scholarly journals Saccharomyces cerevisiae Grx6 and Grx7 Are Monothiol Glutaredoxins Associated with the Early Secretory Pathway

2008 ◽  
Vol 7 (8) ◽  
pp. 1415-1426 ◽  
Author(s):  
Alicia Izquierdo ◽  
Celia Casas ◽  
Ulrich Mühlenhoff ◽  
Christopher Horst Lillig ◽  
Enrique Herrero
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Inhibitory Effect ◽  
Protein Accumulation ◽  
Oxidoreductase Activity ◽  
Early Secretory Pathway ◽  
Glycosylation Inhibitor

ABSTRACT Saccharomyces cerevisiae Grx6 and Grx7 are two monothiol glutaredoxins whose active-site sequences (CSYS and CPYS, respectively) are reminiscent of the CPYC active-site sequence of classical dithiol glutaredoxins. Both proteins contain an N-terminal transmembrane domain which is responsible for their association to membranes of the early secretory pathway vesicles, facing the luminal side. Thus, Grx6 localizes at the endoplasmic reticulum and Golgi compartments, while Grx7 is mostly at the Golgi. Expression of GRX6 is modestly upregulated by several stresses (calcium, sodium, and peroxides) in a manner dependent on the Crz1-calcineurin pathway. Some of these stresses also upregulate GRX7 expression under the control of the Msn2/4 transcription factor. The N glycosylation inhibitor tunicamycin induces the expression of both genes along with protein accumulation. Mutants lacking both glutaredoxins display reduced sensitivity to tunicamycin, although the drug is still able to manifest its inhibitory effect on a reporter glycoprotein. Grx6 and Grx7 have measurable oxidoreductase activity in vivo, which is increased in the presence of tunicamycin. Both glutaredoxins could be responsible for the regulation of the sulfhydryl oxidative state at the oxidant conditions of the early secretory pathway vesicles. However, the differences in location and expression responses against stresses suggest that their functions are not totally overlapping.

scholarly journals The PBN1 Gene of Saccharomyces cerevisiae: An Essential Gene That Is Required for the Post-translational Processing of the Protease B Precursor

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1277-1292 ◽  
Author(s):  
Rajesh R Naik ◽  
Elizabeth W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Essential Gene ◽  
Signal Sequence ◽  
Signal Peptidase ◽  
Amino Terminal ◽  
Autocatalytic Cleavage ◽  
Protease B ◽  
Chromosome Iii

Abstract The vacuolar hydrolase protease B in Saccharomyces cerevisiae is synthesized as an inactive precursor (Prb1p). The precursor undergoes post-translational modifications while transiting the secretory pathway. In addition to N- and O -linked glycosylations, four proteolytic cleavages occur during the maturation of Prb1p. Removal of the signal peptide by signal peptidase and the autocatalytic cleavage of the large aminoterminal propeptide occur in the endoplasmic reticulum (ER). Two carboxy-terminal cleavages of the post regions occur in the vacuole: the first cleavage is catalyzed by protease A and the second results from autocatalysis. We have isolated a mutant, pbn1-1, that exhibits a defect in the ER processing of Prb1p. The autocatalytic cleavage of the propeptide from Prb1p does not occur and Prb1p is rapidly degraded in the cytosol. PBN1 was cloned and is identical to YCL052c on chromosome III. PBN1 is an essential gene that encodes a novel protein. Pbn1p is predicted to contain a sub-C-terminal transmembrane domain but no signal sequence. A functional HA epitope-tagged Pbn1p fusion localizes to the ER. Pbn1p is N-glycosylated in its amino-terminal domain, indicating a lumenal orientation despite the lack of a signal sequence. Based on these results, we propose that one of the functions of Pbn1p is to aid in the autocatalytic processing of Prb1p.

scholarly journals Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
Author(s):  
Fabien Lefèbvre ◽  
Valérie Prouzet-Mauléon ◽  
Michel Hugues ◽  
Marc Crouzet ◽  
Aurélie Vieillemard ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Rho Gtpase ◽  
Secretory Vesicles ◽  
Gtpase Activating Protein ◽  
Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

cAMP modulates glucocorticoid-induced protein accumulation and glucocorticoid receptor in cardiomyocytes

