Biological characterization of Drosophila Rapgap1, a GTPase activating protein for Rap1

Abstract

The activity of Ras family proteins is modulated in vivo by the function of GTPase activating proteins, which increase their intrinsic rate of GTP hydrolysis. We have isolated cDNAs encoding a GAP for the Drosophila Rap1 GTPase. Drosophila Rapgap1 encodes an 850-amino acid protein with a central region that displays substantial sequence similarity to human RapGAP. This domain, when expressed in Escherichia coli, potently stimulates Rap1 GTPase activity in vitro. Unlike Rap1, which is ubiquitously expressed, Rapgap1 expression is highly restricted. Rapgap1 is expressed at high levels in the developing photoreceptor cells and in the optic lobe. Rapgap1 mRNA is also localized in the pole plasm in an oskar-dependent manner. Although mutations that completely abolish Rapgap1 function display no obvious phenotypic abnormalities, overexpression of Rapgap1 induces a rough eye phenotype that is exacerbated by reducing Rap1 gene dosage. Thus, Rapgap1 can function as a negative regulator of Rap1-mediated signaling in vivo.

Figure 1

Structure of the Rapgap1 gene and the protein. (A) Structure of the Rapgap1 gene. Exons are indicated as open boxes. The start codon (ATG) and the stop codon (TAA) are indicated. The region with sequence similarity to human RapGAP is ccrosshatched. (B) Amino acid sequence of Drosophila Rapgap1. Residues 181–640 of Drosophila Rapgap1 are aligned with residues 42–410 of human RapGAP (11). Black shading indicates identical residues. A 9-amino acid motif which is similar to a sequence in human RapGAP is underlined.

Figure 2

Interaction of Rapgap1 with Rap1 in vitro and in vivo. (A) Hydrolysis of GTP by GST–Rap1 in the presence of GST–Rapgap1. Control experiments measuring hydrolysis in the presence of GST alone or in the reaction buffer are shown. (B) Flies expressing three copies of the GMR–Rapgap1 transgene. (C) Flies heterozygous for a loss of function mutation in Rap1. (D) Flies carrying three copies of the GMR-Rapgap1 transgene that are also heterozygous for a loss-of-function mutation in Rap1.

Figure 3

Expression of Rapgap1 protein. (A) Cellular blastoderm embryo. (B) Germ-band extended embryo. (C) Germ-band retracted embryo; focused at the level of the peripheral nervous system. (D) Germ band retracted embryo; midgut primordia in focus. (E) Eye-imaginal disc from late third instar larva. (F) Eye-imaginal disc and optic lobe. The arrow in E and F indicates the morphogenetic furrow. Anterior is to the left in A–E. In F, ed refers to eye-imaginal disc and ol refers to the optic lobe.

Figure 4

Rapgap1 mRNA localization is dependent on posterior group genes. Rapgap1 RNA detected by in situ hybridization. Embryos collected from females of the following genotypes: (A) wild-type, (B) oskar^AK (four copies oskar), (C) oskar-bcd-3′UTR, (D) vasa^D1, (E) tudor^WC8, and (F) nanos^BN. Anterior is to the left.