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dictyNews Volume 17 Number 12
Dicty News
Electronic Edition
Volume 17, number 12
November 17, 2001
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu.
Back issues of Dicty-News, the Dicty Reference database and other useful
information is available at DictyBase--http://dictybase.org.
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Abstracts
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CREATION OF AN ALLOSTERIC PHOSPHOFRUCTOKINASE STARTING WITH A NONALLOSTERIC
ENZYME: THE CASE OF DICTYOSTELIUM DISCOIDEUM PHOSPHOFRUCTOKINASE
Beln Santamara, Antonio M. Estvez, Oscar H. Martnez-Costa and Juan
J. Aragn
Departamento de Bioqumica and Instituto de Investigaciones Biomdicas
Alberto Sols UAM-CSIC, Facultad de Medicina de la Universidad Autnoma,
28029 Madrid, Spain
J. Biol. Chem., accepted for publication, available on line at www.jbc.org
Abstract
An allosteric phosphofructokinase (PFK) was created by sequence manipulation
of the nonallosteric enzyme from the slime mold Dictyostelium discoideum
(DdPFK). Most amino acid residues proposed as important for catalytic and
allosteric sites are conserved in DdPFK except for a few of them, and their
reversion did not modify its kinetic behavior. However, deletions at the
unique C-terminal extension of this PFK produced a markedly allosteric
enzyme. Thus, a mutant lacking the last 26 C-terminal residues exhibited
hysteresis in the time course, intense cooperativity (nH = 3.8) and a
200-fold decrease in the apparent affinity for fructose 6-phosphate
(S0.5 = 4500 mM), strong activation by fructose 2,6-bisphosphate
(Kact = 0.1 mM) and fructose 1,6-bisphosphate (Kact = 40 mM), dependence
on enzyme concentration, proton inhibition and subunit association-
dissociation in response to fructose 6-phosphate, versus the nonhysteretic
and hyperbolic wild-type enzyme (nH = 1.0; Km = 22 mM) that remained as a
stable tetramer. Systematic deletions and point mutations at the C-tail
region of DdPFK identified the last C-terminal residue, Leu834, as critical
to produce a nonallosteric enzyme. All allosteric mutants were practically
insensitive to MgATP inhibition, suggesting that this effect does not
involve the same allosteric transition as that responsible for fructose
6-phosphate cooperativity and fructose bisphosphate activation.
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The Dictyostelium LvsA Protein is Localized on the Contractile Vacuole
and is Required for Osmoregulation.
Noel J. Gerald, Michael Siano and Arturo De Lozanne
Section of Molecular Cell & Developmental Biology
and Institute for Cellular and Molecular Biology,
University of Texas at Austin, Austin, TX 78712.
Accepted into Traffic
LvsA is a Dictyostelium protein that is essential for cytokinesis and that
is related to the mammalian beige/LYST family of proteins. To better
understand the function of this novel protein family we tagged LvsA with
GFP using recombination techniques. GFP-LvsA is primarily associated with
the membranes of the contractile vacuole (CV) system and it also has a
punctate distribution in the cytoplasm. Two markers of the Dictyostelium
CV, the vacuolar proton pump and calmodulin, show extensive colocalization
with GFP-LvsA on CV membranes. Interestingly, the association of lvsA with
CV membranes occurs only during the discharge phase of the vacuole. In
LvsA mutants the CV becomes disorganized and calmodulin dissociates from
the CV membranes. Consequently, the CV is unable to function normally, it
can swell but seems unable to discharge and the LvsA mutants become
osmosensitive. These results demonstrate that LvsA can associate
transiently with the CV membrane compartment and that this association
is necessary for the function of the CV during osmoregulation. This
transient association with specific membrane compartments may be a general
property of other BEACH-domain containing proteins.
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PCR-mediated generation of a gene disruption construct without the use of
DNA ligase and plasmid vectors
Hidekazu Kuwayama, Shinji Obara, Takahiro Morio, Mariko Katoh, Hideko
Urushihara and Yoshimasa Tanaka
Institute of Biological Sciences,0University of Tsukuba,0Tsukuba, Ibaraki
305-8572, Japan.
Nucleic Acid Research, in press
Abstract
We introduce a PCR-based procedure for generating a gene disruption
construct. This method depends on DNA fragment fusion by PCR technique
and requires only two steps of PCR to obtain a sufficient amount of the
gene disruption construct for one transformation experiment. The first
step involves three separate PCR syntheses of a selectable marker cassette
and the 5'- and 3'- regions of a target gene. Of the four primers used in
the amplification of the 5 - and 3 -regions of the target gene, two primers
placed proximal to the site of the marker cassette are designed to have
sequence tags complementary to the 5 - or 3 - side of the marker cassette.
