scholarly journals Direct Sorting of the Yeast Uracil Permease to the Endosomal System Is Controlled by Uracil Binding and Rsp5p-dependent Ubiquitylation

2004 ◽  
Vol 15 (2) ◽  
pp. 883-895 ◽  
Author(s):  
Marie-Odile Blondel ◽  
Joëlle Morvan ◽  
Sophie Dupré ◽  
Danièle Urban-Grimal ◽  
Rosine Haguenauer-Tsapis ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Vacuolar Membrane ◽  
Multivesicular Bodies ◽  
Toxic Level ◽  
Uridine Uptake ◽  
Endosome Trafficking ◽  
Low Levels ◽  
Low Efficiency ◽  
Endosomal System ◽  
In Cells

The yeast uracil permease, Fur4p, is downregulated by uracil, which is toxic to cells with high permease activity. Uracil promotes cell surface Rsp5p-dependent ubiquitylation of the permease, signaling its endocytosis and further vacuolar degradation. We show here that uracil also triggers the direct routing of its cognate permease from the Golgi apparatus to the endosomal system for degradation, without passage via the plasma membrane. This early sorting was not observed for a variant permease with a much lower affinity for uracil, suggesting that uracil binding is the signal for the diverted pathway. The FUI1-encoded uridine permease is similarly sorted for early vacuolar degradation in cells exposed to a toxic level of uridine uptake. Membrane proteins destined for vacuolar degradation require sorting at the endosome level to the intraluminal vesicles of the multivesicular bodies. In cells with low levels of Rsp5p, Fur4p can be still diverted from the Golgi apparatus but does not reach the vacuolar lumen, being instead missorted to the vacuolar membrane. Correct luminal delivery is restored by the biosynthetic addition of a single ubiquitin, suggesting that the ubiquitylation of Fur4p serves as a specific signal for sorting to the luminal vesicles of the multivesicular bodies. A fused ubiquitin is also able to sort some Fur4p from the Golgi to the degradative pathway in the absence of added uracil but the low efficiency of this sorting indicates that ubiquitin does not itself act as a dominant signal for Golgi-to-endosome trafficking. Our results are consistent with a model in which the binding of intracellular uracil to the permease signals its sorting from the Golgi apparatus and subsequent ubiquitylation ensures its delivery to the vacuolar lumen.

scholarly journals Efficacy and Selectivity of FGF2-Saporin Cytosolically Delivered by PCI in Cells Overexpressing FGFR1

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1476
Author(s):  
Aurora K. Vikan ◽  
Michal Kostas ◽  
Ellen Margrethe Haugsten ◽  
Pål K. Selbo ◽  
Jørgen Wesche
Keyword(s):  
Treatment Options ◽  
Current Treatment ◽  
Fibroblast Growth Factor Receptors ◽  
Ribosome Inactivating Protein ◽  
Cytosolic Delivery ◽  
Low Levels ◽  
U2os Cells ◽  
Targeting Effect ◽  
Target Effects ◽  
In Cells

Fibroblast growth factor receptors (FGFRs) have become an attractive target in cancer research and therapy due to their implication in several cancers. Limitations of current treatment options require a need for additional, more specific and potent strategies to overcome cancers driven by FGFRs. Photochemical internalization (PCI) is a light-controlled method for cytosolic delivery of drugs that are entrapped in endosomes and lysosomes. We here evaluated the efficacy and selectivity of PCI of FGF2-saporin (FGF-SAP) in cells overexpressing FGFR1. FGF-SAP is a conjugate of FGF2 and the highly cytotoxic ribosome-inactivating protein (RIP) saporin, which is used as payload to eliminate cancer cells. Evaluation of the targeting effect of PCI of FGF-SAP was done by comparing the cytotoxic response in osteosarcoma cells with very low levels of FGFR1 (U2OS) to cells overexpressing FGFR1 (U2OS-R1). We demonstrate that PCI greatly enhances cytotoxicity of the drug showing efficient cell killing at pM concentrations of the drug in U2OS-R1 cells. However, U2OS cells were also sensitive to the toxin after PCI. Binding experiments using confocal microscopy and Western blotting techniques indicate that FGF-SAP is taken up by cells through heparan sulfate proteoglycans (HSPGs) in U2OS cells. We further show that the cytotoxicity of FGF-SAP in U2OS cells was reduced when cells were co-treated with heparin to compete out binding to HSPG, demonstrating that the cytotoxic effect was due to internalization by HSPGs. We conclude that to prevent off-target effects of FGF-based toxins, it will be necessary to circumvent binding to HSPGs, for example by mutating the binding site of FGF2 to HSPGs.

