TY - JOUR
T1 - Relationship of Subunit 8 of Yeast ATP Synthase and the Inner Mitochondrial Membrane
T2 - Subunit 8 Variants Containing Multiple Lysine Residues in the Central Hydrophobic Domain Retain Function
AU - Papakonstantinou, Theo
AU - Law, Ruby H.P.
AU - Manon, Stephen
AU - Devenish, Rodney J.
AU - Nagley, Phillip
PY - 1995/1/1
Y1 - 1995/1/1
N2 - 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.
AB - 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.
KW - allotopic expression
KW - Mitochondrial ATP synthase
KW - proteolipid
KW - site‐directed mutagenesis
KW - yeast
UR - http://www.scopus.com/inward/record.url?scp=0028801804&partnerID=8YFLogxK
U2 - 10.1111/j.1432-1033.1995.tb20197.x
DO - 10.1111/j.1432-1033.1995.tb20197.x
M3 - Article
C2 - 7867634
AN - SCOPUS:0028801804
SN - 0014-2956
VL - 227
SP - 745
EP - 752
JO - European Journal of Biochemistry
JF - European Journal of Biochemistry
IS - 3
ER -