dictyNews
Electronic Edition
Volume 26, number 3
January 20, 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.


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



Identification and characterization of an 8 kDa light chain associated with 
Dictyostelium discoideum MyoB, a class I myosin 

Scott W. Crawley, Marc A. de la Roche, Sheu-Fen Lee, Zhihao Li, Seth Chityat,
Steven P. Smith, and Graham P. Cote 

Biochemistry Dept., Queen's University, Kingston, Ontario K7L 3N6 


Journal of Biological Chemistry, in press

Dictyostelium discoideum MyoB is a single-headed class I myosin. Analysis of 
purified MyoB by SDS-PAGE indicated the presence of an ~9 kDa light chain. A 
tryptic digest of MyoB yielded a partial sequence for the light chain that 
exactly matched a sequence in a 73-amino acid, 8,296-Da protein (dictyBase 
DDB0188713). This protein, termed MlcB, contains two EF-hand motifs and 
shares ~30% sequence identity with the N- and C-terminal lobes of calmodulin. 
Flag-MlcB expressed in Dictyostelium co-immunoprecipitated with MyoB, but 
not with the related class I myosins MyoC and MyoD. Recombinant MlcB bound 
Ca2+ with a Kd value of 0.2 µM and underwent a Ca2+-induced change in 
conformation that increased alpha-helical content and surface 
hydrophobicity. Mutational analysis showed that the first EF-hand was 
responsible for Ca2+ binding. In the presence and absence of Ca2+ MlcB was 
a monomer in solution and bound to a MyoB IQ motif peptide with a Kd value 
of ~0.5 µM. A MyoB head-neck construct with a Ser to Glu mutation at the 
TEDS site bound MlcB and displayed an actin-activated MgATPase activity 
that was insensitive to Ca2+. We conclude that MlcB represents a novel type 
of small myosin light chain that binds to IQ motifs in a manner comparable 
to a single lobe of a typical four EF-hand protein. 


Submitted by: Graham Cote [coteg@post.queensu.ca]

-----------------------------------------------------------------------------


The C-module DNA-binding factor mediates expression of the Dictyostelium 
aggregation-specific adenylyl cyclase ACA

Oliver Siol (1) Theodor Dingermann (1) and Thomas Winckler (1,2)

(1) Institut fuer Pharmazeutische Biologie, Universitaet Frankfurt/M. 
(Biozentrum), Frankfurt am Main, Germany, and (2) Lehrstuhl fuer 
Pharmazeutische Biologie, Universitaet Jena, Germany


Eukaryot. Cell, in press
Aggregation of Dictyostelium discoideum amoebae into multicellular 
structures is organized by cyclic AMP, which acts both as a chemoattractant, 
second messenger, and morphogen. Aggregation of D. discoideum cells depends 
on the expression of adenylyl cyclase ACA, which provides extracellular cAMP 
for signal relay and intracellular cAMP for the induction of genes required 
at multicellular stages. We have identified a DNA-binding activity specific 
for a highly A+T-enriched motif in the upstream region of the ACA-encoding 
gene, acaA. The factor shows DNA-binding characteristics very similar to 
that of C-module-binding factor (CbfA). Although CbfA was originally 
identified as a putative regulator of the activity of D. discoideum 
retrotransposon TRE5-A, it was also found to be essential for aggregation 
of D. discoideum cells. The identified DNA-binding activity was absent in 
mutant cells depleted for CbfA, and CbfA could be precipitated using an acaA 
promoter fragment. We propose that CbfA binds to the acaA promoter to 
provide a basal transcription activity that is required for induction of 
ACA expression after the onset of D. discoideum development.


Submitted by: Thomas Winckler [t.winckler@uni-jena.de]

==============================================================================
[End dictyNews, volume 26, number 3]