dictyNews
Electronic Edition
Volume 39, number 26
September 13 2013
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Abstracts
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G17-modified Hammerhead Ribozymes are active in vitro and in vivo
Anne Kalweit and Christian Hammann
Ribogenetics@Biochemistry Lab, School of Engineering and Science,
MoLife Research Center, Jacobs University Bremen, 28759 Bremen,
Germany
RNA, in press
Natural hammerhead ribozymes (HHRz) feature tertiary interactions
between hairpin loops or bulges in two of three helices that surround
the catalytic core of conserved nucleotides. Their conservation was
originally established on minimal versions lacking the tertiary
interactions. While those sequence requirements in general also apply
to natural versions, we show here differences for the HHRz cleavage
site N17. A guanosine at this position strongly impairs cleavage
activity in minimal versions, whereas we observe for the G17 variants
of four tertiary stabilized HHRz significant cleavage and ligation
activity in vitro. Kinetic analyses of these variants revealed reduced
rate and extent of cleavage, compared to wild type sequences, while
variants with distorted tertiary interactions cleaved at reduced rate,
but to the same extent. Contrary to this, G17 variants exhibit similar
in vitro ligation activity as compared to the respective wild type motif.
To also address the catalytic performance of these motifs in vivo, we
have inserted HHRz cassettes in the lacZ gene and tested this beta-
galactosidase reporter in Dictyostelium discoideum. In colorimetric
assays, we observe differences in the enzymatic activity of beta-
galactosidase, which correlate well with the activity of the different
HHRz variants in vitro, and which can be unambiguously attributed
to ribozyme cleavage by primer extension analysis.
Submitted by Christian Hammann [[log in to unmask]]
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Title: Abi is required for modulation and stability of the SCAR/WAVE
complex, but not localization or activity.
Andrew J. Davidson, Seiji Ura, Peter A. Thomason, Gabriela Kalna
& Robert H. Insall
Eukaryotic cell, in press
The SCAR/WAVE complex drives actin-based protrusion, cell migration
and cell separation during cytokinesis. However, the contribution of the
individual complex members to the activity of the whole remains a
mystery. This is primarily because complex members depend on one
another for stability, which limits the scope for experimental manipulation.
Several studies suggest that Abi, a relatively small complex member,
connects signalling to SCAR/WAVE complex localization and activation
through its poly-proline C-terminal tail. We generated a deletion series
of the Dictyostelium Abi to investigate its exact role in regulation of the
SCAR complex, and identified a minimal fragment that would stabilize
the complex. Surprisingly, loss of either the N-terminus of Abi or the
C-terminal polyproline tail confers no detectable defect in complex
recruitment to the leading edge or formation of pseudopods. A fragment
containing approximately 20% of Abi - and none of the sites that couple
to known signaling pathways - allowed the SCAR complex to function
with normal localisation and kinetics. However, expression of N-terminal
Abi deletions exacerbated the cytokinesis defect of the Dictyostelium abi
mutant, which was earlier shown to be caused by inappropriate activation
of SCAR. This demonstrates - unexpectedly - that Abi does not mediate
the SCAR complex's ability to make pseudopods, beyond its role in
complex stability. Instead we propose that Abi has a modulatory role
when the SCAR complex is activated through other mechanisms.
Submitted by Andrew Davidson [[log in to unmask]]
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Dictyostelium lipid droplets host novel proteins
Xiaoli Du1, Caroline Barisch1, Peggy Paschke1, Cornelia Herrfurth3,
Oliver Bertinetti2, Nadine Pawolleck1, Heike Otto1, Harald Rühling1,
Ivo Feussner3, Friedrich W. Herberg2, and Markus Maniak1*
1Abt. Zellbiologie, 2Abt. Biochemie, Universität Kassel, Germany
3Abt. Biochemie der Pflanze, Georg-August-Universität Göttingen,
Germany
Eukaryotic Cell, in press
Across all kingdoms of life, cells store energy in a specialized organelle,
the lipid droplet. In general it consists of a hydrophobic core of
triglycerides and steryl-esters surrounded by only one leaflet derived
from the endoplasmic reticulum membrane to which a specific set of
proteins is bound. We have chosen the unicellular organism Dictyostelium
discoideum to establish kinetics of lipid droplet formation and degradation
and to further identify the lipid constituents and proteins of lipid droplets.
Here we show that the lipid composition is similar to what is found in
mammalian lipid droplets. In addition, phospholipids preferentially consist
of mainly saturated fatty acids, whereas neutral lipids are enriched in
unsaturated fatty acids. Among the novel proteins components are LdpA,
a protein specific to Dictyostelium, and Net4, which has strong homologies
to mammalian DUF829/Tmem53/NET4 that was previously only known as
a constituent of the mammalian nuclear envelope. The proteins analyzed
so far appear to move from the endoplasmic reticulum to the lipid droplets,
supporting the concept that lipid droplets are formed on this membrane.
Submitted by Markus Maniak [[log in to unmask]]
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Identification of pentatricopeptide repeat proteins in the model organism
Dictyostelium discoideum.
Sam Manna, Jessica Brewster & Christian Barth
Department of Microbiology, La Trobe University, VIC 3086, Australia.
International Journal of Genomics, vol. 2013, Article ID 586498,
doi:10.1155/2013/586498.
Pentatricopeptide repeat (PPR) proteins are RNA binding proteins with
functions in organelle RNA metabolism. They are found in all eukaryotes,
but have been most extensively studied in plants. We report on the
identification of 12 PPR-encoding genes in the genome of the protist
Dictyostelium discoideum, with potential homologs in other members of
the same lineage and some predicted novel functions for the encoded
gene products in protists. For one of the gene products, we show that it
localises to mitochondria and we also demonstrate that antisense
inhibition of its expression leads to slower growth, a phenotype
associated with mitochondrial dysfunction.
Submitted by Christian Barth [[log in to unmask]]
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Tong Sun, Bohye Kim and Lou W. Kim*
Department of Biological Sciences, Florida International University,
Miami, FL, 33199, USA
Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by
regulating PI3K membrane localization in Dictyostelium
Develop. Growth Differ., in press
Glycogen Synthase Kinase 3 (GSK3) is a multifunctional kinase involved
in diverse cellular activities such as metabolism, differentiation, and
morphogenesis. Recent studies showed that GSK3 in Dictyostelium affects
chemotaxis via TorC2 pathway and Daydreamer. Now we report that GSK3
affects PI3K membrane localization, of which the mechanism has remained
to be fully understood in Dictyostelium. The membrane localization domain
(LD) of Phosphatidylinositol-3-kinase 1 (PI3K1) is phosphorylated on serine
residues in a GSK3 dependent mechanism and PI3K1-LD exhibited biased
membrane localization in gsk3 cells compared to the wild type cells.
Furthermore, multiple GSK3-phosphorylation consensus sites exist in
PI3K1-LD, of which phosphomimetic substitutions restored cAMP induced
transient membrane localization of PI3K1-LD in gsk3 cells. Serine to alanine
substitution mutants of PI3K1-LD, in contrast, displayed constitutive
membrane localization in wild type cells. Biochemical analysis revealed that
GSK3 dependent serine phosphorylation of PI3K1-LD is constitutive during
the course of cAMP stimulation. Together, these data suggest that GSK3
dependent serine phosphorylation is a prerequisite for chemoattractant
cAMP induced PI3K membrane localization.
Submitted by Leung Kim [[log in to unmask]]
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[End dictyNews, volume 39, number 26]
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