scholarly journals The vacuolar DHHC-CRD protein Pfa3p is a protein acyltransferase for Vac8p

2005 ◽  
Vol 170 (7) ◽  
pp. 1091-1099 ◽  
Author(s):  
Jessica E. Smotrys ◽  
Marissa J. Schoenfish ◽  
Monica A. Stutz ◽  
Maurine E. Linder
Keyword(s):  
Membrane Protein ◽  
Normal Growth ◽  
Growth Conditions ◽  
Vacuolar Membrane ◽  
Membrane Association ◽  
Rich Domain ◽  
Vacuole Fusion ◽  
Cysteine Rich Domain

Palmitoylation of the vacuolar membrane protein Vac8p is essential for vacuole fusion in yeast (Veit, M., R. Laage, L. Dietrich, L. Wang, and C. Ungermann. 2001. EMBO J. 20:3145–3155; Wang, Y.X., E.J. Kauffman, J.E. Duex, and L.S. Weisman. 2001. J. Biol. Chem. 276:35133–35140). Proteins that contain an Asp-His-His-Cys (DHHC)–cysteine rich domain (CRD) are emerging as a family of protein acyltransferases, and are therefore candidates for mediators of Vac8p palmitoylation. Here we demonstrate that the DHHC-CRD proteins Pfa3p (protein fatty acyltransferase 3, encoded by YNL326c) and Swf1p are important for vacuole fusion. Cells lacking Pfa3p had fragmented vacuoles when stressed, and cells lacking both Pfa3p and Swf1p had fragmented vacuoles under normal growth conditions. Pfa3p promoted Vac8p membrane association and palmitoylation in vivo and partially purified Pfa3p palmitoylated Vac8p in vitro, establishing Vac8p as a substrate for palmitoylation by Pfa3p. Vac8p is the first N-myristoylated, palmitoylated protein identified as a substrate for a DHHC-CRD protein.

scholarly journals Increased Susceptibility of  Thymocytes to Apoptosis in Mice Lacking AIM, a Novel Murine Macrophage-derived Soluble Factor Belonging to the Scavenger Receptor Cysteine-rich Domain Superfamily

1999 ◽  
Vol 189 (2) ◽  
pp. 413-422 ◽  
Author(s):  
Toru Miyazaki ◽  
Yumiko Hirokami ◽  
Nobuyuki Matsuhashi ◽  
Hisakazu Takatsuka ◽  
Makoto Naito
Keyword(s):  
Scavenger Receptor ◽  
The Body ◽  
Murine Macrophage ◽  
Double Positive ◽  
Differential Signal ◽  
Rich Domain ◽  
Macrophage Scavenger Receptor ◽  
Cysteine Rich Domain

Apoptosis of cells must be regulated both positively and negatively in response to a variety of stimuli in the body. Various environmental stresses are known to initiate apoptosis via differential signal transduction cascades. However, induction of signals that may inhibit apoptosis is poorly understood, although a number of intracellular molecules that mediate inhibition of apoptosis have been identified. Here we present a novel murine macrophage-specific 54-kD secreted protein which inhibits apoptosis (termed AIM, for apoptosis inhibitor expressed by macrophages). AIM belongs to the macrophage scavenger receptor cysteine-rich domain superfamily (SRCR-SF), members of which share a highly homologous conserved cysteine-rich domain. In AIM-deficient mice, the thymocyte numbers were diminished to half those in wild-type mice, and CD4/CD8 double-positive (DP) thymocytes were strikingly more susceptible to apoptosis induced by both dexamethasone and irradiation in vivo. Recombinant AIM protein significantly inhibited cell death of DP thymocytes in response to a variety of stimuli in vitro. These results indicate that in the thymus, AIM functions in trans to induce resistance to apoptosis within DP cells, and thus supports the viability of DP thymocytes before thymic selection.

scholarly journals Isw1 Functions in Parallel with the NuA4 and Swr1 Complexes in Stress-Induced Gene Repression

2006 ◽  
Vol 26 (16) ◽  
pp. 6117-6129 ◽  
Author(s):  
Kimberly C. Lindstrom ◽  
Jay C. Vary ◽  
Mark R. Parthun ◽  
Jeffrey Delrow ◽  
Toshio Tsukiyama
Keyword(s):  
Genetic Interaction ◽  
Normal Growth ◽  
Growth Conditions ◽  
Dna Accessibility ◽  
Modifying Factors ◽  
Specific Subset ◽  
Nuclear Processes ◽  
Dependent Processes