1996 ◽  
Vol 271 (5) ◽  
pp. E827-E833 ◽  
Author(s):  
A. Sato ◽  
K. E. Sheppard ◽  
M. J. Fullerton ◽  
J. W. Funder
Keyword(s):  
Glucocorticoid Receptor ◽  
Second Messengers ◽  
Muscle Growth ◽  
Inhibitory Effect ◽  
Neonatal Rat ◽  
Protein Accumulation ◽  
Rat Cardiomyocytes ◽  
Cyclic Monophosphate ◽  
Protein Levels

Glucocorticoids have complex effects on cardiac muscle growth in vivo, and one possible reason may the regulatory cross talk between glucocorticoids and second messengers. In this study we investigated the effect of adenosine 3',5'-cyclic monophosphate (cAMP), shown to affect cardiomyocyte growth and glucocorticoid action in several systems, on glucocorticoid-induced protein accumulation and glucocorticoid receptor (GR) in neonatal rat cardiomyocytes. Dexamethasone (DEX) decreased the protein-to-DNA ratio, and 8-bromoadenosine 3',5'-cyclic monophosphate (BrcAMP) or forskolin increased this ratio. The inhibitory effect of DEX was potentiated by an elevated cAMP, despite the stimulatory effect of cAMP alone. Nuclear GR binding was increased by BrcAMP, with no change in GR mRNA or protein levels, via increased affinity of nuclear GR. H-89 blocked the effects of BrcAMP. In conclusion, glucocorticoids have an inhibitory effect on protein accumulation in cardiomyocytes via GR, an effect potentiated by elevated cAMP via increased nuclear GR binding. These results suggest that glucocorticoid effects on cardiomyocytes may be modulated by cAMP-mediated mechanisms, which may produce the complex effects of glucocorticoids on cardiomyocyte growth in vivo.

scholarly journals Essential Regions of Saccharomyces cerevisiae Telomerase RNA: Separate Elements for Est1p and Est2p Interaction

2002 ◽  
Vol 22 (7) ◽  
pp. 2366-2374 ◽  
Author(s):  
April J. Livengood ◽  
Arthur J. Zaug ◽  
Thomas R. Cech
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Dna Synthesis ◽  
Active Site ◽  
Telomerase Rna ◽  
Telomeric Dna ◽  
Protein Subunits ◽  
Essential Region ◽  
Selection For

ABSTRACT The Saccharomyces cerevisiae telomerase RNA subunit is encoded by the TLC1 gene. A selection for viable alleles of TLC1 RNA from a large library of random deletion alleles revealed that less than half (∼0.5 kb of the ∼1.3-kb RNA) is required for telomerase function in vivo. The main essential region (430 nucleotides), which contains the template for telomeric DNA synthesis, was required for coimmunoprecipitation with Est1p and Est2p. Furthermore, the subregion required for interaction with Est1p, the telomerase recruitment subunit, differed from those required for interaction with Est2p, the reverse transcriptase subunit. Two regions of the RNA distant from the template in the nucleotide sequence were required for Est2p binding, but the template itself was not. Having the RNA secured to the protein away from the template is proposed to facilitate the translocation of the RNA template through the active site. More generally, our results support a role for the telomerase RNA serving as a scaffold for binding key protein subunits.

scholarly journals Casein Kinase I Regulates Membrane Binding by ARF GAP1

2002 ◽  
Vol 13 (8) ◽  
pp. 2559-2570 ◽  
Author(s):  
Sidney Yu ◽  
Michael G. Roth
Keyword(s):  
Amino Acid ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Membrane Binding ◽  
Casein Kinase ◽  
Protein Domain ◽  
Amino Terminal ◽  
Early Secretory Pathway

ARF GAP1, a 415-amino acid GTPase activating protein (GAP) for ADP-ribosylation factor (ARF) contains an amino-terminal 115-amino acid catalytic domain and no other recognizable features. Amino acids 203–334 of ARF GAP1 were sufficient to target a GFP-fusion protein to Golgi membranes in vivo. When overexpressed in COS-1 cells, this protein domain inhibited protein transport between the ER and Golgi and, in vitro, competed with the full-length ARF GAP1 for binding to membranes. Membrane binding by ARF GAP1 in vitro was increased by a factor in cytosol and this increase was inhibited by IC261, an inhibitor selective for casein kinase Iδ (CKIδ), or when cytosol was treated with antibody to CKIδ. The noncatalytic domain of ARF GAP1 was phosphorylated both in vivo and in vitro by CKI. IC261 blocked membrane binding by ARF GAP1 in vivo and inhibited protein transport in the early secretory pathway. Overexpression of a catalytically inactive CKIδ also inhibited the binding of ARF GAP1 to membranes and interfered with protein transport. Thus, a CKI isoform is required for protein traffic through the early secretory pathway and can modulate the amount of ARF GAP1 that can bind to membranes.

