ABSTRACT
RacE is a small GTPase required for cytokinesis in Dictyostelium discoideum. To investigate RacE's potential binding and signaling interfaces that allow its function in cytokinesis, 10 different chimeras were created between RacE and the closely related small GTPase, RacC. RacE/RacC chimeras, containing various combinations of four RacE regions, E I-IV: E-I (aa 1-67), E-II (aa 68-124), E-III (aa 125-184), and E-IV (aa 185-223), were tested for their ability to rescue the multinucleated, cytokinesis-defective phenotype of RacE null cells grown in suspension. Regions E-II and E-IV were essential but not sufficient for the rescue of RacE null cells. These two regions, in combination with either region E-1 or E-III, resulted in rescue. Results presented here suggest that region E-II contains a crucial, yet incomplete, binding site. Regions E-I or E-III separately provide additional, necessary elements for RacE's function. The extended E tail of RacE (E-IV) may act as a 'sensor' of the bound nucleotide state of RacE and facilitate GDP to GTP exchange (possibly through interactions with a GEF molecule), thereby resulting in activation of RacE. This study provides new evidence for small GTPases engaging several distinct protein interfaces to mediate signaling in various cellular processes.
Subject(s)
Cell Division/physiology , Dictyostelium/physiology , Monomeric GTP-Binding Proteins/physiology , Amino Acid Sequence , Animals , Dictyostelium/cytology , Dictyostelium/genetics , Gene Deletion , Molecular Sequence Data , Monomeric GTP-Binding Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction/physiologyABSTRACT
Restriction enzyme-mediated integration (REMI) has been used to study a number of cellular and developmental processes in Dictyostelium discoideum. In this paper we review the basics of this powerful method of introducing random mutations in Dictyostelium. Here we discuss several mutation screens that have been devised and some of the genes that have been discovered through this approach to mutagenesis. Included in this discussion is how one goes about isolating a gene that has been disrupted by REMI, and how one confirms that this disruption is actually responsible for the observed phenotype. Finally, we describe how REMI can be used as an effective teaching tool in undergraduate cell biology laboratory courses.
