scholarly journals t-SNARE Phosphorylation Regulates Endocytosis in Yeast

2002 ◽  
Vol 13 (5) ◽  
pp. 1594-1607 ◽  
Author(s):  
Sangiliyandi Gurunathan ◽  
Michael Marash ◽  
Adina Weinberger ◽  
Jeffrey E. Gerst
Keyword(s):  
Protein Phosphatase ◽  
Normal Growth ◽  
Gene Inactivation ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutation ◽  
Phosphatase Activation ◽  
Mating Factor ◽  
In Cells

Earlier we demonstrated that activation of a ceramide-activated protein phosphatase (CAPP) conferred normal growth and secretion to yeast lacking their complement of exocytic v-SNAREs (Snc1,2) or bearing a temperature-sensitive mutation in an exocytic t-SNARE (Sso2). CAPP activation led to Sso dephosphorylation and enhanced the assembly of t-SNAREs into functional complexes. Thus, exocytosis in yeast is modulated by t-SNARE phosphorylation. Here, we show that endocytic defects in cells lacking the v- and t-SNAREs involved in endocytosis are also rescued by CAPP activation. Yeast lacking the Tlg1 or Tlg2 t-SNAREs, the Snc v-SNAREs, or both, undergo endocytosis after phosphatase activation. CAPP activation correlated with restored uptake of FM4-64 to the vacuole, the uptake and degradation of the Ste2 receptor after mating factor treatment, and the dephosphorylation and assembly of Tlg1,2 into SNARE complexes. Activation of the phosphatase by treatment with C2-ceramide,VBM/ELO gene inactivation, or by the overexpression of SIT4 was sufficient to confer rescue. Finally, we found that mutation of single PKA sites in Tlg1 (Ser31 to Ala31) or Tlg2 (Ser90 to Ala90) was sufficient to restore endocytosis, but not exocytosis, to snc cells. These results suggest that endocytosis is also modulated by t-SNARE phosphorylation in vivo.

scholarly journals The Skn7 Response Regulator ofSaccharomyces cerevisiaeInteracts with Hsf1 In Vivo and Is Required for the Induction of Heat Shock Genes by Oxidative Stress

2000 ◽  
Vol 11 (7) ◽  
pp. 2335-2347 ◽  
Author(s):  
Desmond C. Raitt ◽  
Anthony L. Johnson ◽  
Alexander M. Erkine ◽  
Kozo Makino ◽  
Brian Morgan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heat Shock ◽  
Response Regulator ◽  
Coiled Coil ◽  
Normal Growth ◽  
Temperature Sensitive ◽  
Regulatory Sequences ◽  
Heat Shock Genes

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein–protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.

scholarly journals Molecular Basis for Rab Prenylation

2000 ◽  
Vol 150 (1) ◽  
pp. 89-104 ◽  
Author(s):  
Christelle Alory ◽  
William E. Balch
Keyword(s):  
Mammalian Cells ◽  
Normal Growth ◽  
Kinetic Properties ◽  
Temperature Sensitive ◽  
Rab Gtpases ◽  
Inherited Disease ◽  
Vesicular Traffic ◽  
Null Strain

Rab escort proteins (REP) 1 and 2 are closely related mammalian proteins required for prenylation of newly synthesized Rab GTPases by the cytosolic heterodimeric Rab geranylgeranyl transferase II complex (RabGG transferase). REP1 in mammalian cells is the product of the choroideremia gene (CHM). CHM/REP1 deficiency in inherited disease leads to degeneration of retinal pigmented epithelium and loss of vision. We now show that amino acid residues required for Rab recognition are critical for function of the yeast REP homologue Mrs6p, an essential protein that shows 50% homology to mammalian REPs. Mutant Mrs6p unable to bind Rabs failed to complement growth of a mrs6Δ null strain and were found to be dominant inhibitors of growth in a wild-type MRS6 strain. Mutants were identified that did not affect Rab binding, yet prevented prenylation in vitro and failed to support growth of the mrs6Δ null strain. These results suggest that in the absence of Rab binding, REP interaction with RabGG transferase is maintained through Rab-independent binding sites, providing a molecular explanation for the kinetic properties of Rab prenylation in vitro. Analysis of the effects of thermoreversible temperature-sensitive (mrs6ts) mutants on vesicular traffic in vivo showed prenylation activity is only transiently required to maintain normal growth, a result promising for therapeutic approaches to disease.