scholarly journals Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases

1994 ◽  
Vol 124 (3) ◽  
pp. 351-363 ◽  
Author(s):  
KW Cunningham ◽  
GR Fink
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Mammalian Cells ◽  
Growth Control ◽  
Inhibitory Effect ◽  
Yeast Growth ◽  
Recessive Mutations ◽  
Calcineurin A ◽  
Dependent Growth

Ca2+ ATPases deplete the cytosol of Ca2+ ions and are crucial to cellular Ca2+ homeostasis. The PMC1 gene of Saccharomyces cerevisiae encodes a vacuole membrane protein that is 40% identical to the plasma membrane Ca2+ ATPases (PMCAs) of mammalian cells. Mutants lacking PMC1 grow well in standard media, but sequester Ca2+ into the vacuole at 20% of the wild-type levels. pmc1 null mutants fail to grow in media containing high levels of Ca2+, suggesting a role of PMC1 in Ca2+ tolerance. The growth inhibitory effect of added Ca2+ requires activation of calcineurin, a Ca2+ and calmodulin-dependent protein phosphatase. Mutations in calcineurin A or B subunits or the inhibitory compounds FK506 and cyclosporin A restore growth of pmc1 mutants in high Ca2+ media. Also, growth is restored by recessive mutations that inactivate the high-affinity Ca(2+)-binding sites in calmodulin. This mutant calmodulin has apparently lost the ability to activate calcineurin in vivo. These results suggest that activation of calcineurin by Ca2+ and calmodulin can negatively affect yeast growth. A second Ca2+ ATPase homolog encoded by the PMR1 gene acts together with PMC1 to prevent lethal activation of calcineurin even in standard (low Ca2+) conditions. We propose that these Ca2+ ATPase homologs are essential in yeast to deplete the cytosol of Ca2+ ions which, at elevated concentrations, inhibits yeast growth through inappropriate activation of calcineurin.

scholarly journals Nicotinamide Clearance by Pnc1 Directly Regulates Sir2-Mediated Silencing and Longevity

2004 ◽  
Vol 24 (3) ◽  
pp. 1301-1312 ◽  
Author(s):  
Christopher M. Gallo ◽  
Daniel L. Smith ◽  
Jeffrey S. Smith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nicotinic Acid ◽  
Life Span ◽  
Histone Deacetylase ◽  
Transcriptional Repression ◽  
Transcriptional Silencing ◽  
Inhibitory Effect ◽  
Salvage Pathway

ABSTRACT The Saccharomyces cerevisiae Sir2 protein is an NAD+-dependent histone deacetylase (HDAC) that functions in transcriptional silencing and longevity. The NAD+ salvage pathway protein, Npt1, regulates Sir2-mediated processes by maintaining a sufficiently high intracellular NAD+ concentration. However, another NAD+ salvage pathway component, Pnc1, modulates silencing independently of the NAD+ concentration. Nicotinamide (NAM) is a by-product of the Sir2 deacetylase reaction and is a natural Sir2 inhibitor. Pnc1 is a nicotinamidase that converts NAM to nicotinic acid. Here we show that recombinant Pnc1 stimulates Sir2 HDAC activity in vitro by preventing the accumulation of NAM produced by Sir2. In vivo, telomeric, rDNA, and HM silencing are differentially sensitive to inhibition by NAM. Furthermore, PNC1 overexpression suppresses the inhibitory effect of exogenously added NAM on silencing, life span, and Hst1-mediated transcriptional repression. Finally, we show that stress suppresses the inhibitory effect of NAM through the induction of PNC1 expression. Pnc1, therefore, positively regulates Sir2-mediated silencing and longevity by preventing the accumulation of intracellular NAM during times of stress.

scholarly journals Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein.

