The yeast mitochondrial intermembrane space: Purification and analysis of two distinct fractions

We have developed a protocol for the sequential release of the intermembran e space (IMS) content of Saccharomyces cerevisiae mitochondria. Two distinc t fractions were obtained: a soluble IMS with cytochrome b(2) as key marker and a salt-extractable IMS with cytochrome c as key marker. The identity o f several proteins was determined by amino-terminal amino acid sequencing. The IMS fractions were devoid of contaminations from cytosol and mitochondr ial outer and inner membranes. By subtraction analysis, the protein profile s of soluble and salt-extractable IMS fractions were depleted of contaminat ing bands derived from matrix proteins. The fractionation method will provi de the basis for the further analysis of IMS proteins and characterization of their functions in bioenergetics, mitochondrial biogenesis, and regulato ry processes. (C) 1998 Academic Press.