In vivo import of yeast adenylate kinase into mitochondria affected by site‐directed mutagenesis

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Abstract
Site-directed mutagenesis and deletions were used to study mitochondrial import or a major yeast adenylate kinase, Aky2p. This enzyme lacks a cleavable presequence and occurs in active and apparently unprocessed form both in mitochondria and cytoplasm. Mutations were applied to regions known to be surface-exposed and to diverge between short and long isoforms. In vertebrates, short adenylate kinase isozymes occur exclusively in the cytoplasm, whereas long versions of the enzyme have mitochondrial locations. Mutations in the extra loop of the yeast (long-form) enzyme did not affect mitochondrial import of the protein, whereas variants altered in the central, N- or C-terminal parts frequently displayed increased or, in the case of a deletion of the 8 N-teminal triplets, decreased import efficiencies. Although the N-terminus is important for targeting adenylate kinase to mitochondria, other parameters like internal sequence determinants and folding velocity of the nascent protein may also play a role.