scholarly journals The subcellular localization of yeast glycogen synthase is dependent upon glycogen content

2010 ◽  
Vol 56 (5) ◽  
pp. 408-420 ◽  
Author(s):  
Wayne A. Wilson ◽  
Michael P. Boyer ◽  
Keri D. Davis ◽  
Michael Burke ◽  
Peter J. Roach
Keyword(s):  
Glycogen Content ◽  
Fluorescent Protein ◽  
Glycogen Synthase ◽  
Glycogen Storage ◽  
Yeast Saccharomyces Cerevisiae ◽  
Localization Pattern ◽  
Green Fluorescent ◽  
Yeast Glycogen ◽  
Storage Polysaccharide ◽  
Glycogen Particles

The budding yeast, Saccharomyces cerevisiae , accumulates the storage polysaccharide glycogen in response to nutrient limitation. Glycogen synthase, the major form of which is encoded by the GSY2 gene, catalyzes the key regulated step in glycogen storage. Here, we utilized Gsy2p fusions to green fluorescent protein (GFP) to determine where glycogen synthase was located within cells. We demonstrated that the localization pattern of Gsy2-GFP depended upon the glycogen content of the cell. When glycogen was abundant, Gsy2-GFP was found uniformly throughout the cytoplasm, but under low glycogen conditions, Gsy2-GFP localized to discrete spots within cells. Gsy2p is known to bind to glycogen, and we propose that the subcellular distribution of Gsy2-GFP reflects the distribution of glycogen particles. In the absence of glycogen, Gsy2p translocates into the nucleus. We hypothesize that Gsy2p is normally retained in the cytoplasm through its interaction with glycogen particles. When glycogen levels are reduced, Gsy2p loses this anchor and can traffic into the nucleus.

scholarly journals Extracellular Vesicles-Encapsulated Yeast Prions and What They Can Tell Us about the Physical Nature of Propagons

2020 ◽  
Vol 22 (1) ◽  
pp. 90
Author(s):  
Mehdi Kabani
Keyword(s):  
Protein Quality ◽  
Fluorescent Protein ◽  
Physical Nature ◽  
Dependent Manner ◽  
Yeast Saccharomyces Cerevisiae ◽  
Yeast Prions ◽  
Daughter Cells ◽  
Cytoplasmic Proteins ◽  
Green Fluorescent ◽  
Functionally Diverse

The yeast Saccharomyces cerevisiae hosts an ensemble of protein-based heritable traits, most of which result from the conversion of structurally and functionally diverse cytoplasmic proteins into prion forms. Among these, [PSI+], [URE3] and [PIN+] are the most well-documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Yeast prions propagate by molecular chaperone-mediated fragmentation of these aggregates, which generates small self-templating seeds, or propagons. The exact molecular nature of propagons and how they are faithfully transmitted from mother to daughter cells despite spatial protein quality control are not fully understood. In [PSI+] cells, Sup35p forms detergent-resistant assemblies detectable on agarose gels under semi-denaturant conditions and cytosolic fluorescent puncta when the protein is fused to green fluorescent protein (GFP); yet, these macroscopic manifestations of [PSI+] do not fully correlate with the infectivity measured during growth by the mean of protein infection assays. We also discovered that significant amounts of infectious Sup35p particles are exported via extracellular (EV) and periplasmic (PV) vesicles in a growth phase and glucose-dependent manner. In the present review, I discuss how these vesicles may be a source of actual propagons and a suitable vehicle for their transmission to the bud.

scholarly journals Two Heteromeric Kinesin Complexes in Chemosensory Neurons and Sensory Cilia of Caenorhabditis elegans

1999 ◽  
Vol 10 (2) ◽  
pp. 345-360 ◽  
Author(s):  
Dawn Signor ◽  
Karen P. Wedaman ◽  
Lesilee S. Rose ◽  
Jonathan M. Scholey
Keyword(s):  
Signal Transduction ◽  
Fluorescent Protein ◽  
Partial Purification ◽  
Localization Pattern ◽  
Chemosensory Neurons ◽  
C Elegans ◽  
Microtubule Motors ◽  
Sensory Cilia ◽  
Green Fluorescent ◽  
Kinesin Family

