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Vol. 10, Issue 4, 1205-1219, April 1999
and§
*Institut für Biochemie I, Medizinische Fakultät,
Universität zu Köln, 50931 Köln,
Germany; Using a PCR approach we have isolated racF1, a novel
member of the Rho family in Dictyostelium. The
racF1 gene encodes a protein of 193 amino acids and is
constitutively expressed throughout the Dictyostelium
life cycle. Highest identity (94%) was found to a RacF2 isoform, to
Dictyostelium Rac1A, Rac1B, and Rac1C (70%), and to Rac
proteins of animal species (64-69%). To investigate the role of RacF1
in cytoskeleton-dependent processes, we have fused it at its
amino-terminus with green fluorescent protein (GFP) and studied the
dynamics of subcellular redistribution using a confocal laser scanning
microscope and a double-view microscope system. GFP-RacF1 was
homogeneously distributed in the cytosol and accumulated at the plasma
membrane, especially at regions of transient intercellular contacts.
GFP-RacF1 also localized transiently to macropinosomes and phagocytic
cups and was gradually released within <1 min after formation of the
endocytic vesicle or the phagosome, respectively. On stimulation with
cAMP, no enrichment of GFP-RacF1 was observed in leading fronts, from
which it was found to be initially excluded. Cell lines were obtained
using homologous recombination that expressed a truncated
racF1 gene lacking sequences encoding the
carboxyl-terminal region responsible for membrane targeting. These
cells displayed normal phagocytosis, endocytosis, and exocytosis rates.
Our results suggest that RacF1 associates with dynamic structures that
are formed during pinocytosis and phagocytosis. Although RacF1 appears
not to be essential, it might act in concert and/or share functions
with other members of the Rho family in the regulation of a subset of
cytoskeletal rearrangements that are required for these processes.
Max-Planck-Institut für Biochemie,
82152 Martinsried, Germany; and Dipartimento di Scienze
Cliniche e Biologiche, Ospedale S. Luigi Gonzaga, 10043 Orbassano-Torino, Italy
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