The two primers used in the PCR synthesis of the marker cassette are
complementary to the tagged primers. By fusion PCR, the 5 - and 3 - PCR
products are linked to the marker cassette via the regions of tagged
primers which overlap. A sufficient amount of the disruption construct
can be directly amplified with the outermost primers. This method is
simple, rapid, and relatively inexpensive. In addition, there is the
freedom of attaching long flanking regions with any selectable marker
cassette.
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Dictyostelium LvsB Mutants Model the Lysosomal Defects Associated with
Chediak-Higashi Syndrome
Edward Harris1, Ning Wang2, Wei-l Wu2, Alisha Weatherford1, Arturo
De Lozanne2, and James Cardelli1*
1Department of Microbiology and Immunology, LSU Health Sciences Center,
Shreveport, LA 71130
2Department of Molecular Cell and Developmental Biology and Institute for
Cell and Molecular Biology, University of Texas, Austin, TX 78712
Accepted Molecular Biology of the Cell
ABSTRACT
Chediak-Higashi Syndrome (CHS) is a genetic disorder caused by mutations
in a gene encoding a protein named LYST in humans (Lysosomal-Trafficking
Regulator) or Beige in mice. A prominent feature of this disease is the
accumulation of enlarged lysosome-related granules in a variety of cells.
The genome of D. discoideum contains 6 genes encoding proteins that are
related to LYST/Beige in amino acid sequence, and disruption of one of these
genes, lvsA (large volume sphere), results in profound defects in cytokinesis.
To better understand the function of this family of proteins in membrane
trafficking, we have analyzed mutants disrupted in lvsA, lvsB, lvsC, lvsD,
lvsE and lvsF. Of all these, only lvsA and lvsB mutants displayed interesting
phenotypes in our assays. LvsA-null cells exhibited defects in phagocytosis,
and contained abnormal looking contractile vacuole membranes. Loss of LvsB,
the Dictyostelium protein most similar to LYST/Beige, resulted in the formation
of enlarged vesicles that by multiple criteria appeared to be acidic
lysosomes. The rates of endocytosis, phagocytosis and fluid phase exocytosis
were normal in lvsB-null cells. Also, the rates of processing and the
efficiency of targeting of lysosomal alpha-mannosidase were normal, although
lvsB mutants inefficiently retained alpha-mannosidase, as well as two other
lysosomal cysteine proteinases. Finally, results of pulse-chase experiments
indicated that an increase in fusion rates accounted for the enlarged
lysosomes in lvsB-null cells, suggesting LvsB acts as a negative regulator
of fusion. Our results support the notion that LvsB/LYST/Beige function in a
similar manner to regulate lysosome biogenesis.
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On the Evolutionary Conservation of the Cell Death Pathway: Mitochondrial
Release of an Apoptosis-inducing Factor during Dictyostelium discoideum Cell
Death
Damien Arnoult,* Irene Tatischeff, Jerome Estaquier,* Mathilde Girard,
Franck Sureau, Jean Pierre Tissier, Alain Grodet,* Marc Dellinger,
FranoisTraincard, Axel Kahn, Jean-Claude Ameisen,* and Patrice Xavier
Petit #
Mol. Bio. Cell 12, 3016-3030
Mitochondria play a pivotal role in apoptosis in multicellular organisms by
releasing apoptogenic factors such as cytochrome c that activate the
caspases effector pathway, and apoptosis-inducing factor (AIF) that is
involved in a caspase-independent cell death pathway. Here we report that
cell death in the single-celled organism Dictyostelium discoideum involves
early disruption of mito-chondrialtransmembrane potential (DYm) that
precedes the induction of several apoptosis-likefeatures, including exposure
of the phosphatidyl residues at the external surface of the plasmamembrane,
an intense vacuolization, a fragmentation of DNA into large fragments, an
autophagy,and the release of apoptotic corpses that are engulfed by
neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF
that is localized into mitochondria and is translocated from the
mitochondria to the cytoplasm and the nucleus after the onset of cell
death. Cytoplasmic extracts from dying Dictyostelium cells trigger the
breakdown of isolated mammalian and Dictyo-stelium nuclei in a cell-free
system, and this process is inhibited by a polyclonal antibody specific
for Dictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting
that DdAIF is involved in DNA degradation during Dictyostelium cell death.
Our findings indicate that the cell death pathway in Dictyostelium involves
mitochondria and an AIF homolog, suggesting the evolutionary conservation
of at least part of the cell death pathway in unicellular and multicellular
organisms.
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[End Dicty News, volume 17, number 12]
