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Fig. 4 Intrinsic and GEF Rin1 catalyzed nucleotide exchange. (A)–(D) Nucleotide exchange rates of 200 nM Rab5c wt and Rab variants in the absence or presence of different concentrations of Rin1 (0–100 nM) as indicated at 20°C. The vertical grey line in (A), (C) and (D) corresponds to the length of the x-axis in (B). (E) Intrinsic nucleotide exchange rates of Rab5C wt and Rab5c variants at 30°C. (F) 1300 nM Rab5c D137N and subsequently 10 mM GPD were added to 200 nM mGDP at 20°C. (G). GTPγS-loaded Rab5c proteins, together with seven different unrelated reference baits (cb01 to cb07), were immobilized and used for precipitation of interacting proteins from HeLa cell lysates, in duplicate (49). The precipitation proteins were identified by mass spectroscopy. The summed peptide intensities of individual identified proteins were logarithmical-transformed to the basis of 10 and are represented by shades of gray. Note, intensities smaller than 104 are not obtained due to technical constraints. The 19 identified known Rab binding proteins (in bold, y-axis) and randomly selected ‘background’ proteins are shown.