dictyNews
Electronic Edition
Volume 40, number 27
October 31, 2014

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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. Hartson‡, Christopher M. West*

*Dept. of Biochemistry & Molecular Biology, Oklahoma Center for 
Medical Glycobiology, University of Oklahoma Health Sciences Center, 
Oklahoma City, OK 73104 USA; ‡Dept. 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 [[log in to unmask]]
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Argonaute proteins affect siRNA levels and accumulation of a novel 
extrachromosomal DNA from the Dictyostelium retrotransposon DIRS-1

Benjamin Boesler1,  Doreen Meier1, Konrad U. Förstner2, 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 
Würzburg, Josef-Schneider-Str. 2/Bau D15, 97080 Würzburg, 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 [[log in to unmask]]   
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[End dictyNews, volume 40, number 27]