CSM News
Electronic Edition
Volume 9, number 2
July 12, 1997

Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to CSM-News@nwu.edu.

Back issues of CSM-News, the CSM Reference database and other useful
information is available at the Dictyostelium Web Page 
"http://dicty.cmb.nwu.edu/dicty/dicty.html"

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 Abstracts
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Chunzhong Yang, Simuran K. Brar*, Laurie Desbarats, and Chi-Hung Siu§

Banting and Best Department of Medical Research and Department of Biochemistry
University of Toronto, Toronto, Ontario M5G 1L6, Canada

Differentiation, in press

ABSTRACT

   In Dictyostelium discoideum, the cadA gene encodes the cell
adhesion molecule DdCAD-1, a protein of Mr 24,000, which mediates
Ca2+-dependent cell-cell adhesion during development.  We have examined the
effects of cAMP, cell-cell contact, and growth conditions on cadA
expression.  cadA has a unique pattern of expression, which appears to be a
combination of the expression patterns of early genes and aggregation stage
genes.  Expression of the cadA gene in bacterially grown cells is activated
at the beginning of the developmental cycle, followed by a period of rapid
DdCAD-1 accumulation.  The mRNA level reaches its maximum at 9 h of
development and then declines to the basal level at ~18 h, while the
protein level remains constant after reaching its maximum at 12 h.
Pulse-chase experiments have demonstrated that DdCAD-1 has a significantly
longer half-life than the average cellular protein.  Transcription of the
cadA gene is stimulated by exogenous cAMP pulses, leading to a 3- to 5-fold
increase in the transcription rate.  In the fgdA mutant which lacks a
functional Ga2, cAMP fails to enhance cadA expression, suggesting that cAMP
stimulates cadA transcription via a G protein-dependent pathway.  However,
inhibition of cell-cell contact has no effect on the synthesis of DdCAD-1.
Growth conditions also have a major influence on cadA expression.
Axenically grown cells produce a high level of cadA transcripts during
vegetative growth.  The mRNA level shows a steady decrease during
development and is reduced to the basal level by 12 h.  In contrast, the
level of DdCAD-1 remains relatively high throughout development, suggesting
that axenic growth affects the accumulation of cadA mRNA but not the
stability of the protein.  These results indicate that multiple mechanisms
are involved to maintain a high level of DdCAD-1 during development.

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The Cell Adhesion Molecule DdCAD-1 in Dictyostelium is Targeted to the Cell
Surface by a Non-classical Transport Pathway Involving Contractile Vacuoles

Hiromi Sesaki*, Estella F. S. Wong*Ý, and Chi-Hung Siu*Ý

*Banting and Best Department of Medical Research and ÝDepartment of
Biochemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada
J. Cell Biol., in press.

ABSTRACT

   DdCAD-1 is a 24-kDa Ca2+-dependent cell-cell adhesion molecule
which is expressed soon after the initiation of development in
Dictyostelium cells.  DdCAD-1 is present on the cell surface as well as in
the cytosol.  However, the deduced amino acid sequence of DdCAD-1 lacks a
hydrophobic signal peptide or any predicted transmembrane domain,
suggesting that it may be presented on the cell surface via a non-classical
transport mechanism.  Here we report that DdCAD-1 is transported to the
cell surface via contractile vacuoles, which are normally involved in
osmoregulation.  Immunofluorescence microscopy and subcellular
fractionation revealed a preferential association of DdCAD-1 with
contractile vacuoles.  Proteolytic treatment of isolated contractile
vacuoles degraded vacuole-associated calmodulin but not DdCAD-1,
demonstrating that DdCAD-1 was present in the lumen.  The use of
hyper-osmotic conditions which suppress contractile vacuole activity led to
a dramatic decrease in DdCAD-1 accumulation on the cell surface and the
absence of cell cohesiveness.  Shifting cells back to a hypotonic condition
after hypertonic treatments induced a rapid increase in DdCAD-1-positive
contractile vacuoles, followed by the accumulation of DdCAD-1 on the cell
membrane. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a specific inhibitor of
vacuolar type H+-ATPase and thus the activity of contractile vacuoles, also
inhibited the accumulation of DdCAD-1 on the cell surface.  Furthermore, an
in vitro reconstitution system was established and isolated contractile
vacuoles were shown to import soluble DdCAD-1 into their lumen in an
ATP-stimulated manner.  Taken together, these data provide the first
evidence for a non-classical protein transport mechanism which utilizes
contractile vacuoles to target a soluble cytosolic protein to the cell
surface.

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[End CSM News, volume 9, number 2]