1997 ◽  
Vol 8 (1) ◽  
pp. 13-31 ◽  
Author(s):  
L Hicke ◽  
B Zanolari ◽  
M Pypaert ◽  
J Rohrer ◽  
H Riezman
Keyword(s):  
Fusion Protein ◽  
Secretory Pathway ◽  
Protein A ◽  
Brefeldin A ◽  
Early Endosome ◽  
Late Endosome ◽  
Endocytic Pathway ◽  
Early Secretory Pathway ◽  
Late Endosomes

Molecules travel through the yeast endocytic pathway from the cell surface to the lysosome-like vacuole by passing through two sequential intermediates. Immunofluorescent detection of an endocytosed pheromone receptor was used to morphologically identify these intermediates, the early and late endosomes. The early endosome is a peripheral organelle that is heterogeneous in appearance, whereas the late endosome is a large perivacuolar compartment that corresponds to the prevacuolar compartment previously shown to be an endocytic intermediate. We demonstrate that inhibiting transport through the early secretory pathway in sec mutants quickly impedes transport from the early endosome. Treatment of sensitive cells with brefeldin A also blocks transport from this compartment. We provide evidence that Sec18p/N-ethylmaleimide-sensitive fusion protein, a protein required for membrane fusion, is directly required in vivo for forward transport early in the endocytic pathway. Inhibiting protein synthesis does not affect transport from the early endosome but causes endocytosed proteins to accumulate in the late endosome. As newly synthesized proteins and the late steps of secretion are not required for early to late endosome transport, but endoplasmic reticulum through Golgi traffic is, we propose that efficient forward transport in the early endocytic pathway requires delivery of lipid from secretory organelles to endosomes.

scholarly journals FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (10) ◽  
pp. 5968-5975 ◽  
Author(s):  
C M Alarcón ◽  
J Heitman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Conformational Changes ◽  
Active Site ◽  
Feedback Inhibition ◽  
Immunosuppressive Drugs ◽  
Intermediate Step ◽  
Active Site Residues ◽  
Peptidyl Prolyl Isomerase

The peptidyl-prolyl isomerase FKBP12 was originally identified as the intracellular receptor for the immunosuppressive drugs FK506 (tacrolimus) and rapamycin (sirolimus). Although peptidyl-prolyl isomerases have been implicated in catalyzing protein folding, the cellular functions of FKBP12 in Saccharomyces cerevisiae and other organisms are largely unknown. Using the yeast two-hybrid system, we identified aspartokinase, an enzyme that catalyzes an intermediate step in threonine and methionine biosynthesis, as an in vivo binding target of FKBP12. Aspartokinase also binds FKBP12 in vitro, and drugs that bind the FKBP12 active site, or mutations in FKBP12 surface and active site residues, disrupt the FKBP12-aspartokinase complex in vivo and in vitro.fpr1 mutants lacking FKBP12 are viable, are not threonine or methionine auxotrophs, and express wild-type levels of aspartokinase protein and activity; thus, FKBP12 is not essential for aspartokinase activity. The activity of aspartokinase is regulated by feedback inhibition by product, and genetic analyses reveal that FKBP12 is important for this feedback inhibition, possibly by catalyzing aspartokinase conformational changes in response to product binding.

scholarly journals The Yeast Saccharomyces cerevisiae Contains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

1998 ◽  
Vol 9 (5) ◽  
pp. 1081-1091 ◽  
Author(s):  
Sandra Luikenhuis ◽  
Gabriel Perrone ◽  
Ian W. Dawes ◽  
Chris M. Grant
Keyword(s):  
Oxidative Stress ◽  
Saccharomyces Cerevisiae ◽  
Mammalian Species ◽  
Glutathione Metabolism ◽  
Yeast Saccharomyces Cerevisiae ◽  
Oxidoreductase Activity ◽  
Heat Stable ◽  
Mixed Disulfides ◽  
Oxidative Damage To Proteins

Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases. Two genes, designatedGRX1 and GRX2, which share 40–52% identity and 61–76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae. Strains deleted for both GRX1 andGRX2 were viable but lacked heat-stable oxidoreductase activity using β-hydroxyethylene disulfide as a substrate. Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1 mutant was unaffected in oxidoreductase activity, whereas the grx2 mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo. Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1 andGRX2. In addition, a grx1 mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide. Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains. The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth. Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins.

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