dictyNews
Electronic Edition
Volume 35, number 13
Nov 5, 2010

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Abstracts
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Functional dissection of adenylate cyclase r, an inducer of spore 
encapsulation.

Zhi-hui Chen, Christina Schilde and Pauline Schaap*
College of Life Sciences, University of Dundee, Dundee, United Kingdom


J.Biol.Chem., in press
	 
Cyclic AMP acting on PKA controls sporulation and encystation in social 
and solitary amoebas. In Dictyostelium discoideum, adenylate cyclase R 
(ACR), is essential for spore encapsulation. In addition to its cyclase (AC) 
domain, ACR harbours seven transmembrane helices, a histidine kinase 
domain and two receiver domains. We investigated the role of these 
domains in the regulation of AC activity. Expression of an ACR-YFP fusion 
protein in acr- cells rescued their sporulation defective phenotype and 
revealed that ACR is associated with the nuclear envelope and 
endoplasmic reticulum. Loss of the transmembrane helices (deltaTM) 
caused 60% reduction of AC activity, but deltaTM-ACR still rescued the 
acr- phenotype. The isolated AC domain was properly expressed but 
inactive. Mutation of three essential ATP-binding residues in the histidine 
kinase domain did not affect the AC activity or phenotypic rescue. 
Mutation of the essential phosphoryl-accepting aspartate in receivers 
1, 2 or both had only modest effects on AC activity and did not affect 
phenotypic rescue, indicating that AC activity is not critically regulated 
by phosphorelay. Remarkably, the dimerizing histidine phospho-acceptor 
subdomain, which in ACR lacks the canonical histidine for 
autophosphorylation, was essential for AC activity. Transformation of 
wild-type cells with an ACR allele (deltaCRA) that is truncated after 
this domain  inhibited AC activity of endogenous ACR and replicated 
the acr- phenotype. Combined with the observation that the isolated AC 
domain was inactive, the dominant-negative effect of deltaCRA strongly 
suggests that the defunct phospho-acceptor domain acquired a novel 
role in enforcing dimerization of the AC domain.  


Submitted by Pauline Schaap [p.schaap@dundee.ac.uk]
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The C-module-binding factor supports the amplification of TRE5-A 
retrotransposons in the Dictyostelium discoideum genome

Annika Bilzer, Heike Dölz, Alexander Reinhardt, Anika Schmith, Oliver Siol, 
Thomas Winckler


Eukaryotic Cell, in press

Retrotransposable elements are molecular parasites that have invaded 
the genomes of virtually all organisms. Although retrotransposons encode 
essential proteins to mediate their amplification, they also require 
assistance by host cell-encoded machineries that perform functions such 
as DNA transcription and repair. The retrotransposon TRE5-A of the 
social amoeba Dictyostelium discoideum generates a notable amount of 
both sense and antisense RNAs, which are generated from 
element-internal promoters located in the A- module and the C-module, 
respectively. We observed that TRE5-A retrotransposons depend on 
the C-module-binding factor (CbfA) to maintain high steady-state levels 
of TRE5-A transcripts and that CbfA supports the retrotransposition 
activity of TRE5-A elements. The carboxy-terminal domain of CbfA was 
found to be required and sufficient to mediate the accumulation of 
TRE5-A transcripts, but it did not support productive retrotransposition 
of TRE5-A. This result suggests different roles for CbfA protein domains 
in the regulation of the TRE5-A retrotransposition frequency in 
D. discoideum cells. Although CbfA binds to the C-module in vitro, the 
factor regulates neither C-module nor A-module promoter activity in vivo. 
We speculate that CbfA supports the amplification of TRE5-A 
retrotransposons by suppressing the expression of a not yet identified 
component of the cellular posttranscriptional gene silencing machinery.  


Submitted by Thomas Winckler [t.winckler@uni-jena.de]
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[End dictyNews, volume 35, number 13]