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J Biol Chem. 2016 Sep 23;291(39):20329-44. doi: 10.1074/jbc.M116.724005. Epub 2016 Aug 5.

A Targeted Mutation Identified through pKa Measurements Indicates a Postrecruitment Role for Fis1 in Yeast Mitochondrial Fission.

Author information

1
From the Department of Biology and.
2
the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and.
3
From the Department of Biology and the Program in Molecular Biophysics, Johns Hopkins University, Baltimore, Maryland 21218.
4
the Department of Pathology, Baylor College of Medicine, Houston, Texas 77030.
5
the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and rbhill@mcw.edu.

Abstract

The tail-anchored protein Fis1 is implicated as a passive tether in yeast mitochondrial fission. We probed the functional role of Fis1 Glu-78, whose elevated side chain pKa suggests participation in protein interactions. Fis1 binds partners Mdv1 or Dnm1 tightly, but mutation E78A weakens Fis1 interaction with Mdv1, alters mitochondrial morphology, and abolishes fission in a growth assay. In fis1Δ rescue experiments, Fis1-E78A causes a novel localization pattern in which Dnm1 uniformly coats the mitochondria. By contrast, Fis1-E78A at lower expression levels recruits Dnm1 into mitochondrial punctate structures but fails to support normal fission. Thus, Fis1 makes multiple interactions that support Dnm1 puncta formation and may be essential after this step, supporting a revised model for assembly of the mitochondrial fission machinery. The insights gained by mutating a residue with a perturbed pKa suggest that side chain pKa values inferred from routine NMR sample pH optimization could provide useful leads for functional investigations.

KEYWORDS:

confocal microscopy; macromolecular machine; mitochondria; mitochondrial dynamics; nuclear magnetic resonance (NMR); pKa measurements; protein assembly; protein-protein interaction; structural model; tetratricopeptide repeat (TPR) domains

PMID:
27496949
PMCID:
PMC5034033
DOI:
10.1074/jbc.M116.724005
[Indexed for MEDLINE]
Free PMC Article

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