scholarly journals The Ran GTPase System in Fission Yeast Affects Microtubules and Cytokinesis in Cells That Are Competent for Nucleocytoplasmic Protein Transport

2002 ◽  
Vol 22 (24) ◽  
pp. 8491-8505 ◽  
Author(s):  
Sandra S. Salus ◽  
Janos Demeter ◽  
Shelley Sazer
Keyword(s):  
Fission Yeast ◽  
Protein Transport ◽  
Temperature Sensitive ◽  
Nucleotide Exchange ◽  
Cellular Processes ◽  
Nucleotide Exchange Factor ◽  
Ring Structures ◽  
Dependent Processes ◽  
In Cells

ABSTRACT Misregulation of the evolutionarily conserved GTPase Ran in fission yeast results in defects in several cellular processes in cells that are competent for nucleocytoplasmic protein transport. These results suggest that transport is neither the only nor the primary Ran-dependent process in living cells. The ability of Ran to independently regulate multiple cellular processes in vivo is demonstrated by showing that (i) eight different transport-competent RanGEF (guanine nucleotide exchange factor) mutants have defects in mitotic spindle formation; (ii) the RanGEF temperature-sensitive mutant pim1-d1 has abnormal actin ring structures at the septum. Overexpression of Imp2p, which specifically destabilizes these structures, restores viability. (iii) Ran-dependent processes differ in their requirements for active Ran in vivo. Microtubule function, cytokinesis, and nuclear envelope structure are the Ran-dependent processes most sensitive to the amount of Ran protein in the cell, whereas nucleocytoplasmic protein transport is the most robust. Therefore, the ability of Ran from Schizosaccharomyces pombe to independently regulate multiple cellular processes may reflect differences in its interactions with the binding proteins that mediate these functions and explain the complex phenotypic consequences of its misregulation in vivo.

scholarly journals Regulation of chromosome segregation by Glc8p, a structural homolog of mammalian inhibitor 2 that functions as both an activator and an inhibitor of yeast protein phosphatase 1.

1995 ◽  
Vol 15 (11) ◽  
pp. 6064-6074 ◽  
Author(s):  
H Y Tung ◽  
W Wang ◽  
C S Chan
Keyword(s):  
Protein Kinase ◽  
Chromosome Segregation ◽  
Protein Phosphatase ◽  
Site Directed Mutagenesis ◽  
Protein Phosphatase 1 ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Yeast Saccharomyces Cerevisiae ◽  
Growth Phenotype

The Ipl1 protein kinase is essential for proper chromosome segregation and cell viability in the budding yeast Saccharomyces cerevisiae. We have previously shown that the temperature-sensitive growth phenotype of conditional ipl1-1ts mutants can be suppressed by a partial loss-of-function mutation in the GLC7 gene, which encodes the catalytic subunit (PP1C) of protein phosphatase 1, thus suggesting that this enzyme acts in opposition to the Ipl1 protein kinase in regulating yeast chromosome segregation. We report here that the Glc8 protein, which is related in primary sequence to mammalian inhibitor 2, also participates in this regulation. Like inhibitor 2, the Glc8 protein is heat stable, exhibits anomalous electrophoretic mobility, and functions in vitro as an inhibitor of yeast as well as rabbit skeletal muscle PP1C. Interestingly, overexpression as well as deletion of the GLC8 gene results in a partial suppression of the temperature-sensitive growth phenotype of ipl1ts mutants and also moderately reduces the amount of protein phosphatase 1 activity which is assayable in crude yeast lysates. In addition, the chromosome missegregation phenotype caused by an increase in the dosage of GLC7 is totally suppressed by the glc8-delta 101::LEU2 deletion mutation. These findings together suggest that the Glc8 protein is involved in vivo in the activation of PP1C and that when the Glc8 protein is overproduced, it may also inhibit PP1C function. Furthermore, site-directed mutagenesis studies of GLC8 suggest that Thr-118 of the Glc8 protein, which is equivalent to Thr-72 of inhibitor 2, may play a central role in the ability of this protein to activate and/or inhibit PP1C in vivo.

