Assembly of functional proton-translocating ATPase complex in yeast mitochondria with cytoplasmically synthesized subunit 8, a polypeptide normally encoded within the organelle

A mitochondrial gene from Saccharomyces cerevisiae encoding a hydrophobic membrane protein, subunit 8 of the F0/F1-type mitochondrial ATPase complex, has been functionally replaced by an artificial nuclear gene specifying an imported version of this protein. The experiments reported here utilized a...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1988-04, Vol.85 (7), p.2091-2095
Main Authors: Nagley, P, Farrell, L.B, Gearing, D.P, Nero, D, Meltzer, S, Devenish, R.J
Format: Article
Language:English
Subjects:
Adenosine triphosphatases
Amino acids
Andromeda Galaxy
Biochemistry
CYTOPLASMIC INHERITANCE
Fungal Proteins - metabolism
GENE
GENES
Genetic vectors
HEREDITE CYTOPLASMIQUE
HERENCIA CITOPLASMATICA
HIDROLASAS
HYDROLASE
HYDROLASES
Intracellular Membranes - metabolism
LEVADURA
LEVURE
Membrane Proteins - metabolism
Mitochondria
Mitochondria - enzymology
Neurospora crassa - genetics
Neurospora crassa - metabolism
PEPTIDE
PEPTIDES
PEPTIDOS
Plasmids
Protein Processing, Post-Translational
Protein Sorting Signals - metabolism
Proteins
Proton-Translocating ATPases - metabolism
Recombinant Fusion Proteins - metabolism
SACCHAROMYCES CEREVISIAE
Saccharomyces cerevisiae - enzymology
Saccharomyces cerevisiae - genetics
YEASTS
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Description
Summary:A mitochondrial gene from Saccharomyces cerevisiae encoding a hydrophobic membrane protein, subunit 8 of the F0/F1-type mitochondrial ATPase complex, has been functionally replaced by an artificial nuclear gene specifying an imported version of this protein. The experiments reported here utilized a multicopy expression vector (pLF1) that replicates in the nucleus of yeast cells and that carries an inserted DNA segment, specifying a precursor protein (N9/Y8) consisting of subunit 8 fused to an N-terminal cleavable transit peptide (the leader sequence from Neurospora crassa ATPase subunit 9). The successful incorporation of the imported subunit 8 into functional ATPase complexes after transformation with pLF1 expressing N9/Y8 was indicated by the efficient genetic complementation of respiratory growth defects of aap1 mit- mutants, which lack endogenous subunit 8. The reconstitution of ATPase function was confirmed by biochemical assays of ATPase performance in mitochondria and by immunochemical analyses that demonstrated the assembly of the cytoplasmically synthesized subunit 8 into the ATPase complex. Reconstitution of ATPase function required the cytoplasmically synthesized subunit to have a transit peptide. The strategy for importation and reconstitution developed for subunit 8 leads to a systematic approach to the directed manipulation of mitochondrially encoded membrane-associated proteins that has general implications for exploring membrane biogenesis mechanistically and evolutionarily.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.85.7.2091