glycolytic isoenzymes by any alternative, functional glycolytic pathway configuration. The potential of this approach was demonstrated by the construction and characterization of S. cerevisiae strains whose growth depended on two nonnative glycolytic pathways: a complete glycolysis from the related yeast Saccharomyces kudriavzevii and a mosaic glycolysis consisting of yeast and human enzymes. This work demonstrates the feasibility and potential of modular, combinatorial approaches to engineering and analysis of core cellular processes.

scholarly journals A Roadmap for Intestinal Regeneration

2020 ◽  
Vol 52 ◽  
Author(s):  
David Quispe-Parra ◽  
Griselle Valentín ◽  
José E. García-Arrarás
Keyword(s):  
Molecular Mechanism ◽  
Sea Cucumber ◽  
Regenerative Process ◽  
Research Model ◽  
Functional Studies ◽  
Cellular Processes ◽  
Sequencing Technologies ◽  
Cellular Events ◽  
Intestinal Regeneration ◽  
Holothuria Glaberrima

Regeneration of lost or injured organs is an intriguing process where numerous cellular events take place to form the new structure. Studies of this process during reconstitution of the intestine have been performed in echinoderms, particularly in holothurians. Many cellular events triggered during regeneration have been described using the sea cucumber Holothuria glaberrima as a research model. More recent experiments have targeted the molecular mechanism behind the process, a task that has been eased by the new sequencing technologies now available. In this review we present the studies involving cellular processes and the genes that have been identified to be associated with the early events of gut regeneration. We also present the ongoing efforts to perform functional studies necessary to establish the role(s) of the identified genes. A synopsis of the studies is given with the course of the regenerative process established so far.

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