dictyNews Volume 40 Number 27

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dictyNews 
Electronic Edition 
Volume 40, number 27 
October 31, 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. 

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========= 
Abstracts 
========= 

Glycosylation of Skp1 Promotes Formation of Skp1/Cullin-1/F-box  
Protein Complexes in Dictyostelium 

M. Osman Sheikh*, Yuechi Xu*, Hanke van der Wel*, Paul Walden*,  
Steven D. Hartsonà, Christopher M. West* 

*Dept. of Biochemistry & Molecular Biology, Oklahoma Center for  
Medical Glycobiology, University of Oklahoma Health Sciences Center,  
Oklahoma City, OK 73104 USA; àDept. of Biochemistry & Molecular  
Biology, Oklahoma State University, Stillwater, OK 74078 USA 

 
Molecular & Cellular Proteomics, in press 

O2-sensing in diverse protozoa depends on the prolyl 4-hydroxylation  
of Skp1 and modification of the resulting hydroxyproline with a series  
of 5 sugars. In yeast, plants and animals, Skp1 is associated with  
F-box proteins. The Skp1/F-box protein heterodimer can, for many  
F-box proteins, dock onto Cullin-1 en route to assembly of the  
Skp1/Cullin-1/F-box protein/Rbx1 subcomplex of E3SCFUb-ligases.  
E3SCFUb-ligases conjugate Lys48-polyubiquitin chains onto targets  
bound to the substrate receptor domains of F-box proteins, preparing  
them for recognition by the 26S-proteasome. In the social amoeba  
Dictyostelium, we show that O2-availability is rate limiting for  
hydroxylation of newly synthesized Skp1. To investigate the effect  
of reduced hydroxylation, we analyzed knock-out mutants of the Skp1  
prolyl hydroxylase and each of the Skp1 glycosyltransferases.  
Proteomic analysis of co-immunoprecipitates showed that wild-type  
cells that can fully glycosylate Skp1 have greater abundance of an  
SCF complex containing the Cullin-1 homolog CulE and FbxD, a newly  
described WD40-type F-box protein, relative to the complexes that  
predominate in cells defective in Skp1 hydroxylation or glycosylation.  
Similarly, the previously described FbxA/Skp1CulA complex was also  
more abundant in glycosylation competent cells. The CulE interactome  
also includes higher levels of proteasomal regulatory particles when  
Skp1 is glycosylated, suggesting increased activity consistent with  
greater association with F-box proteins. Finally, the interactome of  
FLAG-FbxD was modified when it harbored an F-box mutation that  
compromised Skp1 binding consistent with an impact on the abundance  
of potential substrate proteins. We propose that O2-dependent  
posttranslational glycosylation of Skp1 promotes association with  
F-box proteins and their engagement in functional E3SCFUb-ligases  
that regulate O2-dependent developmental progression. 

 
Submitted by Chris West [Cwest2@ouhsc.edu] 
---------------------------------------------------------------------- 

 
Argonaute proteins affect siRNA levels and accumulation of a novel  
extrachromosomal DNA from the Dictyostelium retrotransposon DIRS-1 

Benjamin Boesler1,  Doreen Meier1, Konrad U. Fšrstner2, Michael  
Friedrich1,  Christian Hammann3, Cynthia M. Sharma2 and  
Wolfgang Nellen1,* 

The authors wish it to be known that, in their opinion, the first  
two authors should be regarded as joint first authors. 

1 Department of Genetics, FB10, Kassel University,  
Heinrich-Plett-Str. 40, 34132 Kassel, Germany 
2 Research Center for Infectious Diseases (ZINF), University of  
WŸrzburg, Josef-Schneider-Str. 2/Bau D15, 97080 WŸrzburg, Germany 
3 Ribogenetics Biochemistry Laboratory, School of Engineering and  
Science, Molecular Life Sciences Research Center, Jacobs University,  
Campus Ring 1, DE-28759 Bremen, Germany 

 
J. Biol. Chem., accepted  
doi:10.1074/jbc.M114.612663 

The retrotransposon DIRS-1 is the most abundant retroelement in  
Dictyostelium discoideum and constitutes the pericentromeric  
heterochromatin of the six chromosomes in Dictyostelium discoideum.  
The vast majority of cellular siRNAs is derived from DIRS-1,  
suggesting that the element is controlled by RNAi related  
mechanisms. We investigated the role of two of the five Argonaute  
proteins of D. discoideum, AgnA and AgnB, in DIRS-1 silencing.  
Deletion of agnA resulted in the accumulation of DIRS-1 transcripts,  
the expression of DIRS-1 encoded proteins and the loss of most  
DIRS-1 derived secondary siRNAs. Simultaneously, extrachromosomal  
single stranded DIRS-1 DNA accumulated in the cytoplasm of agnA-  
strains. These DNA molecules appear to be products of reverse  
transcription and thus could represent intermediate structures  
before transposition. We further show that transitivity of  
endogenous siRNAs is impaired in agnA- strains. The deletion of  
agnB alone had no strong effect on DIRS-1 transposon regulation.  
However, in agnA-/agnB- double mutant strains strongly reduced  
accumulation of extrachromosomal DNA compared to the single  
agnA- strains was observed.  

 
Submitted by Wolfgang Nellen [nellen@uni-kassel.de]    
============================================================== 
[End dictyNews, volume 40, number 27]