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.
Highlights
Mitochondrial fission is essential for organelle and cell viability [1], and altered fission contributes to a broad range of diseases, including neurodegeneration, diabetes, and cancer [2,3,4,5,6]
Two proteins necessary for mitochondrial fission are conserved in all species containing mitochondria [7]: the tail-anchored mitochondrial protein Fis1, which exposes a tetratricopeptide repeat (TPR)5 domain to the cytoplasm, and the large
Yeast mitochondrial fission has long been known to depend on Fis1 as a membrane-tethered anchor for recruiting Mdv1 and Dnm1 [8, 44, 46, 47], but the idea that Fis1 acts only as an anchor without further contribution to the mechanism of membrane scission has gained favor with the discovery of other natural anchors (49 –51) and with the demonstration that artificially tethering the C-terminal portion of Mdv1 to mitochondria can partially rescue morphology in yeast cells that lack Fis1, Mdv1, and Caf4 [13]
Summary
The pKa of Fis Glu-78 Is Significantly Perturbed—Protein interfaces are mediated by both polar and nonpolar amino acids, but the majority of buried surface area is nonpolar [21,22,23]. Direct side chain pKa measurements require side chain assignments [24], so we used the known sensitivity of amide backbone NMR chemical shifts to proximal titration events [25, 26] to infer apparent side chain pKa values in yeast Fis. We fit Fis backbone amide proton and nitrogen NMR chemical shift changes as a function of pH (Fig. 1) to the Henderson-Hasselbalch equation, focusing on acidic residues because these were well described in the pH range covered by our titration (Fig. 1D, supplemental Results, and supplemental Fig. 1).
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