scholarly journals p53-dependent association between cyclin G and the B' subunit of protein phosphatase 2A.

1996 ◽  
Vol 16 (11) ◽  
pp. 6593-6602 ◽  
Author(s):  
K Okamoto ◽  
C Kamibayashi ◽  
M Serrano ◽  
C Prives ◽  
M C Mumby ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
The Other ◽  
Temperature Sensitive ◽  
Regulatory Subunits ◽  
B Subunit ◽  
Phosphatase 2A ◽  
Cyclin G

We and others previously showed that cyclin G is a transcriptional target of the p53 tumor suppressor protein. However, cellular proteins which might form a complex with cyclin G have not yet been identified. To gain insight into the biological role of cyclin G, we used the yeast two-hybrid screen and isolated two mouse cDNAs encoding cyclin G-interacting proteins. Interestingly, both positive cDNAs encoded B' regulatory subunits of protein phosphatase 2A (PP2A). One clone encodes B'alpha, while the other clone codes for a new member of the B' family, B'beta. B'beta is 70% identical to other members of the B' family. B'alpha associated both in vitro and in vivo with cyclin G but not with the other mammalian cyclins. Furthermore, cyclin G formed a complex with B'alpha only after induction of p53 in p53 temperature-sensitive cell lines. These results indicate that cyclin G forms a specific complex with the B' subunit of PP2A and that complex formation is regulated by p53. Potential roles for the cyclin G-B' complex in p53-mediated pathways are discussed.

Effect of Some Metabolic Inhibitors on Vinblastine-Induced Ribosomal-Granular Material Complexes

1971 ◽  
Vol 29 ◽  
pp. 548-549
Author(s):  
Awtar Krishan ◽  
Dora Hsu
Keyword(s):  
Granular Material ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Actinomycin D ◽  
Metabolic Inhibitors ◽  
Protein And Rna Synthesis ◽  
Apparent Reduction ◽  
Plant Alkaloids ◽  
In Cells

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).

Biologic Aspects of Expression of Stably Integrated Transgenes in Cells of the Skin In Vitro and In Vivo

1999 ◽  
Vol 111 (3) ◽  
pp. 198-205 ◽  
Author(s):  
Gerald G. Krueger ◽  
Jeffery R. Morgan ◽  
Marta J. Petersen
Keyword(s):  
In Cells

scholarly journals 1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

1997 ◽  
Vol 41 (5) ◽  
pp. 1082-1093 ◽  
Author(s):  
S M Daluge ◽  
S S Good ◽  
M B Faletto ◽  
W H Miller ◽  
M H St Clair ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Oral Bioavailability ◽  
Human Peripheral Blood ◽  
Cross Resistance ◽  
Immunodeficiency Virus ◽  
Carbocyclic Nucleoside ◽  
Hiv 1 ◽  
In Cells

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

scholarly journals The catalytic subunit of protein phosphatase 2A is carboxyl-methylated in vivo.

1994 ◽  
Vol 269 (23) ◽  
pp. 16311-16317 ◽  
Author(s):  
B. Favre ◽  
S. Zolnierowicz ◽  
P. Turowski ◽  
B.A. Hemmings
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Catalytic Subunit ◽  
Phosphatase 2A
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