10.7554/eLife.18675.006Figure 2—figure supplement 2. Interaction of Rab1, Rab35, Rab8, Rab10, Rab13 and Rab15 with Mical-cL.
(a) The ITC data nicely show the relatively weak affinity of Rab1 towards Mical-cL (KD = 5.2 µM) compared to Rab8 (KD = 233 nM), Rab10 (KD = 787 nM), Rab13 (KD = 93 nM) and Rab15 (KD = 33 nM). Rab35 (sometimes also referred to as Rab1c) has an intermediate affinity for Mical-cL (KD = 1.8 µM). Since preparative loading of Rab35 with GppNHp (see below) was not successful, the concentration of the Rab35 protein fraction in the active state was deduced from HPLC analysis (see below) and the ITC data was corrected accordingly. (b) Comparison of the nucleotide status of Rab8 and Rab35 by HPLC analysis (black trace – absorption at 254 nm, red trace – absorption at 280 nm). The lower lane shows the trace of a nucleotide mix consisting of guanosine, GMP, GDP, GppNHp and GTP and a small impurity of GppNH2 as a reference, the retention times (in minutes) for each nucleotide are shown above the peak and in the table on the right. Whereas Rab8 could be preparatively loaded with GppNHp and analysis showed approximately 94% bound GppNHp, Rab35 was only 82% GppNHp-bound and the trace shows a major impurity of GppNH2 (18%). For this reason, the Rab35 protein concentration used in the ITC data in (a) was corrected for this.
DOI: http://dx.doi.org/10.7554/eLife.18675.006