scholarly journals Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway.

1996 ◽  
Vol 7 (12) ◽  
pp. 1909-1919 ◽  
Author(s):  
M Ziman ◽  
J S Chuang ◽  
R W Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Endocytic Pathway ◽  
Chitin Synthase ◽  
Cell Fractionation ◽  
Cell Wall Polysaccharide ◽  
Chitin Synthesis ◽  
A Cell ◽  
Steady State Levels ◽  
Membrane Bound

In Saccharomyces cerevisiae, the synthesis of chitin, a cell-wall polysaccharide, is temporally and spatially regulated with respect to the cell cycle and morphogenesis. Using immunological reagents, we found that steady-state levels of Chs1p and Chs3p, two chitin synthase enzymes, did not fluctuate during the cell cycle, indicating that they are not simply regulated by synthesis and degradation. Previous cell fractionation studies demonstrated that chitin synthase I activity (CSI) exists in a plasma membrane form and in intracellular membrane-bound particles called chitosomes. Chitosomes were proposed to act as a reservoir for regulated transport of chitin synthase enzymes to the division septum. We found that Chs1p and Chs3p resided partly in chitosomes and that this distribution was not cell cycle regulated. Pulse-chase cell fractionation experiments showed that chitosome production was blocked in an endocytosis mutant (end4-1), indicating that endocytosis is required for the formation or maintenance of chitosomes. Additionally, Ste2p, internalized by ligand-induced endocytosis, cofractionated with chitosomes, suggesting that these membrane proteins populate the same endosomal compartment. However, in contrast to Ste2p, Chs1p and Chs3p were not rapidly degraded, thus raising the possibility that the temporal and spatial regulation of chitin synthesis is mediated by the mobilization of an endosomal pool of chitin synthase enzymes.

scholarly journals Cell cycle–dependent phosphorylation of Sec4p controls membrane deposition during cytokinesis

2016 ◽  
Vol 214 (6) ◽  
pp. 691-703 ◽  
Author(s):  
Dante Lepore ◽  
Olya Spassibojko ◽  
Gabrielle Pinto ◽  
Ruth N. Collins
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Membrane Trafficking ◽  
Intracellular Trafficking ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Positive Regulator ◽  
Physiological Relevance ◽  
A Cell ◽  
Cycle Dependent

Intracellular trafficking is an essential and conserved eukaryotic process. Rab GTPases are a family of proteins that regulate and provide specificity for discrete membrane trafficking steps by harnessing a nucleotide-bound cycle. Global proteomic screens have revealed many Rab GTPases as phosphoproteins, but the effects of this modification are not well understood. Using the Saccharomyces cerevisiae Rab GTPase Sec4p as a model, we have found that phosphorylation negatively regulates Sec4p function by disrupting the interaction with the exocyst complex via Sec15p. We demonstrate that phosphorylation of Sec4p is a cell cycle–dependent process associated with cytokinesis. Through a genomic kinase screen, we have also identified the polo-like kinase Cdc5p as a positive regulator of Sec4p phosphorylation. Sec4p spatially and temporally localizes with Cdc5p exclusively when Sec4p phosphorylation levels peak during the cell cycle, indicating Sec4p is a direct Cdc5p substrate. Our data suggest the physiological relevance of Sec4p phosphorylation is to facilitate the coordination of membrane-trafficking events during cytokinesis.

scholarly journals Farnesol-induced growth inhibition in Saccharomyces cerevisiae by a cell cycle mechanism

Microbiology ◽  
1999 ◽  
Vol 145 (2) ◽  
pp. 293-299 ◽  
Author(s):  
Kiyotaka Machida ◽  
Toshio Tanaka ◽  
Yoshihisa Yano ◽  
Shuzo Otani ◽  
Makoto Taniguchi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Growth Inhibition ◽  
A Cell

Chitin synthesis and localization in cell division cycle mutants of Saccharomyces cerevisiae

1983 ◽  
Vol 3 (5) ◽  
pp. 922-930
Author(s):  
R L Roberts ◽  
B Bowers ◽  
M L Slater ◽  
E Cabib
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Wall ◽  
Permissive Temperature ◽  
Wild Type ◽  
Chitin Synthesis ◽  
Specific Staining ◽  
Septal Region ◽  
Wall Components ◽  
Generalized Distribution

Growth of Saccharomyces cerevisiae cell cycle mutants cdc3, cdc4, cdc7, cdc24, and cdc28 at a nonpermissive temperature (37 degrees C) resulted in increased accumulation of chitin relative to other cell wall components, as compared with that observed at a permissive temperature (25 degrees C). Wild-type cells showed the same chitin/carbohydrate ratio at both temperatures, whereas mutants cdc13 and cdc21 yielded only a small increase in the ratio at 37 degrees C. These results confirm and extend those reported by B. F. Sloat and J. R. Pringle (Science 200:1171-1173, 1978) for mutant cdc24. The distribution of chitin in the cell wall was studied by electron microscopy, by specific staining with wheat germ agglutinin-colloidal gold complexes. At the permissive temperature, chitin was restricted to the septal region in all strains, whereas at 37 degrees C a generalized distribution of chitin in the cell wall was observed in all mutants. These results do not support a unique interdependence between the product of the cdc24 gene and localization of chitin deposition; they suggest that unbalanced conditions created in the cell by arresting the cycle at different stages result in generalized activation of the chitin synthetase zymogen. Thus, blockage of an event in the cell cycle may lead to consequences that are not functionally related to that event under normal conditions.

