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dictyNews Volume 27 Number 01
dictyNews
Electronic Edition
Volume 27, number 1
July 14, 2006
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
useful information is available at dictyBase - http://dictybase.org.
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Abstracts
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Detection of Cytoplasmic Glycosylation Associated with Hydroxyproline
Christopher M. West(1), Hanke van der Wel(1), and Ira J. Blader(2)
(1)Department of Biochemistry & Molecular Biology, Oklahoma Center for
Medical Glycobiology, and the (2)Department of Microbiology and Immunology,
University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma,
73104 USA
Methods in Enzymology, in press.
A special class of glycosylation occurs on a proline residue of the
cytoplasmic/nuclear protein Skp1 in the social amoeba Dictyostelium. In
order for this glycosylation to occur, the proline must first be
hydroxylated by the action of a soluble prolyl 4-hydroxylase acting on the
protein. Cytoplasmic prolyl 4-hydroxylases are dioxygen-dependent enzymes
that have low affinity for their O2 substrate and therefore have been
implicated in O2-sensing in Dictyostelium as well as in vertebrates and
invertebrates. The sugar-hydroxyproline linkage has low abundance, is
resistant to alkali cleavage and known glycosidases, and does not bind
known lectins. However, initial screens for this modification can be made
by assessing changes in electrophoretic mobility of candidate proteins after
treatment of cells with prolyl hydroxylase inhibitors, and/or by metabolic
labeling with [3H]sugar precursors. In addition, cytoplasmic
hydroxylation/glycosylation can be assessed by assaying for cytoplasmic
glycosyltransferases. Here we describe these methods and examples of their
use in analyzing Skp1 glycosylation in Dictyostelium and the apicomplexan
Toxoplasma gondii, the causative agent of toxoplasmosis in humans.
Submitted by: Chris West [Cwest2@ouhsc.edu]
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A 60 kDa protein component of the counting factor complex regulates group
size in Dictyostelium
Debra A. Brock1, Wouter N. van Egmond2, Yousif Shamoo3, R. Diane Hatton1,
and Richard H. Gomer1
1Howard Hughes Medical Institute and 3Department of Biochemistry and Cell
Biology, MS-140, Rice University, Houston, TX 77005-1892
2Department of Biochemistry University of Groningen Kerklaan 30 9750
AA Haren The Netherlands
Eukaryotic Cell, in press
Much remains to be understood about how a group of cells or a tissue senses
and regulates its size. Dictyostelium cells sense and regulate the size of
groups and fruiting bodies using a secreted 450 kDa complex of proteins
called counting factor (CF). Low levels of CF result in large groups, and
high levels of CF result in small groups. We previously found three
components of CF. We describe here a fourth component, CF60. CF60 has
similarity to acid phosphatases, although it has very little, if any, acid
phosphatase activity. CF60 is secreted by starving cells and is lost from
the 450 kDa CF when a different CF component, CF50, is absent. Although we
were unable to obtain cells lacking CF60, decreasing CF60 levels by
antisense resulted in large groups, and overexpressing CF60 resulted in
small groups. When added to wild-type cells, conditioned starvation medium
from CF60 overexpressor cells as well as recombinant CF60 caused the
formation of small groups. The ability of recombinant CF60 to decrease
group size did not require the presence of the CF components CF45-1 or
countin, but did require the presence of CF50. Recombinant CF60 does not
have acid phosphatase activity, indicating that the CF60 bioactivity is not
due to a phosphatase activity. Together, the data suggest that CF60 is a
component of CF, and thus this secreted signal has four different protein
components.
Submitted by: Richard Gomer [richard@rice.edu]
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[End dictyNews, volume 27, number 1] 
