Abstract
Subunits e and g of Saccharomyces cerevisiae ATP synthase are required to maintain ATP synthase dimeric forms. Mutants devoid of these subunits display anomalous mitochondrial morphologies. An expression system regulated by doxycycline was used to modulate the expression of the genes encoding the subunits e and g. A decrease in the amount of subunit e induces a decrease in the amount of subunit g, but a decrease in the amount of subunit g does not affect subunit e. The loss of subunit e or g leads to the loss of supramolecular structures of ATP synthase, which is fully reversible upon removal of doxycycline. In the absence of doxycycline, mitochondria present poorly defined cristae. In the presence of doxycycline, onion-like structures are formed after five generations. When doxycycline is removed after five generations, cristae are mainly observed. The data demonstrate that the inner structure of mitochondria depends upon the ability of ATP synthase to make supramolecular structures.
Highlights
F0F1-ATP synthase is a molecular rotary motor that is responsible for aerobic synthesis of ATP
The Decrease in the Amount of Subunits e and g Destabilizes the Supramolecular Structures of the Yeast ATP Synthase—To modulate the expression of the TIM11 and ATP20 genes that encode subunits e and g, respectively, we used an expression system in which transcription was driven by a hybrid tetOCYC11 promoter through the action of a tetR-VP16 activator [21]
We have previously reported that null mutants in either TIM11 or ATP20 genes converted into rhoϪ cells with high frequency [15]
Summary
Materials—Doxycycline was purchased from ICN Biochemicals Inc. All other reagents were of reagent grade quality. The integrative vectors were cleaved by NheI and inserted in the his 3 locus of the respective null mutants ⌬TIM11 and ⌬ATP20, This paper is available on line at http://www.jbc.org. Mitochondria were isolated from cells, grown with or without doxycycline, and harvested at an A550 nm of 2–3 corresponding to the logarithmic growth phase. They were prepared from protoplasts as described previously [25]. Ultrastructural Studies—Freezing and freeze substitution of yeast cell pellets were performed as described previously [15]. Grids were examined at 120 kV using a Philips Tecnai 12 Biotwin microscope
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
