Topology and proximity relationships of yeast mitochondrial ATP synthase subunit 8 determined by unique introduced cysteine residues

We have used site-directed chemical labelling to demonstrate the membrane t opology and to identify neighbouring subunits of subunit 8 (Y8) in yeast mi tochondrial ATP synthase (mtATPase). Unique cysteine residues were introduc ed at the N or C-terminus of Y8 by site-directed mutagenesis. Expression an d targeting to mitochondria in vivo of each of these variants in a yeast Y8 null mutant was able to restore activity to an otherwise nonfunctional ATP synthase complex. The position of each introduced cysteine relative to the inner mitochondrial membrane was probed with thiol-specific nonpermeant an d permeant reagents in both intact and lysed mitochondria. The data indicat e that the N-terminus of Y8 is located in the intermembrane space of mitoch ondria whereas the C-terminus is located within the mitochondrial matrix. T he proximity of Y8 to other proteins of mtATPase was tested using heterobif unctional cross-linking reagents, each with one thiol-specific reactive gro up and one nonspecific, photoactivatible reactive group. These experiments revealed the proximity of the C-terminal domain of Y8 to subunits d and f, and that of the N-terminal domain to subunit f. It is concluded that Y8 pos sesses a single transmembrane domain which extends across the inner membran e of intact mitochondria. As subunit d is a likely component of the stator stalk of mitochondrial ATP synthase, we propose, on the basis of the observ ed cross-links, that Y8 may also be part of the stator stalk.