scholarly journals Effects of Temperature on the Meiotic Recombination Landscape of the Yeast Saccharomyces cerevisiae

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Ke Zhang ◽  
Xue-Chang Wu ◽  
Dao-Qiong Zheng ◽  
Thomas D. Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Conversion ◽  
Meiotic Recombination ◽  
Hot Spots ◽  
Genetic Maps ◽  
Dna Breaks ◽  
Low Levels ◽  
C 30 ◽  
Recombination Hot Spots ◽  
In Cells

ABSTRACT Although meiosis in warm-blooded organisms takes place in a narrow temperature range, meiosis in many organisms occurs over a wide variety of temperatures. We analyzed the properties of meiosis in the yeast Saccharomyces cerevisiae in cells sporulated at 14°C, 30°C, or 37°C. Using comparative-genomic-hybridization microarrays, we examined the distribution of Spo11-generated meiosis-specific double-stranded DNA breaks throughout the genome. Although there were between 300 and 400 regions of the genome with high levels of recombination (hot spots) observed at each temperature, only about 20% of these hot spots were found to have occurred independently of the temperature. In S. cerevisiae , regions near the telomeres and centromeres tend to have low levels of meiotic recombination. This tendency was observed in cells sporulated at 14°C and 30°C, but not at 37°C. Thus, the temperature of sporulation in yeast affects some global property of chromosome structure relevant to meiotic recombination. Using single-nucleotide polymorphism (SNP)-specific whole-genome microarrays, we also examined crossovers and their associated gene conversion events as well as gene conversion events that were unassociated with crossovers in all four spores of tetrads obtained by sporulation of diploids at 14°C, 30°C, or 37°C. Although tetrads from cells sporulated at 30°C had slightly (20%) more crossovers than those derived from cells sporulated at the other two temperatures, spore viability was good at all three temperatures. Thus, despite temperature-induced variation in the genetic maps, yeast cells produce viable haploid products at a wide variety of sporulation temperatures. IMPORTANCE In the yeast Saccharomyces cerevisiae , recombination is usually studied in cells that undergo meiosis at 25°C or 30°C. In a genome-wide analysis, we showed that the locations of genomic regions with high and low levels of meiotic recombination (hot spots and cold spots, respectively) differed dramatically in cells sporulated at 14°C, 30°C, and 37°C. Thus, in yeast, and likely in other non-warm-blooded organisms, genetic maps are strongly affected by the environment.

Development of an RNA-protein crosslinker to capture protein interactions with diverse RNA structures in cells

RNA ◽  
2021 ◽  
pp. rna.078896.121
Author(s):  
Yan Han ◽  
Xuzhen Guo ◽  
Tiancai Zhang ◽  
Jiangyun Wang ◽  
Keqiong Ye
Keyword(s):  
Protein Interactions ◽  
Rna Structure ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
High Specificity ◽  
Ester Group ◽  
Yeast Cells ◽  
Primary Amines ◽  
Low Efficiency ◽  
In Cells

Characterization of RNA-protein interaction is fundamental for understanding metabolism and function of RNA. UV crosslinking has been widely used to map the targets of RNA-binding proteins, but is limited by low efficiency, requirement for zero-distance contact and biases for single-stranded RNA structure and certain residues of RNA and protein. Here, we report the development of an RNA-protein crosslinker (AMT-NHS) composed of a psoralen derivative and an N-hydroxysuccinimide ester group, which react with RNA bases and primary amines of protein, respectively. We show that AMT-NHS can penetrate into living yeast cells and crosslink Cbf5 to H/ACA snoRNAs with high specificity. The crosslinker induced different crosslinking patterns than UV and targeted both single- and double-stranded regions of RNA. The crosslinker provides a new tool to capture diverse RNA-protein interactions in cells.

