Response to Yang-Yen: Does N-terminal Processing of Mcl-1 Occur at Mitochondrial Outer Membrane or Matrix?

This is a response to a letter by Yang-Yen (1)

As Hsin-Fang Yang-Yen points out, processing of Mcl-1 to Mcl-1ΔN depends on the electrochemical potential (Δψ) across the mitochondrial inner membrane. Despite the fact that Mcl-1 is an outer membrane protein, targeted and inserted into this location via its transmembrane segment, the Δψ requirement suggests the possibility of import to the inner membrane or matrix. Nevertheless, as we discussed in our article other possibilities exist to explain the requirement for Δψ. Uniquely, Mcl-1 has a long flexible N-terminal region (2), which could potentially gain (reversible) Δψ-dependent access to processing machinery inside the organelle while anchored via its transmembrane (TM) at the outer membrane. Another possibility is that Δψ at the inner membrane influences events at the outer membrane. For example, loss of Δψ converts Pink1 from a fate of degradation at the inner membrane to a location at the outer membrane, where it can influence mitophagy (3). Given these possibilities, we elected to rigorously examine the mitochondrial location(s) of Mcl-1 and Mcl-1ΔN utilizing multiple approaches and focusing on the endogenous proteins as opposed to ectopically expressed mutants and constructs. The results consistently indicated that both Mcl-1 and Mcl-1ΔN are located in the outer membrane.

In the individual experiments referred to by Hsin-Fang Yang-Yen, they were each conducted on the same cell culture samples and cell extracts within the same experiment. However, because we were measuring small changes in the migration behavior of the test proteins, gel systems needed to be optimized for each protein. Thus, for example, Mcl-1 isoforms were resolved on one gel system whereas Bak and modified Bak were analyzed on a different system. Had they been analyzed on the same gel, erroneous interpretations would have resulted.