(A) NF1 and related proteins have a characteristic domain organisation. The RasGAP domain and adjacent CRAL/TRIO and PH-like domains can be used to identify NF1-like proteins, although the PH-like domain is divergent. Approximate locations of mutations identified in axenic mutants are indicated with arrows; these are described precisely in Table 1. (B) The D. discoideum (Dd) NF1 sequence shows homology to the Homo sapiens protein along its entire length: the sequence of the Hs protein was split into segments with a sliding window of 200 amino acids, and these globally aligned to the Dd, Takifugu rubripes, and Drosophila melanogaster NF1 orthologues, and the Saccharomyces cerevisiae Ira1p sequence. Dashed lines mark the outermost windows containing parts of the central domains. (C) NF1 protein sequences from Takifugu rubripes, Drosophila melanogaster, Trichinella spiralis, Trichoplax adhaerens, Salpingoeca rosetta, Capsaspora owczarzaki, Mortierella verticillata, Saccharomyces cerevisiae (Ira1p), Dd, and Naegleria gruberi (EFC40840.1) were globally aligned with the Homo sapiens NF1 sequence. The bars display the percentage similarity and identity of the protein to the human sequence. (D) Phylogram of NF1 and MNF homologues; the Dictyostelium AxeB protein is an NF1 homologue, while homologues of NfaA form the MNF class of RasGAP, defined here. The presence of NF1 and MNF in Naegleria and Thecamonas as well as amoebozoans indicates that MNF was ancestral and then lost in a common ancestor of the Holozoa and Holomycota after the divergence of apusozoans. The scale shows substitutions/site. See Figure 2—figure supplement 1 for a version with all species labelled, and also Figure 2—figure supplements 2 and Figure 2—source data 1 for illustration of the wider pattern of conservation of RasGAPs.