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dictyNews Volume 40 Number 06
dictyNews
Electronic Edition
Volume 40, number 6
February 21, 2014
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu
or by using the form at
http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit.
Back issues of dictyNews, the Dicty Reference database and other
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Abstracts
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Glycosylation of Skp1 affects its conformation and promotes binding
to a model F-box protein
M. Osman Sheikh, Christopher M. Schafer, John T. Powell,
Karla K. Rodgers, Blaine H. M. Mooers, and Christopher M. West
Department of Biochemistry & Molecular Biology, Oklahoma Center
for Medical Glycobiology, University of Oklahoma Health Sciences
Center, 975 NE 10th St., BRC 415, OUHSC, Oklahoma City,
OK 73104 USA
telephone 1-405-271-4147; email Cwest2@ouhsc.edu
Biochemistry, in press
In the social amoeba Dictyostelium, Skp1 is hydroxylated on proline-143
and further modified by three cytosolic glycosyltransferases to yield an
O-linked pentasaccharide that contributes to O2-regulation of
development. Skp1 is an adapter in the Skp1/cullin1/F-box protein family
of E3 ubiquitin ligases that targets specific proteins for polyubiquitination
and subsequent proteasomal degradation. To investigate the biochemical
consequences of glycosylation, untagged full-length Skp1 and several of
its posttranslationally modified isoforms were expressed and purified to
near homogeneity using recombinant and in vitro strategies. Interaction
studies with the soluble mammalian F-box protein Fbs1/Fbg1/OCP1
revealed preferential binding to the glycosylated isoforms of Skp1. This
difference correlated with increased alpha-helical and decreased
beta-sheet content of glycosylated Skp1s based on circular dichroism,
and increased folding order based on small-angle X-ray scattering. A
comparison of the molecular envelopes of fully glycosylated Skp1 and
the apoprotein indicated that both isoforms exist as an anti-parallel dimer
that is more compact and extended in the glycosylated state. Analytical
gel filtration and chemical cross-linking studies showed an increasing
tendency of less modified isoforms to dimerize. Considering that regions
of free Skp1 are intrinsically disordered and Skp1 can adopt distinct folds
when bound to F-box proteins, we propose that glycosylation, which
occurs adjacent to the F-box binding site, influences the spectrum of
energetically similar conformations which vary inversely in their
propensity to dock with Fbs1 or another Skp1. Glycosylation may thus
influence Skp1 function by modulating F-box protein binding in cells..
Submitted by Chris West [Cwest2@ouhsc.edu]
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Novel Regulation of Skp1 by the Dictyostelium AgtA
alpha-Galactosyltransferase Involves the Skp1-Binding Activity of its
WD40-Repeat Domain
Christopher M. Schafer, M. Osman Sheikh, Dongmei Zhang, and
Christopher M. West
Department of Biochemistry & Molecular Biology, Oklahoma Center
for Medical Glycobiology, University of Oklahoma Health Sciences
Center, 975 NE 10th St., BRC 415, OUHSC, Oklahoma City,
OK 73104 USA
telephone 1-405-271-4147; email Cwest2@ouhsc.edu
J. Biol. Chem., in press.
The role of Skp1 as an adaptor protein that links Cullin-1 to F-box
proteins in E3 Skp1/Cullin-1/F-box protein (SCF) ubiquitin ligases is
well characterized. In the social amoeba Dictyostelium and probably
many other unicellular eukaryotes, Skp1 is modified by a
pentasaccharide attached to a hydroxyproline near its C-terminus.
This modification is important for oxygen-sensing during Dictyostelium
development and is mediated by a HIF-alpha type prolyl 4-hydroxylase
and five sequentially acting cytoplasmic glycosyltransferase activities.
Gene disruption studies show that AgtA, the enzyme responsible for
addition of the final two galactose residues, in alpha-linkages to the
Skp1 core trisaccharide, is unexpectedly critical for oxygen-dependent
terminal development. AgtA possesses a WD40-repeat domain
C-terminal to its single catalytic domain and, by use of domain deletions,
binding studies, and enzyme assays, we find that the WD40-repeats
confer a salt-sensitive second-site binding interaction with Skp1 that
mediates novel catalytic activation in addition to simple substrate
recognition. In addition, AgtA binds similarly well to precursor isoforms
of Skp1 by a salt-sensitive mechanism that competes with binding to
an F-box protein and recognition by early modification enzymes, and
the effect of binding is diminished when AgtA modifies Skp1. Genetic
studies show that loss of AgtA is more severe when an earlier
glycosylation step is blocked, and overexpressed AgtA is deleterious
if catalytically inactivated. Together, the findings suggest that AgtA
mediates non-enzymatic control of unmodified and substrate precursor
forms of Skp1 by a binding mechanism that is normally relieved by
switch-like activation of its glycosylation function.