Chemosensation in the nervous system of the nematodeCaenorhabditis elegans depends on sensory cilia, whose assembly and maintenance requires the transport of components such as axonemal proteins and signal transduction machinery to their site of incorporation into ciliary structures. Members of the heteromeric kinesin family of microtubule motors are prime candidates for playing key roles in these transport events. Here we describe the molecular characterization and partial purification of two heteromeric kinesin complexes from C. elegans, heterotrimeric CeKinesin-II and dimeric CeOsm-3. Transgenic worms expressing green fluorescent protein driven by endogenous heteromeric kinesin promoters reveal that both CeKinesin-II and CeOsm-3 are expressed in amphid, inner labial, and phasmid chemosensory neurons. Additionally, immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites. These results, together with the phenotypes of known mutants in the pathway of sensory ciliary assembly, suggest that CeKinesin-II and CeOsm-3 drive the transport of ciliary components required for sequential steps in the assembly of chemosensory cilia.

scholarly journals Saccharomyces cerevisiae contains a Type II phosphoinositide 4-kinase

2003 ◽  
Vol 371 (2) ◽  
pp. 533-540 ◽  
Author(s):  
Shary N. SHELTON ◽  
Barbara BARYLKO ◽  
Derk D. BINNS ◽  
Bruce F. HORAZDOVSKY ◽  
Joseph P. ALBANESI ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Distinct Type ◽  
Type Ii ◽  
Yeast Saccharomyces Cerevisiae ◽  
Molar Ratios ◽  
Ionic Detergent ◽  
Km Value ◽  
Green Fluorescent ◽  
Vacuolar Membranes

The yeast Saccharomyces cerevisiae contains two known phosphoinositide 4-kinases (PI 4-kinases), which are encoded by PIK1 and STT4; both are essential. Pik1p is important for exocytic transport from the Golgi, whereas Stt4p plays a role in cell-wall integrity and cytoskeletal rearrangements. In the present study, we report that cells have a third PI 4-kinase activity encoded by LSB6, a protein identified previously in a two-hybrid screen as interacting with LAS17p. Although Pik1p and Stt4p are closely related members of the Type III class of PI 4-kinases, Lsb6p belongs to the distinct Type II class, based on its amino acid sequence, its sensitivity to inhibition by adenosine and its insensitivity to wortmannin. Lsb6p is the first fungal Type II enzyme cloned. The protein was expressed and purified from Sf9 cells and used to define kinetic parameters. As commonly observed for surface-active enzymes, activities varied both with substrate concentration and lipid/detergent molar ratios. Maximal activities of approx. 100min−1 were obtained at the PI/Triton X-100 ratio of 1:5. The Km value for ATP was 266μM, intermediate between the values reported for mammalian Type II and III kinases. Epitope-tagged protein, expressed in yeast, was entirely particulate, and about half of it could be extracted with non-ionic detergent. Lsb6p–green fluorescent protein was found both on vacuolar membranes and on the plasma membrane, suggesting a role in endocytic or exocytic pathways.

scholarly journals LZTS2 Is a Novel β-Catenin-Interacting Protein and Regulates the Nuclear Export of β-Catenin

2006 ◽  
Vol 26 (23) ◽  
pp. 8857-8867 ◽  
Author(s):  
Gregory Thyssen ◽  
Tzu-Huey Li ◽  
Lynn Lehmann ◽  
Ming Zhuo ◽  
Manju Sharma ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Glycogen Synthase ◽  
Nuclear Export Signal ◽  
Point Mutations ◽  
Cellular Level ◽  
Interacting Protein ◽  
Nuclear Exclusion ◽  
Green Fluorescent ◽  
Wnt Signal

ABSTRACT β-Catenin plays multiple roles in cell-cell adhesion and Wnt signal transduction. Through the Wnt signal, the cellular level of β-catenin is constitutively regulated by the multicomponent destruction complex containing glycogen synthase kinase 3β, axin, and adenomatous polyposis coli. Here, we present multiple lines of evidence to demonstrate that LZTS2 (lucine zipper tumor suppressor 2) interacts with β-catenin, represses the transactivation of β-catenin, and affects the subcellular localization of β-catenin. The LZTS2 gene is located at 10q24.3, which is frequently lost in a variety of human tumors. A functional nuclear export signal (NES) was identified in the C terminus of the protein (amino acids 631 to 641). Appending this motif to green fluorescent protein (GFP) induced nuclear exclusion of the GFP fusion protein. However, introducing point mutations in either one or two leucine residues of this NES sequence abolished the nuclear exclusion of the LZTS2 protein. The nuclear export of LZTS2 can be blocked by leptomycin B (LMB), an inhibitor of the CRM1/exportin-alpha pathway. Intriguingly, β-catenin colocalizes with LZTS2 in the cytoplasm of cells in the absence of LMB but in the nuclei of cells in the presence of LMB. Increasing the LZTS2 protein in cells reduces the level of nuclear β-catenin in SW480 cells. Taken together, these data demonstrate that LZTS2 is a β-catenin-interacting protein that can modulate β-catenin signaling and localization.

