Localization elements and zip codes in the intracellular transport and localization of messenger RNAs in Saccharomyces cerevisiae

2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Anusha Chaudhuri ◽  
Subhadeep Das ◽  
Biswadip Das
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Intracellular Transport ◽  
Messenger Rnas ◽  
Localization Elements

scholarly journals Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
Harmen M. van Rossum ◽  
Barbara U. Kozak ◽  
Matthijs S. Niemeijer ◽  
James C. Dykstra ◽  
Marijke A. H. Luttik ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Lipoic Acid ◽  
Intracellular Transport ◽  
Pyruvate Dehydrogenase Complex ◽  
Constitutive Expression ◽  
Growth Media ◽  
Acetyl Coa ◽  
Mitochondrial Fatty Acid

ABSTRACTIn many eukaryotes, the carnitine shuttle plays a key role in intracellular transport of acyl moieties. Fatty acid-grownSaccharomyces cerevisiaecells employ this shuttle to translocate acetyl units into their mitochondria. Mechanistically, the carnitine shuttle should be reversible, but previous studies indicate that carnitine shuttle-mediated export of mitochondrial acetyl units to the yeast cytosol does not occurin vivo. This apparent unidirectionality was investigated by constitutively expressing genes encoding carnitine shuttle-related proteins in an engineeredS. cerevisiaestrain, in which cytosolic acetyl coenzyme A (acetyl-CoA) synthesis could be switched off by omitting lipoic acid from growth media. Laboratory evolution of this strain yielded mutants whose growth on glucose, in the absence of lipoic acid, wasl-carnitine dependent, indicating thatin vivoexport of mitochondrial acetyl units to the cytosol occurred via the carnitine shuttle. The mitochondrial pyruvate dehydrogenase complex was identified as the predominant source of acetyl-CoA in the evolved strains. Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty acid synthesis (MCT1), nuclear-mitochondrial communication (RTG2), and encoding a carnitine acetyltransferase (YAT2). Introduction of these mutations into the nonevolved parental strain enabledl-carnitine-dependent growth on glucose. This study indicates intramitochondrial acetyl-CoA concentration and constitutive expression of carnitine shuttle genes as key factors in enablingin vivoexport of mitochondrial acetyl units via the carnitine shuttle.IMPORTANCEThis study demonstrates, for the first time, thatSaccharomyces cerevisiaecan be engineered to employ the carnitine shuttle for export of acetyl moieties from the mitochondria and, thereby, to act as the sole source of cytosolic acetyl-CoA. Further optimization of this ATP-independent mechanism for cytosolic acetyl-CoA provision can contribute to efficient, yeast-based production of industrially relevant compounds derived from this precursor. The strains constructed in this study, whose growth on glucose depends on a functional carnitine shuttle, provide valuable models for further functional analysis and engineering of this shuttle in yeast and other eukaryotes.

scholarly journals Inferring translational heterogeneity from ribosome profiling data

2019 ◽  
Author(s):  
Pedro do Couto Bordignon ◽  
Sebastian Pechmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Biosynthesis ◽  
Ribosome Profiling ◽  
Translation Regulation ◽  
Messenger Rnas ◽  
Translation Elongation ◽  
Cellular Homeostasis ◽  
Ribosome Density ◽  
Experimental Variability ◽  
Underlying Mechanisms

Translation of messenger RNAs into proteins by the ribosome is the most important step of protein biosynthesis. Accordingly, translation is tightly controlled and heavily regulated to maintain cellular homeostasis. Ribosome profiling (Ribo-seq) has revolutionized the study of translation by revealing many of its underlying mechanisms. However, equally many aspects of translation remain mysterious, in part also due to persisting challenges in the interpretation of data obtained from Ribo-seq experiments. Here, we show that some of the variability observed in Ribo-seq data has biological origins and reflects programmed heterogeneity of translation. To systematically identify sequences that are differentially translated (DT) across mRNAs beyond what can be attributed to experimental variability, we performed a comparative analysis of Ribo-seq data from Saccharomyces cerevisiae and derived a consensus ribosome density profile that reflects consistent signals in individual experiments. Remarkably, the thus identified DT sequences link to mechanisms known to regulate translation elongation and are enriched in genes important for protein and organelle biosynthesis. Our results thus highlight examples of translational heterogeneity that are encoded in the genomic sequences and tuned to optimizing cellular homeostasis. More generally, our work highlights the power of Ribo-seq to understand the complexities of translation regulation.

