[19]
PREPARATION OF CONTROLLED YEAST MITOCHONDRIA
149
[19] P r e p a r a t i o n o f Y e a s t M i t o c h o n d r i a ( S a c c h a r o m y c e s cerevisiae) with Good P/O and Respiratory Control Ratios B y BERNARD GUI~RIN, PIERRE LABBE a n d MARIE SOMLO
As described in a previous volume of Methods in Enzymology, 1"the foremost experimental obstacle in the preparation of intact yeast mitochondria is the need for efficiently breaking the refractory cell wall without extensively damaging the liberated mitochondria." Two methods have been used and described in the literature: (1) mechanical cell rupture 1-5 and (2) cell wall digestion by snail gut juice. 6-1° This article gives an account of new modifications of the two methods, the advantages over the previously described versions being an increased yield, greater simplicity, and shorter manipulation time. Futher advantages are (1) use of the mechanical methods for studying proton translocation and oligomycin-sensitivity of oxidative phosphorylation in S. cerevisiae mutants (p. 153), as well as application toCandida lipolytica (p. 152), and (2) use of the enzyme method for the study of mitochondrial membrane permeability and localization of loosely bound enzymes (p. 157). Since we have not experimented with other enzymatic methods not using the snail enzyme, these are not discussed in this article. As emphasized by Mattoon and Balcavage, 1 both the mechanical and the enzymatic methods have specific advantages and drawbacks. The main advantage of the mechanical method is speed; this is of critical importance in certain cases, e.g., when a long physiological study of freshly prepared mitochondria is planned. The preparation of mitochondria by the mechanical methods described in this article takes only 2 hr, as compared to about 6 hr for methods involving enzymatic cell wall digestion. The main advantage of the latter over the mechanical method is that mitochondria of higher integrity are obtained. In fact, as is shown by the systematic comparison below, Qo2 values and respiratory control 1 j. R. Mattoon and W. X. Balcavage, this series, Vol. 10 [26]. 2 M. Guarnieri, J. R. Mattoon, W. X. Balcavage, and C. Payne, Anal. Biochem. 34, 39 (1970). 3 G. Schatz and E. Racker, Biochem. Biophys. Res. Commun. 22, 579 (1966). 4 C. Spencer, S. A. Symons, and R. V. Brunt, Arch. Mikrobiol. 75, 246 (1971). 5 B. Mackler and B. Haynes, Biochim. Biophys. Acta 292, 88 (1973). 6 E. A. Duell, S. I n o u r , and M. F. Utter, J. Bacteriol. 88, 1762 (1964). 7 T. Ohnishi, K. Kawaguchi, and B. Hagihara, J. Biol. Chem. 241, 1797 (1966). 8 L. Kovac, G. S. P. Groot, and E. Racker, Biochim. Biophys. Acta 256, 55 (1972). 9G. Schatz and L. Kovac, this series, Vol. 31 [65]. 10 M. Briquet, N. Sabadie-Pialoux, and A. Goffeau, Arch. Biochem. Biophys. 174, 684 (1976). METHODS IN ENZYMOLOGY,VOL. LV
Copyright O 1979by Academic Press. Inc. All fights of reproduction in any formreserved. ISBN 0-12-181955-8
150
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PREPARATION OF CONTROLLED YEAST MITOCHONDRIA
149
[19] P r e p a r a t i o n o f Y e a s t M i t o c h o n d r i a ( S a c c h a r o m y c e s cerevisiae) with Good P/O and Respiratory Control Ratios B y BERNARD GUI~RIN, PIERRE LABBE a n d MARIE SOMLO
As described in a previous volume of Methods in Enzymology, 1"the foremost experimental obstacle in the preparation of intact yeast mitochondria is the need for efficiently breaking the refractory cell wall without extensively damaging the liberated mitochondria." Two methods have been used and described in the literature: (1) mechanical cell rupture 1-5 and (2) cell wall digestion by snail gut juice. 6-1° This article gives an account of new modifications of the two methods, the advantages over the previously described versions being an increased yield, greater simplicity, and shorter manipulation time. Futher advantages are (1) use of the mechanical methods for studying proton translocation and oligomycin-sensitivity of oxidative phosphorylation in S. cerevisiae mutants (p. 153), as well as application toCandida lipolytica (p. 152), and (2) use of the enzyme method for the study of mitochondrial membrane permeability and localization of loosely bound enzymes (p. 157). Since we have not experimented with other enzymatic methods not using the snail enzyme, these are not discussed in this article. As emphasized by Mattoon and Balcavage, 1 both the mechanical and the enzymatic methods have specific advantages and drawbacks. The main advantage of the mechanical method is speed; this is of critical importance in certain cases, e.g., when a long physiological study of freshly prepared mitochondria is planned. The preparation of mitochondria by the mechanical methods described in this article takes only 2 hr, as compared to about 6 hr for methods involving enzymatic cell wall digestion. The main advantage of the latter over the mechanical method is that mitochondria of higher integrity are obtained. In fact, as is shown by the systematic comparison below, Qo2 values and respiratory control 1 j. R. Mattoon and W. X. Balcavage, this series, Vol. 10 [26]. 2 M. Guarnieri, J. R. Mattoon, W. X. Balcavage, and C. Payne, Anal. Biochem. 34, 39 (1970). 3 G. Schatz and E. Racker, Biochem. Biophys. Res. Commun. 22, 579 (1966). 4 C. Spencer, S. A. Symons, and R. V. Brunt, Arch. Mikrobiol. 75, 246 (1971). 5 B. Mackler and B. Haynes, Biochim. Biophys. Acta 292, 88 (1973). 6 E. A. Duell, S. I n o u r , and M. F. Utter, J. Bacteriol. 88, 1762 (1964). 7 T. Ohnishi, K. Kawaguchi, and B. Hagihara, J. Biol. Chem. 241, 1797 (1966). 8 L. Kovac, G. S. P. Groot, and E. Racker, Biochim. Biophys. Acta 256, 55 (1972). 9G. Schatz and L. Kovac, this series, Vol. 31 [65]. 10 M. Briquet, N. Sabadie-Pialoux, and A. Goffeau, Arch. Biochem. Biophys. 174, 684 (1976). METHODS IN ENZYMOLOGY,VOL. LV
Copyright O 1979by Academic Press. Inc. All fights of reproduction in any formreserved. ISBN 0-12-181955-8
150
ORGANELLES
AND
[19]
MEMBRANES
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