ABSTRACT The packaging of DNA into chromatin allows eukaryotic cells to organize and compact their genomes but also creates an environment that is generally repressive to nuclear processes that depend upon DNA accessibility. There are several classes of enzymes that modulate the primary structure of chromatin to regulate various DNA-dependent processes. The biochemical activities of the yeast Isw1 ATP-dependent chromatin-remodeling enzyme have been well characterized in vitro, but little is known about how these activities are utilized in vivo. In this work, we sought to discern genetic backgrounds that require Isw1 activity for normal growth. We identified a three-way genetic interaction among Isw1, the NuA4 histone acetyltransferase complex, and the Swr1 histone replacement complex. Transcription microarray analysis revealed parallel functions for these three chromatin-modifying factors in the regulation of TATA-containing genes, including the repression of a large number of stress-induced genes under normal growth conditions. In contrast to a recruitment-based model, we find that the NuA4 and Swr1 complexes act throughout the genome while only a specific subset of the genome shows alterations in transcription.

scholarly journals PolB1 Is Sufficient for DNA Replication and Repair Under Normal Growth Conditions in the Extremely Thermophilic Crenarchaeon Sulfolobus acidocaldarius

2020 ◽  
Vol 11 ◽  
Author(s):  
Hiroka Miyabayashi ◽  
Rupal Jain ◽  
Shoji Suzuki ◽  
Dennis W. Grogan ◽  
Norio Kurosawa
Keyword(s):  
Dna Replication ◽  
Normal Growth ◽  
Growth Conditions ◽  
Sulfolobus Acidocaldarius ◽  
Single Deletion ◽  
Dna Replication And Repair ◽  
Normal Sensitivity ◽  
Y Family

The thermophilic crenarchaeon Sulfolobus acidocaldarius has four DNA polymerases (DNAPs): PolB1, PolB2, PolB3, and Dbh (PolY). Previous in vitro studies suggested that PolB1 is the main replicative DNAP of Sulfolobales whereas PolB2 and Y-family polymerases Dpo4 (Saccharolobus solfataricus) or Dbh are involved in DNA repair and translesion DNA synthesis. On the other hand, there are various opinions about the role of PolB3, which remains to be clearly resolved. In order to examine the roles of the DNAPs of S. acidocaldarius through in vivo experiments, we constructed polB2, polB3, and dbh deletion strains and characterized their phenotypes. Efforts to construct a polB1 deletion strain were not successful; in contrast, it was possible to isolate triple gene-deletion strains lacking polB2, polB3, and dbh. The growth of these strains was nearly the same as that of the parent strains under normal growth conditions. The polB2, polB3, and dbh single-deletion strains were sensitive to some types of DNA-damaging treatments, but exhibited normal sensitivity to UV irradiation and several other damaging treatments. Overall, the genotype which exhibited the greatest sensitivity to the DNA-damaging treatments we tested was the ΔpolB2 ΔpolB3 combination, providing the first evidence of overlapping function for these two DNAPs in vivo. The results of our study strongly suggest that PolB1 is responsible for the DNA replication of both the leading and lagging strands and is sufficient to complete the repair of most DNA damage under normal growth conditions in S. acidocaldarius.

scholarly journals The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase

2002 ◽  
Vol 159 (1) ◽  
pp. 23-28 ◽  
Author(s):  
Amy F. Roth ◽  
Ying Feng ◽  
Linyi Chen ◽  
Nicholas G. Davis
Keyword(s):  
Reaction Mechanism ◽  
Enzymatic Reaction ◽  
Conserved Sequence ◽  
Rich Domain ◽  
Protein Palmitoylation ◽  
Domain Protein ◽  
Polytopic Membrane Protein ◽  
Cysteine Rich Domain

Protein palmitoylation has been long appreciated for its role in tethering proteins to membranes, yet the enzymes responsible for this modification have eluded identification. Here, experiments in vivo and in vitro demonstrate that Akr1p, a polytopic membrane protein containing a DHHC cysteine-rich domain (CRD), is a palmitoyl transferase (PTase). In vivo, we find that the casein kinase Yck2p is palmitoylated and that Akr1p function is required for this modification. Akr1p, purified to near homogeneity from yeast membranes, catalyzes Yck2p palmitoylation in vitro, indicating that Akr1p is itself a PTase. Palmitoylation is stimulated by added ATP. Furthermore, during the reaction, Akr1p is itself palmitoylated, suggesting a role for a palmitoyl-Akr1p intermediate in the overall reaction mechanism. Mutations introduced into the Akr1p DHHC-CRD eliminate both the trans- and autopalmitoylation activities, indicating a central participation of this conserved sequence in the enzymatic reaction. Finally, our results indicate that palmitoylation within the yeast cell is controlled by multiple PTase specificities. The conserved DHHC-CRD sequence, we propose, is the signature feature of an evolutionarily widespread PTase family.