scholarly journals The F-Box Protein Rcy1p Is Involved in Endocytic Membrane Traffic and Recycling Out of an Early Endosome in Saccharomyces cerevisiae

2000 ◽  
Vol 149 (2) ◽  
pp. 397-410 ◽  
Author(s):  
Andreas Wiederkehr ◽  
Sandrine Avaro ◽  
Cristina Prescianotto-Baschong ◽  
Rosine Haguenauer-Tsapis ◽  
Howard Riezman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Trafficking ◽  
Fluorescent Dyes ◽  
Lucifer Yellow ◽  
Endocytic Pathway ◽  
Major Fraction ◽  
Membrane Bound ◽  
Recycling Pathway ◽  
F Box Protein ◽  
Vacuolar Protein

In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole. In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1). Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway. Mutation of RCY1 leads to the accumulation of an enlarged compartment that contains the t-SNARE Tlg1p and lies close to areas of cell expansion. In addition, endocytic markers such as Ste2p and the fluorescent dyes, Lucifer yellow and FM4-64, were found in a similar enlarged compartment after their internalization. To determine whether rcy1Δ is defective for recycling, we have developed an assay that measures the recycling of previously internalized FM4-64. This method enables us to follow the recycling pathway in yeast in real time. Using this assay, it could be demonstrated that recycling of membranes is rapid in S. cerevisiae and that a major fraction of internalized FM4-64 is secreted back into the medium within a few minutes. The rcy1Δ mutant is strongly defective in recycling.

Independent regulation of chitin synthase and chitinase activity in Candida albicans and Saccharomyces cerevisiae

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 921-928 ◽  
Author(s):  
Serena Selvaggini ◽  
Carol A. Munro ◽  
Serge Paschoud ◽  
Dominique Sanglard ◽  
Neil A. R. Gow
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
De Novo ◽  
Gene Families ◽  
Chitinase Activity ◽  
Chitin Synthase ◽  
Chitin Synthesis ◽  
Growing Cell ◽  
Chitinase Genes

Chitin is an essential structural polysaccharide in fungi that is required for cell shape and morphogenesis. One model for wall synthesis at the growing cell surface suggests that the compliance that is necessary for turgor-driven expansion of the cell wall involves a delicate balance of wall synthesis and lysis. Accordingly, de novo chitin synthesis may involve coordinated regulation of members of the CHS chitin synthase and CHT chitinase gene families. To test this hypothesis, the chitin synthase and chitinase activities of cell-free extracts were measured, as well as the chitin content of cell walls isolated from isogenic mutant strains that contained single or multiple knock-outs in members of these two gene families, in both Candida albicans and Saccharomyces cerevisiae. However, deletion of chitinase genes did not markedly affect specific chitin synthase activity, and deletion of single CHS genes had little effect on in vitro specific chitinase activity in either fungus. Chitin synthesis and chitinase production was, however, regulated in C. albicans during yeast–hypha morphogenesis. In C. albicans, the total specific activities of both chitin synthase and chitinase were higher in the hyphal form, which was attributable mainly to the activities of Chs2 and Cht3, respectively. It appeared, therefore, that chitin synthesis and hydrolysis were not coupled, but that both were regulated during yeast–hypha morphogenesis in C. albicans.

scholarly journals Targeting of Chitin Synthase 3 to Polarized Growth Sites in Yeast Requires Chs5p and Myo2p

1997 ◽  
Vol 136 (1) ◽  
pp. 95-110 ◽  
Author(s):  
Beatriz Santos ◽  
Michael Snyder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Temperature Shift ◽  
Chitin Synthase ◽  
Chitin Synthesis ◽  
Localization Pattern ◽  
Spatial Regulation ◽  
Single Nucleus ◽  
Temporal And Spatial ◽  
Bud Site ◽  
In Cells

