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Fig. 2. Differential involvement of 9 reveals that the early and late phases of Bax motion are distinct. (A) Coexpression of EYFP-BaxC and ECFP-Bax shows that 9 deletion prevents the early redistribution phase detected at the level of a single mitochondrion. Arrow points to a single mitochondrion. Scale bars: 1 µm. (B) Enforced dimerization of CopGFP-Bax provokes early mitochondrial relocalization without directly causing CCR. Deleting 9 (CopGreen-BaxC) prevents this relocalization. Note the appearance of inclusion bodies that are clearly distinguishable from mitochondria. Cc, cytochrome c. Scale bars: 10 µm. (C) 9 constitutively targets EGFP to the MOM in live yeast cells (EGFP-9). Mitochondria are labeled with DiOC₆. Scale bars: 1 µm. (D) Effect of 9 deletion or 9 immobilization by cyclization (cycloBax) (supplementary material Fig. S1) on the proapoptotic potency of Bax. Loss of mitochondrial membrane potential (low) was used as a late apoptosis reporter (Goldstein et al., 2000). ^**Statistically significant P<0.001 (n=5), t-test. (E) After STS challenge (1 µM, 5 hours), EGFP-BaxC bypasses the early relocalization phase and directly undergoes the late one (compare with t-HcRed–Bax in the two upper rows). Direct late relocalization of EGFP-BaxC is observed in the absence of full-length Bax (HCT116 Bax^–/– cells, lower row); cycloBax (revealed by anti-myc) (supplementary material Fig. S1) behaves similarly to EGFP-BaxC. Scale bars: 10 µm. (F) First updated model of Bax relocalization: the early and late relocalization phases are disconnected; CCR is not associated with the early phase.

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