Figure 7: Alex3 is a component of the protein complex responsible for mitochondrial transport.

(a–f) A GFP construct was used as control. (a–c) Co-immunoprecipitation of Alex3–GFP with Miro1myc, Miro2myc, KIF5Cmyc or Trak2myc results in the specific interaction of Alex3–GFP with Miro1myc, Miro2myc (a) and Trak2myc (c) but no interaction is detected between Alex3–GFP and KIF5Cmyc (b). (d) The interaction between Alex3–GFP and Miro1myc and Miro2myc is reduced in the presence of 2 mM of calcium. (e) When the EF-hand mutant form of Miro1myc is co-expressed with Alex3–GFP, the interaction between the two components is no longer sensitive to Ca²⁺ levels. (f) The association between Alex3–GFP and Trak2myc is also dependent on the presence of Ca²⁺, thereby indicating that the whole complex could be dismantled when Miro1/2 recognizes an increment in Ca²⁺ levels. (g) Pull-down assays using purified GST–Miro1 and GST–Miro1ΔEF proteins as bait and showing interaction with Alex3 (in brain lysates) and its dependence on Ca²⁺concentration (top panel). The lack of the EF-hand domain in Miro-1 protein abolishes Ca²⁺ dependence (bottom panel).