Subunit 8 of Yeast Mitochondrial ATP Synthase: Biochemical Genetics and Membrane Assembly

Nagley, Phillip; Devenish, Rodney J.; Law, Ruby H. P.; Maxwell, Ronald J.; Nero, Debra; Linnane, Anthony W.

doi:10.1007/978-1-4684-5835-0_30

Cited by 8 publications

(7 citation statements)

References 47 publications

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“…The impaired oxidative metabolism of strain T10-1 is indicated by its slow respirationdependent growth rate (comparable to that of T475) and by the nature of its mitochondrial bioenergetic profile (Table 2). From immunochemical data on assembly in vivo in strain T10-1 [6] and by formal genetic criteria discussed above (data not shown), the partiaI rescue of the aapl mit-host is demonstrably dependent upon the nuclearly expressed and imported…”

Section: The In Vitro Assembly Assay Detects Assembly ?Fafurther Y8 Dmentioning

confidence: 99%

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

Grasso¹,

Nero²,

Law³

et al. 1991

European Journal of Biochemistry

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This paper deals with a truncated derivative of subunit 8 of yeast mitochondrial ATP synthase in which a conserved positively charged residue (Lys47) has been removed by site-directed mutagenesis together with the Cterminal residue (Leu48). This derivative has been expressed as a chimaeric precursor N9L/Y8-1 (K47-STP) carrying an N-terminal cleavable leader sequence (N9L), fused by a short bridging sequence to the truncated subunit-8 passenger protein. Allotopic expression of N9L/YS-l(K47-STP) in vivo in an aapl mit-host yeast strain lacking endogenous subunit 8 leads to partial restoration of bioenergetic function in the transformant strain denoted T475. Import and assembly studies were carried out in vitro using target mitochondria from strain YGL-1 partially depleted in subunit 8 ; such controlled depletion has been previously shown to be required for the efficient assembly (monitored immunochemically) of full-length subunit 8 imported in vitro as the precursor N9L/Y8-1. It was found that N9L/YS-l(K47-STP) synthesized in vitro was imported successfully into YGL-1 mitochondria, but no significant assembly of the truncated subunit 8 was observed in these or any other mitochondria tested. The bioenergetic defects in T475 mitochondria are ascribed to the impaired assembly of the subunit-8 variant in vivo, resulting from the truncation at Lys47. In consequence, T475 mitochondria behave as though partially depleted of subunit 8. This conclusion was supported by the ability of isolated T475 mitochondria to provide a vehicle for the efficient import and assembly of subunit 8 processed form full-length N9L/Y8-1. Two related aspects of import and assembly have been addressed as part of the analysis of truncated subunit 8. First, mitochondria from strain T2-1, an aapl mit-mutant genetically reconstituted by allotopic expression of N9L/ Y8-1, were also found to be effective in the in vitro assembly of subunit 8 derived from imported N9L/Y8-1. This suggests an intramitochondrial shortage of subunit 8 delivered by allotopic expression of N9L/Y8-1 in vivo, which may underlie the incomplete restoration of energy coupling in T2-1 mitochondria compared to those of wild-type yeast. Second, on allotopic expression of N9L/Y8-2 (containing subunit 8 directly fused to N9L) in the aapl mithost, a rescued transformant strain T10-1 was generated which displays bioenergetic defects superficially similar to those of T475. Processed subunit 8 clearly assembled into the ATP synthase of isolated YGL-1 mitochondria, in spite of the relatively weak import of N9L/Y8-2 in vitro. This demonstrates the ability of the in vitro assembly system to distinguish assembly properties of variants of subunit 8, such as N9L/YS-l(K47-STP) and N9LIY8-2.The Fa sector of the yeast mitochondrial ATP synthase (mtATPase) provides a versatile system for the study of the assembly of integral membrane proteins into energy-transducing enzyme complexes [l]. Previous studies of mutants of Saccharomyces cerevisiae, affected in the three mitochondrial genes encoding Fa s...

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Section: The In Vitro Assembly Assay Detects Assembly ?Fafurther Y8 Dmentioning

confidence: 99%

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

Grasso¹,

Nero²,

Law³

et al. 1991

European Journal of Biochemistry

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“…Whereas divergent in amino acid composition, Y8 and its other eukaryotic homologs exhibit three highly conserved regions: an N-terminal MPQL motif, a central hydrophobic domain (CHD), and a C-terminal positively charged region (21)(22)(23). Allotopic expression (24), whereby a mitochondrial gene is recoded for nuclear expression with subsequent delivery of the protein back to mitochondria, has enabled our laboratory to undertake a detailed molecular genetic approach to investigate the structure and function of Y8.…”

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The Molecular Neighborhood of Subunit 8 of Yeast Mitochondrial F1F0-ATP Synthase Probed by Cysteine Scanning Mutagenesis and Chemical Modification

Stephens

Khan

Roucou³

et al. 2003

Journal of Biological Chemistry

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“…Yeast subunit 8 (Y8) is a 48‐amino acid polypeptide essential for the assembly and function of the F 0 sector of yeast mitochondrial F 1 F 0 ‐ATP synthase (mtATPase) [7]. Hydropathy plots have suggested that the central hydrophobic domain (CHD) of Y8 constitutes a unique transmembrane domain [10]. The ability of Y8 to tolerate the presence of charged amino acids within this domain, however, is well documented [11–13].…”

mentioning

confidence: 99%

“…This result unequivocally demonstrates the transmembrane nature of Y8. The requirement of Y8 for both enzyme assembly and function [10] also suggests that this protein is in close proximity to other F 0 subunits of the intact enzyme complex. We show here that the C‐terminus of Y8, located in the matrix compartment, is in close proximity to a putative component of the stator stalk, subunit d, and to subunit f. The N‐terminus of Y8, located in the intermembrane space, is in close proximity to subunit f also.…”

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

Stephens

Roucou

Artika

et al. 2000

European Journal of Biochemistry

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We have used site-directed chemical labelling to demonstrate the membrane topology and to identify neighbouring subunits of subunit 8 (Y8) in yeast mitochondrial ATP synthase (mtATPase). Unique cysteine residues were introduced at the N or C-terminus of Y8 by site-directed mutagenesis. Expression and 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 and permeant reagents in both intact and lysed mitochondria. The data indicate that the N-terminus of Y8 is located in the intermembrane space of mitochondria whereas the C-terminus is located within the mitochondrial matrix. The proximity of Y8 to other proteins of mtATPase was tested using heterobifunctional cross-linking reagents, each with one thiol-specific reactive group 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 possesses a single transmembrane domain which extends across the inner membrane 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 observed cross-links, that Y8 may also be part of the stator stalk.

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Subunit 8 of Yeast Mitochondrial ATP Synthase: Biochemical Genetics and Membrane Assembly

Cited by 8 publications

References 47 publications

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

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

The Molecular Neighborhood of Subunit 8 of Yeast Mitochondrial F1F0-ATP Synthase Probed by Cysteine Scanning Mutagenesis and Chemical Modification

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

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Subunit 8 of Yeast Mitochondrial ATP Synthase: Biochemical Genetics and Membrane Assembly

Cited by 8 publications

References 47 publications

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

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

The Molecular Neighborhood of Subunit 8 of Yeast Mitochondrial F1F0-ATP Synthase Probed by Cysteine Scanning Mutagenesis and Chemical Modification

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

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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

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