Each of three positively-charged amino acids in the C-terminal region of yeast mitochondrial ATP synthase subunit 8 is required for assembly
| Autor: | Maria Galanis, Theo Papakonstantinou, Phillip Nagley, Rodney J. Devenish |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Recombinant Fusion Proteins
Protein subunit Molecular Sequence Data Saccharomyces cerevisiae Biophysics Protein Sorting Signals Mitochondrion Biochemistry Structure-Activity Relationship Allotopic expression Site-directed mutagenesis chemistry.chemical_classification Base Sequence biology ATP synthase Biological Transport Cell Biology biology.organism_classification Mitochondria Amino acid Proton-Translocating ATPases chemistry Mutagenesis Site-Directed biology.protein Isoleucine |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1144:22-32 |
| ISSN: | 0005-2728 |
| DOI: | 10.1016/0005-2728(93)90026-c |
| Popis: | Each of three conserved positively-charged residues in the C-terminal region of subunit 8 of yeast ( Saccharomyces cerevisiae ) mitochondrial ATP synthase was replaced with isoleucine. The assembly and functional properties of the resulting variants (substituted at Arg-37, Arg-42 and Lys-47) were examined using in-vitro systems to assay import into isolated mitochondria and to monitor assembly into ATP synthase, as well as an in-vivo rescue system using host yeast cells lacking endogenous subunit 8. Each such variant was found to be impaired in assembly in vitro, after import in the form of a chimaeric protein bearing a leader sequence with mitochondrial targeting function. Import precursors bearing a duplicated-leader sequence, engendering enhanced delivery to mitochondria of the passenger variant subunit-8 proteins, enabled assembly of the (Lys-47 → Ile) variant to be detected in vitro but not that of (Arg-37 → Ile) or (Arg-42 → Ile) variants. The respiratory growth of subunit 8-deficient host cells could be rescued with the (Lys-47 → Ile) variant expressed allotopically in the nucleus. Such rescued cells were found to have an enhanced growth rate (comparable to that produced by non-mutagenized parental subunit 8) when delivered to mitochondria with the duplicated-leader sequence, as compared to the single-leader sequence. This confirms that the impediment in the (Lys-47 → Ile) variant lies in the efficiency of its assembly, rather than a functional defect, as such, arising from the loss of that positive charge. In contrast, host cells were unable to be rescued by the (Arg-37 → Ile) and (Arg-42 → Ile) variants, even when they were endowed with the duplicated leader sequence. It is concluded that the positively-charged C-terminal domain of subunit 8, common to fungal and mammalian homologues of this protein, plays a key role in its assembly into mitochondrial ATP synthase. |
| Databáze: | OpenAIRE |
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