Response to Yang-Yen: Does N-terminal Processing of Mcl-1 Occur at Mitochondrial Outer Membrane or Matrix?
Bibliographic record
Abstract
Does N-terminal Processing of Mcl-1 Occur at Mitochondrial Outer Membrane or Matrix?Journal of Biological ChemistryVol. 286Issue 30PreviewWarr et al. (1) concluded in their paper that the Mcl-1ΔN isoform resides in the mitochondrial outer membrane. The major problem with this conclusion is that the controls for all three experiments attempting to address this issue (Fig. 2) were not handled properly, i.e. all controls for assigning the submitochondrial location of Mcl-1ΔN clearly were done in a separate experiment. For example, in Fig. 2A, Western blots for Tom20 and cytochrome c were not from the same samples analyzed for Mcl-1; in Fig. Full-Text PDF Open Access This is a response to a letter by Yang-Yen (1Yang-Yen H.-F. J. Biol. Chem. 2011; (http://www.jbc.org/cgi/content/full/286/30/le15)PubMed Google Scholar) 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 (2Liu Q. Gehring K. J. Biol. Chem. 2010; 285: 41202-41210Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), 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 (3Jin S.M. Lazarou M. Wang C. Kane L.A. Narendra D.P. Youle R.J. J. Cell Biol. 2010; 191: 933-942Crossref PubMed Scopus (908) Google Scholar). 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.005 | 0.018 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.002 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.003 | 0.003 |
| Scholarly communication | 0.003 | 0.003 |
| Open science | 0.002 | 0.002 |
| Research integrity | 0.026 | 0.036 |
| Insufficient payload (model declined to judge) | 0.011 | 0.011 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".