scholarly journals She3p Possesses a Novel Activity Required for ASH1 mRNA Localization in Saccharomyces cerevisiae

2009 ◽  
Vol 8 (7) ◽  
pp. 1072-1083 ◽  
Author(s):  
Sharon M. Landers ◽  
Michelle R. Gallas ◽  
Jaime Little ◽  
Roy M. Long
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Binding ◽  
Adaptor Protein ◽  
Mrna Localization ◽  
Rna Localization ◽  
The Novel ◽  
Specific Proteins ◽  
Type V ◽  
Localization Elements ◽  
Ash1 Mrna

ABSTRACT Intracellular and intercellular polarity requires that specific proteins be sorted to discreet locations within and between cells. One mechanism for sorting proteins is through RNA localization. In Saccharomyces cerevisiae, ASH1 mRNA localizes to the distal tip of the bud, resulting in the asymmetric sorting of the transcriptional repressor Ash1p. ASH1 mRNA localization requires four cis-acting localization elements and the trans-acting factors Myo4p, She3p, and She2p. Myo4p is a type V myosin motor that functions to directly transport ASH1 mRNA to the bud. She2p is an RNA-binding protein that directly interacts with the ASH1 mRNA cis-acting elements. Currently, the role for She3p in ASH1 mRNA localization is as an adaptor protein, since it can simultaneously associate with Myo4p and She2p. Here, we present data for two novel mutants of She3p, S348E and the double mutant S343E S361E, that are defective for ASH1 mRNA localization, and yet both of these mutants retain the ability to associate with Myo4p and She2p. These observations suggest that She3p possesses a novel activity required for ASH1 mRNA localization, and our data imply that this function is related to the ability of She3p to associate with ASH1 mRNA. Interestingly, we determined that She3p is phosphorylated, and global mass spectrometry approaches have determined that Ser 343, 348, and 361 are sites of phosphorylation, suggesting that the novel function for She3p could be negatively regulated by phosphorylation. The present study reveals that the current accepted model for ASH1 mRNA localization does not fully account for the function of She3p in ASH1 mRNA localization.

scholarly journals Prm1 Targeting to Contact Sites Enhances Fusion during Mating in Saccharomyces cerevisiae

2010 ◽  
Vol 9 (10) ◽  
pp. 1538-1548 ◽  
Author(s):  
Valerie N. Olmo ◽  
Eric Grote
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Fusion ◽  
Intracellular Transport ◽  
Plasma Membranes ◽  
Yeast Cells ◽  
Fusion Event ◽  
Mating Activity ◽  
Content Type ◽  
Contact Sites

ABSTRACT Prm1 is a pheromone-regulated membrane glycoprotein involved in the plasma membrane fusion event of Saccharomyces cerevisiae mating. Although this function suggests that Prm1 should act at contact sites in pairs of mating yeast cells where plasma membrane fusion occurs, only a small percentage of the total Prm1 was actually detected on the plasma membrane. We therefore investigated the intracellular transport of Prm1 and how this transport contributes to cell fusion. Two Prm1 chimeras that were sorted away from the contact site had reduced fusion activity, indicating that Prm1 indeed functions at contact sites. However, most Prm1 is located in endosomes and other cytoplasmic organelles and is targeted to vacuoles for degradation. Mutations in a putative endocytosis signal in a cytoplasmic loop partially stabilized the Prm1 protein and caused it to accumulate on the plasma membrane, but this endocytosis mutant actually had reduced mating activity. When Prm1 was expressed from a galactose-regulated promoter and its synthesis was repressed at the start of mating, vanishingly small amounts of Prm1 protein remained at the time when the plasma membranes came into contact. Nevertheless, this stable pool of Prm1 was retained at polarized sites on the plasma membrane and was sufficient to promote plasma membrane fusion. Thus, the amount of Prm1 expressed in mating yeast is far in excess of the amount required to facilitate fusion.

scholarly journals Mitochondrial Biogenesis Is Positively Regulated by Casein Kinase I Hrr25 Through Phosphorylation of Puf3 in Saccharomyces cerevisiae

