FIGURE 1:

Human Spindly interacts with and is modified by farnesyltransferase. (A) Results of mass spectrometric analysis of immunoprecipitates from mitotic HeLa cells with two affinity-purified antibodies raised against nonoverlapping N- and C-terminal epitopes of Spindly (amino acids 2–254 and 450–605). Sequence coverage and spectrum count for selected proteins are shown. (B) Sequence alignment of Spindly C-termini in eukaryotes showing widespread conservation of a putative farnesylation motif that deviates from the canonical CAAX box (Hougland et al., 2009). The amino acid number of the fifth residue from the C-terminus is indicated. The farnesyl cysteine is present in vertebrates (represented by Homo sapiens, Mus musculus, Gallus gallus, Xenopus laevis, Danio rerio). In the two invertebrate chordate groups, the motif is present in tunicates (Ciona intestinalis) but is absent in amphioxus (B. floridae). Of the two closest nonchordate invertebrate relatives of amphioxus, echinoderms (Strongylocentrotus purpuratus) have the motif but hemichordates do not (S. kowalevskii). We could not find the motif in insects (D. melanogaster), nematodes (C. elegans), or cnidaria (H. magnipapillata). However, the motif appears in the primitive multicellular animal Trichoplax adhaerens, the only extant member of Placozoa, as well as in choanoflagellates (Monosiga brevicollis, Salpingoeca spinifera), the closest unicellular relatives of multicellular animals. The motif is also present in the chytrid fungus Batrachochytrium dendrobatidis. (C) Schematic of experimental approach for the detection of farnesylated Spindly based on in vivo labeling with the unnatural farnesyl diphosphate analogue AGOH and AG-specific antibodies. (D) Immunoblots detecting endogenous and transgenic Spindly and corresponding AG signals in cell lysate input fractions (I) and fractions obtained after immunoprecipitation with anti-Myc tag antibody (Myc) or beads-only controls (Con).