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Fig. 4 The conserved “Y[R/K]” motif in Rab GTPase switch I is required for their activation by TLD RabGEFs.a 2 μM Mant-GDP-loaded GST-tagged RAB38 was added, and nucleotide exchange was triggered by adding 200 μM GDP and 0, 0.125, or 0.25 μM GEF complex. The decrease of fluorescence was measured over time and normalized to fluorescence prior to GEF addition. Error bars represent the standard error of the mean (SEM) from three technical replicates. n = 3. b Nucleotide exchange rates of HPS1-HPS4FL and HPS1-HPS4Δloop are plotted against the concentration of HPS1-HPS4, and the catalytic efficiency (kcat/KM) in M−1s−1 was determined as the slope of the linear fit to the equation y = A*x + B. Error bars represent the SEM of three independent biological repeats. The catalytic efficiency (kcat/KM) of HPS1-HPS4FL and HPS1-HPS4Δloop is shown. n = 3. c Identification of the putative RAB32/38 binding site in BLOC-3. Model of the respective LD1 domain of HPS1 (purple) and HPS4 (pink), and RAB38 (green) complex based on an AlphaFold2 prediction. (d A zoom-in view of the HPS1-HPS4-RAB38 binding site superposed with the crystal structure of RAB38-GTP (magenta, PDB: 6hdu). HPS1 and HPS4 are shown in hydrophobic surface potential, and Y38 and R39 of RAB38 are shown in sticks. The predicted model is colored as in (c). e, f Nucleotide exchange rates of RAB38 and RAB32 are plotted against the concentration of HPS1-HPS4, and the catalytic efficiency (kcat/KM) in M−1s−1 was determined as the slope of the linear fit to the equation y = A*x + B. Error bars represent the SEM of three independent biological repeats. The catalytic efficiency (kcat/KM) of HPS1-HPS4Δloop towards RAB32, RAB38, and indicated mutants is shown. N.d., not determined. n = 3. g Sequence alignment of the switch I regions of Rab GTPases. The Y[R/K] motif is highlighted in red, with * indicating highly conserved residues and: representing residues that are moderately conserved.