scholarly journals Promoter Selectivity of the Bradyrhizobium japonicum RpoH Transcription Factors In Vivo and In Vitro

1998 ◽  
Vol 180 (9) ◽  
pp. 2395-2401 ◽  
Author(s):  
Franz Narberhaus ◽  
Michael Kowarik ◽  
Christoph Beck ◽  
Hauke Hennecke
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Bradyrhizobium Japonicum ◽  
Normal Growth ◽  
Growth Conditions ◽  
Start Site ◽  
E Coli ◽  
Soluble C

ABSTRACT Expression of the dnaKJ andgroESL 1 heat shock operons ofBradyrhizobium japonicum depends on a ς32-like transcription factor. Three such factors (RpoH1, RpoH2, and RpoH3) have previously been identified in this organism. We report here that they direct transcription from some but not all ς32-type promoters when the respective rpoH genes are expressed inEscherichia coli. All three RpoH factors were purified as soluble C-terminally histidine-tagged proteins, although the bulk of overproduced RpoH3 was insoluble. The purified proteins were recognized by an anti-E. coli ς32 serum. While RpoH1 and RpoH2 productively interacted with E. coli core RNA polymerase and produced E. coli groE transcript in vitro, RpoH3 was unable to do so.B. japonicum core RNA polymerase was prepared and reconstituted with the RpoH proteins. Again, RpoH1 and RpoH2 were active, and they initiated transcription at theB. japonicum groESL 1 and dnaKJpromoters. In all cases, the in vitro start site was shown to be identical to the start site determined in vivo. Promoter competition experiments revealed that the B. japonicum dnaKJ andgroESL 1 promoters were suboptimal for transcription by RpoH1- or RpoH2-containing RNA polymerase from B. japonicum. In a mixture of different templates, the E. coli groESL promoter was preferred over any other promoter. Differences were observed in the specificities of both sigma factors toward B. japonicum rpoH-dependent promoters. We conclude that the primary function of RpoH2is to supply the cell with DnaKJ under normal growth conditions whereas RpoH1 is responsible mainly for increasing the level of GroESL1 after a heat shock.

scholarly journals Anaerobic Conditions Induce Expression of Polysaccharide Intercellular Adhesin in Staphylococcus aureus and Staphylococcus epidermidis

2001 ◽  
Vol 69 (6) ◽  
pp. 4079-4085 ◽  
Author(s):  
Sarah E. Cramton ◽  
Martina Ulrich ◽  
Friedrich Götz ◽  
Gerd Döring
Keyword(s):  
Staphylococcus Aureus ◽  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Extracellular Polysaccharide ◽  
Growth Conditions ◽  
Anaerobic Conditions ◽  
Anaerobic Environment ◽  
Specific Mrna

ABSTRACT Products of the intercellular adhesion (ica) operon in Staphylococcus aureus and Staphylococcus epidermidis synthesize a linear β-1,6-linked glucosaminylglycan. This extracellular polysaccharide mediates bacterial cell-cell adhesion and is required for biofilm formation, which is thought to increase the virulence of both pathogens in association with prosthetic biomedical implants. The environmental signal(s) that triggers ica gene product and polysaccharide expression is unknown. Here we demonstrate that anaerobic in vitro growth conditions lead to increased polysaccharide expression in both S. aureus and S. epidermidis, although the regulation is less stringent inS. epidermidis. Anaerobiosis also dramatically stimulates ica-specific mRNA expression inica- and polysaccharide-positive strains of both S. aureus and S. epidermidis.These data suggest a mechanism whereby ica gene expression and polysaccharide production may act as a virulence factor in an anaerobic environment in vivo.

ADR1c mutations enhance the ability of ADR1 to activate transcription by a mechanism that is independent of effects on cyclic AMP-dependent protein kinase phosphorylation of Ser-230

1992 ◽  
Vol 12 (4) ◽  
pp. 1507-1514
Author(s):  
C L Denis ◽  
S C Fontaine ◽  
D Chase ◽  
B E Kemp ◽  
L T Bemis
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Growth Conditions ◽  
Dependent Protein Kinase ◽  
Inactive Form ◽  
Dependent Protein ◽  
Kinase Phosphorylation