Submitted by Chris West [Cwest2@ouhsc.edu]
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Role of PKD2 in Rheotaxis in Dictyostelium
Lima WC, Vinet A, Pieters J, Cosson P.
PLoS One. 2014 Feb 10;9(2):e88682.
The sensing of mechanical forces modulates several cellular responses
as adhesion, migration and differentiation. Transient elevations of calcium
concentration play a key role in the activation of cells following mechanical
stress, but it is still unclear how eukaryotic cells convert a mechanical
signal into an ion flux. In this study, we used the model organism
Dictyostelium discoideum to assess systematically the role of individual
calcium channels in mechanosensing. Our results indicate that PKD2 is
the major player in the cell response to rheotaxis (i.e., shear-flow induced
mechanical motility), while other putative calcium channels play at most
minor roles. Mutant pkd2 KO cells lose the ability to orient relative to a
shear flow, whereas their ability to move towards a chemoattractant is
unaffected. PKD2 is also important for calcium-induced lysosome
exocytosis: WT cells show a transient, 2-fold increase in lysosome
secretion upon sudden exposure to high levels of extracellular calcium,
but pkd2 KO cells do not. In Dictyostelium, PKD2 is specifically localized
at the plasma membrane, where it may generate calcium influxes in
response to mechanical stress or extracellular calcium changes.
Submitted by Wanessa Lima [wanessa.delima@unige.ch]
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The essential function of Dictyostelium Dgat1 in triglyceride production,
but not in ether lipid synthesis, can be substituted by Dgat2
Xiaoli Du1, Cornelia Herrfurth2, Thomas Gottlieb1, Steffen Kawelke2,
Kristin Feussner2, Harald Rhling1, Ivo Feussner2, and Markus Maniak1
Eukaryotic Cell, in press
Triacylglycerol (TAG), the common energy storage molecule is formed
from diacylglycerol and a coenzyme A-activated fatty acid by the action
of an acyl-coenzyme A:diacylglycerol acyltransferase (DGAT). In order
to conduct this step, most organisms rely on more than one enzyme. The
two main candidates in Dictyostelium are Dgat1 and Dgat2. We show, by
creating single and double knockout mutants, that the ER-localized Dgat1
enzyme provides the predominant activity, whereas the lipid droplet
constituent Dgat2 contributes less activity. This situation may be opposite
to what is seen in mammalian cells. Dictyostelium Dgat2 is specialized for
the synthesis of TAG, as is the mammalian enzyme. In contrast, mammalian
DGAT1 is more promiscuous regarding its substrates, producing
diacylglycerol, retinyl esters, and waxes in addition to TAG. The
Dictyostelium Dgat1, however, produces TAG, wax esters, and, most
interestingly also neutral ether lipids, which represent a significant
constituent of lipid droplets. Ether lipids had also been found in
mammalian lipid droplets, but the role of DGAT1 in their synthesis was
unknown. The ability to form TAG either through Dgat1 or Dgat2 activity is
essential for Dictyostelium to grow on bacteria, their natural food substrate.
Submitted by Markus Maniak [maniak@uni-kassel.de]
---------------------------------------------------------------------------
SILAC-based proteomic quantification of chemoattractant-induced
cytoskeleton dynamics on a second to minute timescale
Grzegorz J. Sobczyk, Jun Wang, & Cornelis J. Weijer
Nature Communications, in press
Cytoskeletal dynamics during cell behaviours ranging from endocytosis
and exocytosis to cell division and movement is controlled by a complex
network of signalling pathways, the full details of which are as yet
unresolved. Here we show that SILAC-based proteomic methods can be
used to characterize the rapid chemoattractant-induced dynamic changes
in the actinÐmyosin cytoskeleton and regulatory elements on a proteome-
wide scale with a second to minute timescale resolution. This approach
provides novel insights in the ensemble kinetics of key cytoskeletal
constituents and association of known and novel identified binding
proteins. We validate the proteomic data by detailed microscopy-based
analysis of in vivo translocation dynamics for key signalling factors. This
rapid large-scale proteomic approach may be applied to other situations
where highly dynamic changes in complex cellular compartments are
expected to play a key role.
Submitted by Grzegorz Sobczyk [g.j.sobczyk@dundee.ac.uk]
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[End dictyNews, volume 40, number 6]
