dictyNews Volume 27 Number 01

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Dicty News

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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]