In S. cerevisiae, a single translation product of the FUM1 gene is distributed between the cytosol and the mitochondria (Stein et al, 1994). Fumarase precursors harbour a 24-amino-acid-long N-terminal presequence, which is removed by the mitochondrial matrix peptidase (MPP) on import into mitochondria. Mitochondrial and cytosolic isoenzymes of fumarase share identical N-termini that correspond to the mature processed form (Sass et al, 2001). In vivo, when processing is inhibited either by blocking import with the uncoupler CCCP or by inactivating MPP, all fumarase polypeptides accumulate as precursors (Stein et al, 1994). When fumarase is translated in vitro in the presence of mitochondria, correctly processed molecules are found not only within, but also outside, mitochondria (Knox et al, 1998). Thus, before distribution, all fumarase polypeptides are directed to mitochondria and processed by MPP. A subset of the processed fumarase molecules is then fully imported into the matrix, whereas the majority is released back into the cytosol. Folding of fumarase appears to be the driving force for its retrograde movement into the cytosol. Rapid folding impedes fumarase import into mitochondria post-translationally, both in vivo and in vitro (Stein et al, 1994; Knox et al, 1998). Mutations throughout the coding sequence of fumarase that alter its conformation do not impair targeting to mitochondria, but cause a loss of retrograde movement and distribution (Sass et al, 2003). Overexpression of a cytosolic Hsp70 (Ssa1) causes a shift of fumarase distribution to mitochondria, whereas partially impairing mitochondrial Hsp70 (Ssc1 conditional mutation) diminishes full import and allows more fumarase molecules to reside in the cytosol. Finally, mass spectrometry analysis revealed that mature cytosolic and mitochondrial fumarase molecules are identical, and contain no post-translational modifications. These results support the idea that folding per se is the driving force for the distribution of fumarase (Sass et al, 2003).