Aux1p/Swa2p Is Required for Cortical Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae

2001 ◽  
Vol 12 (9) ◽  
pp. 2614-2628 ◽  
Author(s):  
Yunrui Du ◽  
Marc Pypaert ◽  
Peter Novick ◽  
Susan Ferro-Novick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bifunctional Protein ◽  
Daughter Cells ◽  
Bud Neck ◽  
J Domain ◽  
Green Fluorescent ◽  
Cortical Endoplasmic Reticulum

In the yeast Saccharomyces cerevisiae, the endoplasmic reticulum (ER) is found at the periphery of the cell and around the nucleus. The segregation of ER through the mother-bud neck may occur by more than one mechanism because perinuclear, but not peripheral ER, requires microtubules for this event. To identify genes whose products are required for cortical ER inheritance, we have used a Tn3-based transposon library to mutagenize cells expressing a green fluorescent protein-tagged ER marker protein (Hmg1p). This approach has revealed that AUX1/SWA2plays a role in ER inheritance. The COOH terminus of Aux1p/Swa2p contains a J-domain that is highly related to the J-domain of auxilin, which stimulates the uncoating of clathrin-coated vesicles. Deletion of the J-domain of Aux1p/Swa2p leads to vacuole fragmentation and membrane accumulation but does not affect the migration of peripheral ER into daughter cells. These findings suggest that Aux1p/Swa2p may be a bifunctional protein with roles in membrane traffic and cortical ER inheritance. In support of this hypothesis, we find that Aux1p/Swa2p localizes to ER membranes.

Liver and peripheral tissue glycogen metabolism in obese mice: effect of a mixed meal

1993 ◽  
Vol 265 (5) ◽  
pp. E743-E751
Author(s):  
C. Chen ◽  
P. F. Williams ◽  
I. D. Caterson
Keyword(s):  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Cardiac Muscle ◽  
White Adipose Tissue ◽  
Glycogen Content ◽  
Glycogen Synthase ◽  
Glycogen Metabolism ◽  
Glycogen Storage ◽  
Obese Mice ◽  
Entire Study

Glycogen metabolism in the liver, skeletal muscle, cardiac muscle, and white adipose tissue was studied in gold thioglucose (GTG) obese mice after fasting and during refeeding. Prolonged (48 h) fasted control and GTG mice were refed with standard laboratory diet for 24 h. During fasting and refeeding, the changes in glycogen content and the activity of glycogen synthase I and R and phosphorylase alpha in the liver were similar in lean and GTG mice. However, the glycogen storage in the livers from GTG mice was always greater than that in lean animals. In GTG mice the activity of liver glycogen synthase I and R was significantly higher than that in lean animals 3 and 6 h after refeeding. The activity of liver phosphorylase alpha in GTG mice was higher than that in lean mice after refeeding. There were no significant differences in the glycogen content of white adipose tissue, cardiac muscle, and skeletal muscle from lean and GTG mice during the entire study. The results of this study suggest that increased glycogen storage in the liver is a major alteration in nonoxidative glucose metabolism and contributes to the development of insulin resistance and glucose intolerance in GTG obese mice.

scholarly journals The Cortical Localization of the Microtubule Orientation Protein, Kar9p, Is Dependent upon Actin and Proteins Required for Polarization

1999 ◽  
Vol 144 (5) ◽  
pp. 963-975 ◽  
Author(s):  
Rita K. Miller ◽  
Dina Matheos ◽  
Mark D. Rose
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Nuclear Positioning ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cytoplasmic Microtubule ◽  
Microtubule Orientation ◽  
Cortical Localization ◽  
Cytoplasmic Microtubules ◽  
Green Fluorescent ◽  
Mitotic Cells

In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)– Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2Δ, pea2Δ, bud6Δ, and bni1Δ, required for normal polarization and actin cytoskeleton functions and, of these, bni1Δ affected Kar9p localization most severely. Like kar9Δ, bni1Δ mutants exhibited nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Δ, the bni1Δ mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Δ bni1Δ double mutants suggested that Kar9p retained some function in bni1Δ mitotic cells. Unlike the polarization mutants, kar9Δ shmoos had a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localized normally in kar9Δ. We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks.

