Assembly of imported subunit 8 into the ATP synthase complex of isolated yeast mitochondria

Abstract

This study concerns the assembly into a multisubunit enzyme complex of a small hydrophobic protein imported into isolated mitochondria. Subunit 8 of yeast mitochondrial ATPase (normally a mitochondrial gene product) was expressed in vitro as a chimaeric precursor N9L/Y 8-1, which includes an N-terminal-cleavable transit peptide to direct its import into mitochondria. Assembly into the enzyme complex of the imported subunit 8 was monitored by immunoadsorption using an immobilized anti-F l-p monoclonal antibody. Preliminary experiments showed that N9L/Y8-1 imported into normal rho' mitochondria, with its complement of fully assembled ATPase, did not lead to an appreciable assembly of the exogenous subunit 8. With the expectation that mitochondria previously depleted of subunit 8 could allow such assembly in vitro, target mitochondria were prepared from genetically modified yeast cells in which synthesis of subunit 8 was specifically blocked. Initially, mitochondria were prepared from strain M31, a mit-mutant completely incapable of intramitochondrial biosynthesis of subunit 8. These mit-mitochondria however were unsuitable for assembly studies because they could not import protein in vitro. A controlled depletion strategy was then evolved. An artificial nuclear gene encoding N9L/Y8-1 was brought under the control of a inducible promoter GALI. This regulated gene construct, in a low copy number yeast expression vector, was introduced into strain M31 to generate strain YGL-1. Galactose control of the expression of N9L/Y8-1 was demonstrated by the ability of strain YGL-1 to grow vigorously on galactose as a carbon source, and by the inability to utilize ethanol alone for prolonged periods of growth. The measurement of bioenergetic parameters in mitochondria from YGL-1 cells experimentally depleted of subunit 8, by transferring growing cells from galactose to ethanol, was consistent with the presence in mitochondria of a mosaic of ATPase, namely fully assembled functional ATPase complexes and partially assembled complexes with defective Fo sectors. These mitochondria demonstrated very efficient import of N9L/Y8-1 and readily incorporated the imported processed subunit 8 protein into ATPase. Comparison of the kinetics of import and assembly of subunit 8 showed that assembly was noticeably delayed with respect to import. These findings open the way to a new systematic analysis of the assembly of imported proteins into multisubunit mitochondrial enzyme complexes.A major question in bioenergetics concerns the mechanism of assembly of multisubunit enzyme complexes in energytransducing membranes [l -31. A key enzyme that functions in a range of biological contexts, including bacteria, chloroplasts and mitochondria, is the proton-translocating ATP synthase complex [4, 51. These H+-ATPases typically have a membrane Fo sector with proton-conducting activity that is functionally linked to the ATP-synthesizing activity located on the soluble Fl sector. In the yeast, Succharomyces cerevisiue, the mitochondrial proton-translocatin...

The C‐terminal positively charged region of subunit 8 of yeast mitochondrial ATP synthase is required for efficient assembly of this subunit into the membrane F0sector

“…In general, cells were grown in a supplemented medium containing 2% ethanol as carbon source (medium E) [13]. Strain YGL-1 was grown on medium F [13] containing 2% ethanol and 0.5% galactose.…”

“…Strain YGL-1 was grown on medium F [13] containing 2% ethanol and 0.5% galactose. For some experiments, in order to reduce the time of culturing, cells of strain T475 were grown on medium E with additional glucose (0.5%) and harvested during the catabolite-derepressed late logarithmic phase of growth.…”

“…The assembly assays were performed with the anti-(F, a subunit) monoclonal antibody RH51 (which is efficient in immunoprecipitation of the entire assembled mtATPase complex) conjugated to Sepharose 4B beads. The RH51 antibody was initially classified as recognizing F1 p subunit [13] but was later shown to be specific for F1 c1 subunit (D. G. Grasso, unpublished results). Various mitochondrial fractions were separated during assembly assays according to the following fractionation scheme.…”

“…Mitochondria from the remaining 80 p1 were washed twice as above except that 500 p1 buffer A was used in the first wash. The mitochondrial pellet was solubilized in 40 p1 cholate extraction buffer [13] and, after incubation on ice for 30 min, this mixture was centrifuged at 105000 x g for 30 min. The pellet was resuspended directly in 80 p1 buffer S (fraction 5).…”

“…Expression of the chimaeric precursors in pLFl is under control of the PGKl promoter (essentially constitutive); in pED121, expression is depenent on the GAL1 promoter and is thus induced by the presence of galactose of imported components such as allotopically expressed Y8 1131. Target mitochondria that had been partially depleted of endogenous Y8 by deliberately regulated allotopic expression in vivo [13], when incubated with radiolabelled N9L/Y8-1, were able to assemble imported Y8, as judged by its immunoadsorption to an anti-(F, p subunit) monoclonal antibody [14]. Such in vitro assembly assays exemplify the analysis of mtATPase assembly in isolated mitochondria prepared from yeast cells subjected to various genetic or physiological manipulations.…”