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UPTAKE OF APOCYTOCHROME C INTO YEAST MITOCHONDRIA: PREPARATION OF MITOCHONDRIA WITH INTACT OUTER MEMBRANES

Student’s name

MS Program in Biology

Uptake of apocytochrome c into mitochondria requires an intact outer membrane. Current research has focused on the uptake of apocytochrome c into yeast, mouse and rat liver mitochondria, but little attention has been paid to differences in the integrity of the outer membranes among these species. We have optimized the isolation of mitochondria from yeast by modifying established procedures, including increasing incubation time during spheroplast formation, as well as using a different mitochondrial isolation buffer

Using this modified procedure we have compared mitochondria from wild type yeast (Strain#9763, ATTC) with mitochondria from rat liver before and after exhaustive exercise. Mitochondrial intactness was measured by determining oxygen consumption (mmole/min/mg) over time, before and after the addition of exogenous cytochrome c. My results indicate that, like rat liver mitochondria , yeast mitochondria are intact using the modified isolation procedure (78-88% and 95-98%, respectively). Interestingly, rat liver mitochondria from exercised rats are broken after isolation, (5-15 % intact) suggesting these mitochondria are much more fragile. Furthermore, preliminary results obtained in collaboration with Dr. C. Murray Ardies suggests that a diet containing alcohol and the folic acid analogue, methotrexate, in varying combinations, have a deleterious effect on rat liver mitochondrial function and that pyrimidine nucleotide precursors protect mitochondria from these effects.

Currently we are examining the effects of folic acid analogues on yeast mitochondrial function. Preliminary results indicate that the outer mitochondrial membranes of methotrexate treated yeast are 70-80% intact compared to untreated yeast, which are 95-98% intact. This is similar to the effect on rat liver mitochondria, in which methotrexate reduced intactness from 89% to 68%. We hope to examine the effects of alcohol and pyrimidine nucleotide intermediates on yeast mitochondrial function. These effects will be compared to similar treatments of rat liver mitochondria. It is hoped that the examination of mitochondrial function in yeast, mouse and rat will provide a better understanding of mitochondrial biosynthesis.