Fig. 5: Choanoflagellate SPIRE interacts with RAB8.

a Multiple protein sequence alignment of the FYVE_2 domains of the indicated exocytic proteins. Blue coloring indicates amino acid conservation at specific positions ranging from high (dark blue) to low (light blue) conservation. The eight cysteines for Zn²⁺ ion binding and the hydrophobic turret loop are labelled in red and cyan, respectively. SPIRE protein sequences corresponding to the synaptotagmin-like protein (SLP) homology domains SHD1 and SHD2 are highlighted in light orange, sequences corresponding to the FYVE domain are highlighted in orange (consistent with the coloring in panels c–e). Large loop sequences within SPIRE and RIM1 proteins are hidden for clarity and shown in brackets. Numbering indicates amino acid residues. b Multiple protein sequence alignment of RAB8 proteins from Mus musculus (Mm), Monosiga brevicollis (Mb) and Salpingoeca rosetta (Sr). Coloring indicates amino acid conservation at specific positions ranging from high (dark blue) to low (light blue) amino acid conservation at specific positions. The highly conserved G motifs (G1 to G5) are indicated, as well as the residues forming switch 1 (green) and switch 2 (blue) regions. Numbering indicates amino acid residues. c Protein structure alignment of the predicted (ColabFold/AlphaFold2^66,67) FYVE_2 domains of Mb-SPIRE (orange) and Mm-SPIRE1 (grey). Indicated are the synaptotagmin-like protein homology domains (SHD1, SHD2), the FYVE-type zinc finger and the Zn²⁺ ion binding cysteines (C1-C8, labelled in red). d Predicted protein complex (ColabFold/AlphaFold-multimer^66,67,68) formed by Monosiga brevicollis Mb-SPIRE-FYVE_2 (orange) and Mb-RAB8 (grey). The switch 1 (green) and switch 2 (blue) interaction surfaces of RAB8 are indicated. The Zn²⁺ ion binding cysteines of Mb-SPIRE-FYVE_2 are colored in red. e Crystal structure of the protein complex formed by the melanophilin (MLPH, SlaC2-a) FYVE_2 domain and RAB27B (PDB-ID: 2ZET⁶⁹) is shown. SHD1, SHD2 and FYVE-type zinc finger of MLPH as well as switch 1 and switch 2 regions of RAB27B are indicated. f GST-pulldown assay with purified GTP-locked GST-Mb-RAB8-Q67L and GDP-locked GST-Mb-RAB8-T22N proteins, respectively, from lysates of HEK 293 cells transiently expressing C-terminal AcGFP1-tagged Mb-SPIRE (GFP-Mb-SPIRE-ΔKW, input). AcGFP1 (GFP) was used as negative control and Ponceau S staining shows equal amounts of GST-tagged proteins. N = 2 experimental repeats. g Localisation of transiently co-expressed tagged C-terminal Mb-SPIRE (AcGFP1; GFP-Mb-SPIRE-ΔKW; green) and Mb-RAB8 (mRuby3; Ruby3-Mb-RAB8; red) in human HeLa cells was analysed by fluorescence microscopy. AcGFP1 (GFP) and mRuby3 (Ruby3) expressing cells were used as controls. Deconvoluted images indicate the localisation of the proteins on vesicular structures. Scale bars represent 5 µm. At least six cells from two distinct experiments were imaged for each condition and the cytoplasmic region of one representative cell is presented here.