Cloning and molecular analysis of the HAP2 locus: a global regulator of respiratory genes in Saccharomyces cerevisiae

1985 ◽  
Vol 5 (12) ◽  
pp. 3410-3416
Author(s):  
J L Pinkham ◽  
L Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Analysis ◽  
Carbon Sources ◽  
Direct Integration ◽  
Global Regulator ◽  
Low Levels ◽  
Gene Maps ◽  
Derivatives Of ◽  
In Cells

We report here the cloning of the HAP2 gene, a locus required for the expression of many cytochromes and respiratory functions in Saccharomyces cerevisiae. The cloned sequences were found to direct integration of a marked vector to the chromosomal HAP2 locus, and derivatives of these sequences were shown to yield chromosomal disruptions with a Hap2- phenotype. The gene maps 18 centimorgans centromere proximal to ade5 on the left arm of chromosome VII, distinguishing it from any other previously characterized nuclear petite locus. The HAP2 locus encodes a 1.3-kilobase transcript which is present at extremely low levels and which is derepressed in cells grown in media containing nonfermentable carbon sources. Levels of HAP2 mRNA are not reduced in strains bearing a mutation at the HAP3 locus, which is also required for expression of respiratory functions. Models outlining possible interactions of the products of the HAP2 and HAP3 genes are presented.

Active HCO3− transport in cyanobacteria

1991 ◽  
Vol 69 (5) ◽  
pp. 936-944 ◽  
Author(s):  
George S. Espie ◽  
Anthony G. Miller ◽  
Ramani A. Kandasamy ◽  
David T. Canvin
Keyword(s):  
Inorganic Carbon ◽  
Dissolved Inorganic Carbon ◽  
Reaction Medium ◽  
Transport Systems ◽  
Bicarbonate Transport ◽  
Key Words Cyanobacteria ◽  
Sodium Dependent ◽  
Rate Of Photosynthesis ◽  
Low Levels ◽  
In Cells

Cyanobacteria possess systems for the active transport of both CO2 and HCO3−. While the active CO2 transport system seems to be present in cells grown on all levels of CO2 or dissolved inorganic carbon, the bicarbonate transport systems are only present in cells grown on low levels of CO2 or dissolved inorganic carbon (air levels or lower). Active bicarbonate transport can be shown to occur when the rate of photosynthesis exceeds that which could be sustained by the production of CO2 from the dehydration of bicarbonate or when CO2 transport is inhibited with carbon oxysulfide or hydrogen sulfide. Two systems for active bicarbonate transport have been identified: one is dependent on the presence of millimolar concentrations of sodium, and the other is independent of the sodium requirement. Cells grown with air bubbling normally possess the first whereas cells grown in standing culture normally possess the second. The sodium-dependent bicarbonate transport can be inhibited by omitting sodium from the reaction medium or competitively with lithium when sodium is present. Monensin and amiloride also inhibit sodium-dependent bicarbonate transport. It does not appear to be inhibited by ethoxyzolamide. The inhibition of sodium-independent bicarbonate transport is not yet established. Bicarbonate transport appears to have no effect on CO2 transport and CO2 transport appears to have no effect on bicarbonate transport. Hence, the transport systems seems to be independent. Although a number of mechanisms have been proposed for bicarbonate transport, the experimental data are not sufficient to clearly distinguish between them. Key words: cyanobacteria, active CO2 transport, active HCO3− transport, photosynthesis, sodium.

scholarly journals Tubular lysosomes in ruffle-ended ameloblasts associated with enamel maturation in rat incisor.

1989 ◽  
Vol 37 (6) ◽  
pp. 801-811 ◽  
Author(s):  
A H Salama ◽  
A E Zaki ◽  
D R Eisenmann
Keyword(s):  
Granular Material ◽  
Multivesicular Bodies ◽  
Rat Incisor ◽  
Early Maturation ◽  
Membrane Bound ◽  
Lysosomal System ◽  
Endosomal System ◽  
Fine Granular Material

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were localized to investigate the lysosomal system, particularly tubular lysosomes, in ruffle-ended ameloblasts associated with maturation of enamel in rat incisor. Demineralized specimens were incubated for TMPase and for CMPase in a modified medium where cerium was used as the capture ion. Ruffle-ended ameloblasts showed distal invaginations and membrane-bound bodies filled with fine granular material, some of which displayed CMPase reaction product. Elongated tubular configurations 80-140 nm wide were distributed throughout the cytoplasm and were reactive with both TMPase and CMPase, thus characterizing these structures as lysosomes. They often contained fine granular material morphologically similar to that present in multivesicular bodies. During late enamel maturation, fewer tubular lysosomes were observed when compared to early maturation. These cytochemical results demonstrate the presence of tubular lysosomes in ruffle-ended ameloblasts, and it is suggested that they are elements of the endosomal system in these cells. These findings are also consistent with a resorptive function for ruffle-ended ameloblasts during enamel maturation.

