A molecular genetic approach has been used to test the proposition that the central hydrophobic domain of yeast mitochondrial ATP synthase subunit 8 represents a transmembrane stem in contact with the lipid bilayer. The rationale for this approach is the general inability of membrane bilayers to accomodate unshielded charged residues of polypeptide chains. Non-polar residues at several positions within the central hydrophobic domain of subunit 8 were replaced with the positively charged amino acid lysine. This was done in an attempt to disrupt subunit 8 function, and thereby determine the boundaries of the putative transmembrane stem. Each subunit 8 variant was allotopically expressed in vivo as a mitochondrial import precursor encoded by a nuclear gene. It was found that all variants, which included proteins carrying two lysines at various positions in the hydrophobic domain, exhibited the ability to restore growth of subunit-8-deficient cells on the non-fermentable substrate ethanol. This indicated that the function of none of these subunit 8 variants was severely compromised. There was also no detectable change in the proteolipid characteristics of subunit 8, as defined by the chloroform/methanol solubility properties of variant proteins extracted from membranes following import into isolated mitochondria. These data suggest that subunit 8 is located in a hydrophobic niche in the mitochondrial ATP synthase, probably in contact with other protein subunits of the complex. We conclude that the function of subunit 8 does not necessarily require it to be integrated within the inner mitochondrial membrane, in contact with the lipid bilayer. Our findings also suggest that hydropathy plots, indicating hydrophobic domains within polypeptides, cannot reliably be interpreted as transmembrane helices in the absence of independent evidence.

• PMID: 7867634
• DOI full text
• PubMed
• PubTator

Reference Type

Journal Article | Research Support, Non-U.S. Gov't

Authors

Papakonstantinou T, Law RH, Manon S, Devenish RJ, Nagley P

Primary Lit For

Additional Lit For

Review For