Lipid-Dependent Bimodal MCL1 Membrane Activity

Abstract

Increasing evidence indicates that the mitochondrial lipid membrane environment directly modulates the BCL2 family protein function, but the underlying mechanisms are still poorly understood. Here, we used minimalistic reconstituted systems to examine the influence of mitochondrial lipids on MCL1 activity and conformation. Site-directed mutagenesis and fluorescence spectroscopic analyses revealed that the BCL2 homology region of MCL1 (MCL1ΔNΔC) inhibits permeabilization of MOM-like membranes exclusively via canonical BH3-into-groove interactions with both cBID-like activators and BAX-like effectors. Contrary to currently popular models, MCL1ΔNΔC did not require becoming embedded into the membrane to inhibit membrane permeabilization, and interaction with cBID was more productive for MCL1ΔNΔC inhibitory activity than interaction with BAX. We also report that membranes rich in cardiolipin (CL), but not phosphatidylinositol (PI), trigger a profound conformational change in MCL1ΔNΔC leading to membrane integration and unleashment of an intrinsic lipidic pore-forming activity of the molecule. Cholesterol (CHOL) reduces both the conformational change and the lipidic pore-forming activity of MCL1ΔNΔC in CL-rich membranes, but it does not affect the interaction of MCL1ΔNΔC with proapoptotic partners in MOM-like liposomes. In addition, we identified MCL1α5 as the minimal domain of the protein responsible for its membrane-permeabilizing function both in model membranes and at the mitochondrial level. Our results provide novel mechanistic insight into MCL1 function in the context of a membrane milieu and add significantly to a growing body of evidence supporting an active role of mitochondrial membrane lipids in BCL2 protein function.

Minimalist Model Systems Reveal Similarities and Differences between Membrane Interaction Modes of MCL1 and BAK

“…Recombinant mouse MCL1 lacking the N-terminal 151 amino acids and the C-terminal 23 amino acids (MCL1⌬N151⌬C23) and its S177C and R244E mutants, recombinant human BAK lacking the carboxyl-terminal 21 amino acids (BAK⌬C21) and its C166S and R127E mutants, and recombinant caspase-8-cleaved mouse BID (cBID) and its C30S or C30S/D95A mutants were all expressed and purified as described previously (20,21). Mutations were carried out by site-directed mutagenesis using the following primers: MCL1 S177C, 5Ј-GCTCCAAGGACTGCAAGCCTCTGGGCGA-3Ј (forward) and 5Ј-TCGCCCAGAGGCTTGCAGTCCTTG-GAGC-3Ј (reverse); BID C30S, 5Ј-CTCCAAAGCTCTG-GCAGTACTCGCCAAGAGC-3Ј (forward) and 5Ј-GCTCT-TGGCGAGTACTGCCAGAGCTTTGGAG-3Ј (reverse).…”

The Incomplete Puzzle of the BCL2 Proteins

“…In addition, retrotranslocation or inhibition MODE 0 postulates that BCL2-type proteins inhibit apoptosis by keeping BAX-type proteins inactive through continuous retrotranslocation from the mitochondrial surface into the cytosol [15,16,17,18,19]. These models, however, do not consider an enigmatic property shared by all BCL2-type proteins, which is their ability to promote, rather than inhibit, apoptosis under specific conditions (PRODEATH MODE) [15,20,21,22]. The complex interaction network that orchestrates these proteins’ actions is commonly termed the BCL2 interactome, which constitutes an intricate puzzle yet unresolved (Figure 1).…”

“…Concerning the antiapoptotic members of the BCL2 family, membrane lipid composition can enhance their binding affinity for the proapoptotic members [24,75] or ablate their inhibition capacity and release a hidden pore forming activity [15,20,21,76]. Although the transition of antiapoptotic BCL2 members to pro-death molecules remains poorly understood, the therapeutic potential of this phenotypic reversion should not be neglected, given that their overexpression is key in promoting resistance to chemotherapy.…”

“…The membrane permeabilizing activity of BCL2-type proteins has similarities and differences to that of BAX-type proteins. Structurally, the pores formed by BCL2-type proteins are smaller, and do not require canonical BH3:groove interactions for oligomerization and pore opening [15,20,77]. Similarly to BAX-type proteins, the amphipathic alpha helix 5 of some antiapoptotic member has been reported to mediate their membrane-permeabilizing function [15,20].…”

Mcl-1 and Bcl-xL sequestration of Bak confers differential resistance to BH3-only proteins

“…In mixtures of Bid or Bim, Bcl-x L or Mcl-1, and Bak or Bax, most resistance occurred via MODE 1 (refs. [21,22]). However, MODE 2 was enhanced when mutation decreased the affinity of BH3-only protein for prosurvival proteins [13,21].…”