Four ADR1c mutations that occur close to Ser-230 of the Saccharomyces cerevisiae transcriptional activator ADR1 and which greatly enhance the ability of ADR1 to activate ADH2 expression under glucose-repressed conditions have been shown to reduce or eliminate cyclic AMP-dependent protein kinase (cAPK) phosphorylation of Ser-230 in vitro. In addition, unregulated cAPK expression in vivo blocks ADH2 depression in an ADR1-dependent fashion in which ADR1c mutations display decreased sensitivity to unregulated cAPK activity. Taken together, these data have suggested that ADR1c mutations enhance ADR1 activity by blocking cAPK phosphorylation and inactivation of Ser-230. We have isolated and characterized an additional 17 ADR1c mutations, defining 10 different amino acid changes, that were located in the region defined by amino acids 227 through 239 of ADR1. Three observations, however, indicate that the ADR1c phenotype is not simply equivalent to a lack of cAPK phosphorylation. First, only some of these newly isolated ADR1c mutations affected the ability of yeast cAPK to phosphorylate corresponding synthetic peptides modeled on the 222 to 234 region of ADR1 in vitro. Second, we observed that strains lacking cAPK activity did not display enhanced ADH2 expression under glucose growth conditions. Third, when Ser-230 was mutated to a nonphosphorylatable residue, lack of cAPK activity led to a substantial increase in ADH2 expression under glucose-repressed conditions. Thus, while cAPK controls ADH2 expression and ADR1 is required for this control, cAPK acts by a mechanism that is independent of effects on ADR1 Ser-230. It was also observed that deletion of the ADR1c region resulted in an ADR1c phenotype. The ADR1c region is, therefore, involved in maintaining ADR1 in an inactive form. ADR1c mutations may block the binding of a repressor to ADR1 or alter the structure of ADR1 so that transcriptional activation regions become unmasked.

scholarly journals The Escherichia coli cysG promoter belongs to the ‘extended −10’ class of bacterial promoters

1993 ◽  
Vol 296 (3) ◽  
pp. 851-857 ◽  
Author(s):  
T Belyaeva ◽  
L Griffiths ◽  
S Minchin ◽  
J Cole ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Point Mutations ◽  
Growth Conditions ◽  
Upstream Region ◽  
E Coli ◽  
Run Off ◽  
A Site ◽  
Specific Sequences

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5′-TGN-3′ motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the ‘extended -10’ class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.

scholarly journals Leishmania mexicana Mutants Lacking Glycosylphosphatidylinositol (GPI):Protein Transamidase Provide Insights into the Biosynthesis and Functions of GPI-anchored Proteins

2000 ◽  
Vol 11 (4) ◽  
pp. 1183-1195 ◽  
Author(s):  
James D. Hilley ◽  
Jody L. Zawadzki ◽  
Malcolm J. McConville ◽  
Graham H. Coombs ◽  
Jeremy C. Mottram
Keyword(s):  
Normal Growth ◽  
Surface Proteins ◽  
Host Cells ◽  
Mammalian Host ◽  
Putative Protein ◽  
Major Class ◽  
Major Surface Proteins ◽  
Major Surface

The major surface proteins of the parasitic protozoonLeishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (ΔGPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein–anchor precursors. However, the synthesis and cellular levels of other non–protein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the ΔGPI8 mutant. Significantly, the ΔGPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth.

scholarly journals Interaction between the DrosophilaCAF-1 and ASF1 Chromatin Assembly Factors

2001 ◽  
Vol 21 (19) ◽  
pp. 6574-6584 ◽  
Author(s):  
Jessica K. Tyler ◽  
Kimberly A. Collins ◽  
Jayashree Prasad-Sinha ◽  
Elizabeth Amiott ◽  
Michael Bulger ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Polytene Chromosomes ◽  
Normal Growth ◽  
Chromatin Assembly ◽  
Truncated Form ◽  
Assembly Factor ◽  
Development And Differentiation ◽  
Growth Development

ABSTRACT The assembly of newly synthesized DNA into chromatin is essential for normal growth, development, and differentiation. To gain a better understanding of the assembly of chromatin during DNA synthesis, we identified, cloned, and characterized the 180- and 105-kDa polypeptides of Drosophila chromatin assembly factor 1 (dCAF-1). The purified recombinant p180+p105+p55 dCAF-1 complex is active for DNA replication-coupled chromatin assembly. Furthermore, we have established that the putative 75-kDa polypeptide of dCAF-1 is a C-terminally truncated form of p105 that does not coexist in dCAF-1 complexes containing the p105 subunit. The analysis of native and recombinant dCAF-1 revealed an interaction between dCAF-1 and theDrosophila anti-silencing function 1 (dASF1) component of replication-coupling assembly factor (RCAF). The binding of dASF1 to dCAF-1 is mediated through the p105 subunit of dCAF-1. Consistent with the interaction between dCAF-1 p105 and dASF1 in vitro, we observed that dASF1 and dCAF-1 p105 colocalized in vivo inDrosophila polytene chromosomes. This interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed between RCAF and dCAF-1 during the assembly of newly synthesized DNA into chromatin.

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