Development and implementation of split-GFP-based bimolecular fluorescence complementation (BiFC) assays in yeast

2008 ◽  
Vol 36 (3) ◽  
pp. 479-482 ◽  
Author(s):  
Emma Barnard ◽  
Neil V. McFerran ◽  
Alan Trudgett ◽  
John Nelson ◽  
David J. Timson
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Subcellular Location ◽  
Bimolecular Fluorescence Complementation ◽  
Protein Protein Interactions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Green Fluorescent ◽  
Fluorescence Complementation ◽  
Bimolecular Fluorescence

BiFC (bimolecular fluorescence complementation) is a tool for investigating interactions between proteins. Non-fluorescent fragments of, for example, GFP (green fluorescent protein) are fused to the interacting partners. The interaction brings the fragments together, which then fold, reassemble and fluoresce. This process can be carried out in living cells and provides information both on the interaction and its subcellular location. We have developed a split-GFP-based BiFC assay for use in the budding yeast Saccharomyces cerevisiae in which the modifications are carried out at the genomic level, thus resulting in the tagged yeast proteins being expressed at wild-type levels. The system is capable of detecting interactions in all subcellular compartments tested (the cytoplasm, mitochondria and nucleus) and makes a valuable addition to techniques for the investigation of protein–protein interactions in this model organism.

scholarly journals Coordinated Spindle Assembly and Orientation Requires Clb5p-Dependent Kinase in Budding Yeast

2000 ◽  
Vol 148 (3) ◽  
pp. 441-452 ◽  
Author(s):  
Marisa Segal ◽  
Duncan J. Clarke ◽  
Paul Maddox ◽  
E.D. Salmon ◽  
Kerry Bloom ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Spindle Assembly ◽  
Time Lapse ◽  
Spindle Pole ◽  
Cell Cortex ◽  
Dynamic Interactions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Green Fluorescent

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Δ cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Δ cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly.

scholarly journals Tripterine Treatment Improves Endothelial Progenitor Cell Function via Integrin-Linked Kinase

2015 ◽  
Vol 37 (3) ◽  
pp. 1089-1103 ◽  
Author(s):  
Chenhui Lu ◽  
Xixiang Yu ◽  
Keqiang Zuo ◽  
Xiaoping Zhang ◽  
Chuanwu Cao ◽  
...  
Keyword(s):  
Vascular Function ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Glycogen Synthase ◽  
Tube Formation ◽  
Dominant Negative ◽  
Endothelial Progenitor ◽  
Integrin Linked Kinase ◽  
Gsk 3Β ◽  
Green Fluorescent

Background/Aims: Atherosclerosis is associated with dysfunction of endothelial progenitor cells (EPCs). Tripterine, a chemical compound derived from the Chinese medicinal plant Tripterygium wilfordii Hook, displays anti-inflammatory properties in several animal models. We hypothesized that tripterine can improve EPC function and thus the efficiency of EPC transplantation. Methods and Results: Tripterine preconditioning (2.5 μM, 4 h) improved EPC proliferation, tube formation, migration, and adhesion, and reduced apoptosis in cells cultured in ox-LDL (200 µg/ml). Tripterine restored integrin-linked kinase (ILK) levels downregulated by ox-LDL in EPCs, suggesting the involvement of the ILK/Akt pathway. Small interfering RNA-mediated depletion of ILK and dominant-negative ILK transduction inhibited the phosphorylation of the ILK downstream signaling targets protein kinase B/Akt and glycogen synthase kinase 3-beta (GSK-3β), and reduced β-catenin and cyclin D1 expression. In atherosclerotic mice injected with green fluorescent protein-labeled EPCs to evaluate EPC function, tripterine decreased aortic lesions and plaque deposition, and injection of tripterine-treated EPCs restored ILK levels. Conclusion: The present results suggest that tripterine improves vascular function in atherosclerosis by enhancing EPC function through a mechanism involving the ILK signaling pathway.

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