The coiled-coil domains of the putative yeast cargo receptors Emp46p and Emp47p are responsible for their complex-formation in the Endoplasmic Reticulum. In vitro experiments using coiled-coil domains (Emp46pcc/47pcc) have indicated that formation of the hetero-complex is pH-dependent and that amino acid Glu303 of Emp46pcc is a key residue in this process. In this study, we investigated the effects of various mutations on complex formation and discovered the mechanism for its pH-dependency, which is that dissociation of the complex at low pH arises mainly from stabilization of Emp46pcc itself. Moreover, destabilization by the introduction of a histidine residue in Emp46pcc to repel a lysine residue in Emp47pcc, caused an upward shift in the pH profile of complex formation. Another mutation in Emp46pcc, a proline to an alanine (P291A), increased the stability of the helical structure, especially at low pH and shifted the transition pH upward. Combination of these pH-shifting mutations had an additive effect on the pH profile of complex formation. Thus, we successfully constructed coiled-coils that can react to a wide range of pH, encompassing more appropriate values for use in sensing physiological pH changes in the cell.

• PMID: 29361014
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Journal Article

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Kato K, Furuhashi T, Kato K, Oda A, Kurimoto E

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