Genetics ◽  
2020 ◽  
Vol 215 (2) ◽  
pp. 463-482
Author(s):  
Manika Bhondeley ◽  
Zhengchang Liu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Biogenesis ◽  
Rna Binding ◽  
Casein Kinase ◽  
Mitochondrial Proteins ◽  
Messenger Rnas ◽  
Gfp Expression ◽  
Casein Kinase I ◽  
Link Type ◽  
Gfp Reporter

Mitochondrial biogenesis requires coordinated expression of genes encoding mitochondrial proteins, which in Saccharomyces cerevisiae is achieved in part via post-transcriptional control by the Pumilio RNA-binding domain protein Puf3. Puf3 binds to the 3′-UTR of many messenger RNAs (mRNAs) that encode mitochondrial proteins, regulating their turnover, translation, and/or mitochondrial targeting. Puf3 hyperphosphorylation correlates with increased mitochondrial biogenesis; however, the kinase responsible for Puf3 phosphorylation is unclear. Here, we show that the casein kinase I protein Hrr25 negatively regulates Puf3 by mediating its phosphorylation. An hrr25 mutation results in reduced phosphorylation of Puf3in vivo and a puf3 deletion mutation reverses growth defects of hrr25 mutant cells grown on medium with a nonfermentable carbon source. We show that Hrr25 directly phosphorylates Puf3, and that the interaction between Puf3 and Hrr25 is mediated through the N-terminal domain of Puf3 and the kinase domain of Hrr25. We further found that an hrr25 mutation reduces GFP expression from GFP reporter constructs carrying the 3′-UTR of Puf3 targets. Downregulation of GFP expression due to an hrr25 mutation can be reversed either by puf3Δ or by mutations to the Puf3-binding sites in the 3′-UTR of the GFP reporter constructs. Together, our data indicate that Hrr25 is a positive regulator of mitochondrial biogenesis by phosphorylating Puf3 and inhibiting its function in downregulating target mRNAs encoding mitochondrial proteins.

scholarly journals Saccharomyces cerevisiae: First Steps to a Suitable Model System To Study the Function and Intracellular Transport of Human Kidney Anion Exchanger 1

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Hasib A. M. Sarder ◽  
Xiaobing Li ◽  
Charlotta Funaya ◽  
Emmanuelle Cordat ◽  
Manfred J. Schmitt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Anion Exchanger ◽  
Intracellular Transport ◽  
Human Kidney ◽  
Model System ◽  
Full Length ◽  
Anion Exchanger 1 ◽  
Content Type ◽  
Renal Tubular

ABSTRACT Saccharomyces cerevisiae has been frequently used to study biogenesis, functionality, and intracellular transport of various renal proteins, including ion channels, solute transporters, and aquaporins. Specific mutations in genes encoding most of these renal proteins affect kidney function in such a way that various disease phenotypes ultimately occur. In this context, human kidney anion exchanger 1 (kAE1) represents an important bicarbonate/chloride exchanger which maintains the acid-base homeostasis in the human body. Malfunctions in kAE1 lead to a pathological phenotype known as distal renal tubular acidosis (dRTA). Here, we evaluated the potential of baker's yeast as a model system to investigate different cellular aspects of kAE1 physiology. For the first time, we successfully expressed yeast codon-optimized full-length versions of tagged and untagged wild-type kAE1 and demonstrated their partial localization at the yeast plasma membrane (PM). Finally, pH and chloride measurements further suggest biological activity of full-length kAE1, emphasizing the potential of S. cerevisiae as a model system for studying trafficking, activity, and/or degradation of mammalian ion channels and transporters such as kAE1 in the future. IMPORTANCE Distal renal tubular acidosis (dRTA) is a common kidney dysfunction characterized by impaired acid secretion via urine. Previous studies revealed that α-intercalated cells of dRTA patients express mutated forms of human kidney anion exchanger 1 (kAE1) which result in inefficient plasma membrane targeting or diminished expression levels of kAE1. However, the precise dRTA-causing processes are inadequately understood, and alternative model systems are helpful tools to address kAE1-related questions in a fast and inexpensive way. In contrast to a previous study, we successfully expressed full-length kAE1 in Saccharomyces cerevisiae. Using advanced microscopy techniques as well as different biochemical and functionality assays, plasma membrane localization and biological activity were confirmed for the heterologously expressed anion transporter. These findings represent first important steps to use the potential of yeast as a model organism for studying trafficking, activity, and degradation of kAE1 and its mutant variants in the future.

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