Identification of a sensitive anti-erythropoietin receptor monoclonal antibody allows detection of low levels of EpoR in cells

2010 ◽  
Vol 352 (1-2) ◽  
pp. 126-139 ◽  
Author(s):  
Steve Elliott ◽  
Leigh Busse ◽  
Ian McCaffery ◽  
John Rossi ◽  
Angus Sinclair ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Erythropoietin Receptor ◽  
Low Levels ◽  
Receptor Monoclonal Antibody ◽  
In Cells

scholarly journals The Yeast vps Class E Mutants: The Beginning of the Molecular Genetic Analysis of Multivesicular Body Biogenesis

2010 ◽  
Vol 21 (23) ◽  
pp. 4057-4060 ◽  
Author(s):  
Emily M. Coonrod ◽  
Tom H. Stevens
Keyword(s):  
Membrane Proteins ◽  
Molecular Genetic ◽  
Multivesicular Body ◽  
Molecular Genetic Analysis ◽  
Vacuolar Membrane ◽  
Multivesicular Bodies ◽  
Golgi Membrane ◽  
Cargo Proteins ◽  
Vacuolar Protein ◽  
Class E

In 1992, Raymond et al. published a compilation of the 41 yeast vacuolar protein sorting (vps) mutant groups and described a large class of mutants (class E vps mutants) that accumulated an exaggerated prevacuolar endosome-like compartment. Further analysis revealed that this “class E compartment” contained soluble vacuolar hydrolases, vacuolar membrane proteins, and Golgi membrane proteins unable to recycle back to the Golgi complex, yet these class E vps mutants had what seemed to be normal vacuoles. The 13 class E VPS genes were later shown to encode the proteins that make up the complexes required for formation of intralumenal vesicles in late endosomal compartments called multivesicular bodies, and for the sorting of ubiquitinated cargo proteins into these internal vesicles for eventual delivery to the vacuole or lysosome.

scholarly journals Langerhans' Cells in Equine Cutaneous Papillomas and Normal Skin

1992 ◽  
Vol 29 (2) ◽  
pp. 152-160 ◽  
Author(s):  
M. Hamada ◽  
M. Takechi ◽  
C. Itakura
Keyword(s):  
Major Histocompatibility Complex ◽  
T Lymphocytes ◽  
Golgi Apparatus ◽  
Langerhans Cells ◽  
Normal Skin ◽  
Multivesicular Bodies ◽  
Major Histocompatibility Complex Antigen ◽  
Histocompatibility Complex ◽  
Thoroughbred Horses ◽  
Normal Epidermis

Langerhans' cells (LC) were investigated immunohistochemically and electron microscopically in normal equine epidermis and 133 equine cutaneous papillomas experimentally induced in five 2-year-old Thoroughbred horses. Class II major histocompatibility complex antigen-positive dendritic LC were found in the normal epidermis and ultrastructurally had the characteristic Birbeck's granules. In the developing phase of the papillomas, LC were significantly decreased in number and size, indicative of a hypofunctional state. In the regressing phase of the papillomas, LC were markedly increased in number, especially at the epidermis-dermis junction. LC with long dendrites were rich in cytoplasm with well-developed cytoplasmic organelles, including Golgi apparatus, lysosomes, Birbeck's granules, and multivesicular bodies. These LC were hyperfunctional. An infiltration of many T lymphocytes was also observed at the epidermis-dermis junction.

scholarly journals The MATCHIT Automaton: Exploiting Compartmentalization for the Synthesis of Branched Polymers

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Mathias S. Weyland ◽  
Harold Fellermann ◽  
Maik Hadorn ◽  
Daniel Sorek ◽  
Doron Lancet ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Linear Time ◽  
Optimal Path ◽  
Theoretical Framework ◽  
Branched Polymers ◽  
Branched Polymer ◽  
Maximal Yield ◽  
Polymer Reactions ◽  
In Cells

We propose an automaton, a theoretical framework that demonstrates how to improve the yield of the synthesis of branched chemical polymer reactions. This is achieved by separating substeps of the path of synthesis into compartments. We use chemical containers (chemtainers) to carry the substances through a sequence of fixed successive compartments. We describe the automaton in mathematical terms and show how it can be configured automatically in order to synthesize a given branched polymer target. The algorithm we present finds an optimal path of synthesis in linear time. We discuss how the automaton models compartmentalized structures found in cells, such as the endoplasmic reticulum and the Golgi apparatus, and we show how this compartmentalization can be exploited for the synthesis of branched polymers such as oligosaccharides. Lastly, we show examples of artificial branched polymers and discuss how the automaton can be configured to synthesize them with maximal yield.

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