Chitin is an essential structural component of the yeast cell wall whose deposition is regulated throughout the yeast life cycle. The temporal and spatial regulation of chitin synthesis was investigated during vegetative growth and mating of Saccharomyces cerevisiae by localization of the putative catalytic subunit of chitin synthase III, Chs3p, and its regulator, Chs5p. Immunolocalization of epitope-tagged Chs3p revealed a novel localization pattern that is cell cycledependent. Chs3p is polarized as a diffuse ring at the incipient bud site and at the neck between the mother and bud in small-budded cells; it is not found at the neck in large-budded cells containing a single nucleus. In large-budded cells undergoing cytokinesis, it reappears as a ring at the neck. In cells responding to mating pheromone, Chs3p is found throughout the projection. The appearance of Chs3p at cortical sites correlates with times that chitin synthesis is expected to occur. In addition to its localization at the incipient bud site and neck, Chs3p is also found in cytoplasmic patches in cells at different stages of the cell cycle. Epitope-tagged Chs5p also localizes to cytoplasmic patches; these patches contain Kex2p, a late Golgi-associated enzyme. Unlike Chs3p, Chs5p does not accumulate at the incipient bud site or neck. Nearly all Chs3p patches contain Chs5p, whereas some Chs5p patches lack detectable Chs3p. In the absence of Chs5p, Chs3p localizes in cytoplasmic patches, but it is no longer found at the neck or the incipient bud site, indicating that Chs5p is required for the polarization of Chs3p. Furthermore, Chs5p localization is not affected either by temperature shift or by the myo2-66 mutation, however, Chs3p polarization is affected by temperature shift and myo2-66. We suggest a model in which Chs3p polarization to cortical sites in yeast is dependent on both Chs5p and the actin cytoskeleton/Myo2p.

scholarly journals Saccharomyces cerevisiae cdc15 mutants arrested at a late stage in anaphase are rescued by Xenopus cDNAs encoding N-ras or a protein with beta-transducin repeats.

1993 ◽  
Vol 13 (8) ◽  
pp. 4953-4966 ◽  
Author(s):  
W Spevak ◽  
B D Keiper ◽  
C Stratowa ◽  
M J Castañón
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Yeast Cells ◽  
Cell Cycle Gene ◽  
Intracellular Camp ◽  
Temperature Sensitive ◽  
Ras Genes ◽  
Phosphorylation Event ◽  
Dependent Protein ◽  
A Cell

We have constructed a Xenopus oocyte cDNA library in a Saccharomyces cerevisiae expression vector and used this library to isolate genes that can function in yeast cells to suppress the temperature sensitive [corrected] defect of the cdc15 mutation. Two maternally expressed Xenopus cDNAs which fulfill these conditions have been isolated. One of these clones encodes Xenopus N-ras. In contrast to the yeast RAS genes, Xenopus N-ras rescues the cdc15 mutation. Moreover, overexpression of Xenopus N-ras in S. cerevisiae does not activate the RAS-cyclic AMP (cAMP) pathway; rather, it results in decreased levels of intracellular cAMP in both mutant cdc15 and wild-type cells. Furthermore, we show that lowering cAMP levels is sufficient to allow cells with a nonfunctional Cdc15 protein to complete the mitotic cycle. These results suggest that a key step of the cell cycle is dependent upon a phosphorylation event catalyzed by cAMP-dependent protein kinase. The second clone, beta TrCP (beta-transducin repeat-containing protein), encodes a protein of 518 amino acids that shows significant homology to the beta subunits of G proteins in its C-terminal half. In this region, beta Trcp is composed of seven beta-transducin repeats. beta TrCP is not a functional homolog of S. cerevisiae CDC20, a cell cycle gene that also contains beta-transducin repeats and suppresses the cdc15 mutation.

scholarly journals The properties of adenosine triphosphatase from exponential and synchronous cultures of Alcaligenes eutrophus H16

1978 ◽  
Vol 172 (2) ◽  
pp. 253-260 ◽  
Author(s):  
Clive Edwards ◽  
Juliet A. Spode ◽  
Colin W. Jones
Keyword(s):  
Cell Cycle ◽  
Adenosine Triphosphatase ◽  
Alcaligenes Eutrophus ◽  
Chain Structure ◽  
Whole Cells ◽  
Synchronous Cultures ◽  
Maximum Inhibition ◽  
A Cell ◽  
Membrane Bound ◽  
Endogenous Substrates

The properties of Alcaligenes eutrophus ATPase (adenosine triphosphatase) were investigated by using subcellular fractions prepared from cells growing in exponential and synchronous cultures. Both the soluble and membrane-bound forms of the ATPase were inhibited non-competitively (Ki 142μm) by Nbf-Cl (4-chloro-7-nitrobenzofurazan), whereas only the membrane-bound enzyme was inhibited (non-competitive; Ki 750μm) by NN′-dicyclohexylcarbodi-imide. Neither the activity of the ATPase nor its sensitivity to these two inhibitors varied during exponential growth. However, marked variations in ATPase activity were observed during synchronous growth, which were characterized by maxima at approx. 0.4 and 0.9 of a cell cycle and minima at approx. 0.1 and 0.6 of a cycle. Sensitivity to Nbf-Cl and NN′-dicyclohexylcarbodi-imide also varied during the cell cycle; maximum inhibition by the former occurred at approx. 0.4 and 0.9 of a cell cycle, whereas maximum inhibition by the latter was located at approx. 0.1 and 0.6 of a cell cycle. Proton conductance by whole cells was also periodic during the cell cycle, the lowest rates occurring at approx. 0.15 and 0.55 of a cycle and the highest rates at approx. 0.4 and 0.9 of a cycle, but →H+/O quotients for the oxidation of endogenous substrates remained relatively constant and indicated the presence of four proton-translocating respiratory segments throughout the cell cycle. These results are discussed in terms of ATPase and respiratory-chain structure and function during the cell cycle of Alcaligenes eutrophus.

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