INFLUENCE OF N-TERMINAL SEQUENCE VARIATION ON THE SORTING OF MAJOR ADENYLATE KINASE TO THE MITOCHONDRIAL INTERMEMBRANE SPACE IN YEAST

Major adenylate kinase (Aky2p) from yeast has no cleavable presequence and occurs in identical form in the mitochondrial intermembrane space (6-8 %) and in the cytoplasm (approx. 90 %). To identify the signal(s ) on Aky2p that might be required for mitochondrial import, the N-term inal region was examined. The N-terminus of Aky2p can guide at least t wo cytoplasmic passengers, dihydrofolate reductase from mouse and UMP kinase (Ura6p) from yeast, to the intermembrane space in vivo, showing that the N-terminus harbours import information. In contrast, deletio n of the eight N-terminal amino acid residues or the introduction of t wo compensating frameshifts into this segment does not abolish translo cation into the organelle's intermembrane space. Thus internal targeti ng and sorting information must be present in Aky2p as well. Neither a pronounced amphiphilic alpha-helical moment nor positive charges in t he N-terminal region is a necessary prerequisite for Aky2p to reach th e intermembrane space. Even a surplus of negative charges in mutant N- termini does not impede basal import into the correct submitochondrial compartment. The potential to form an amphipathic alpha-helical struc ture of five to eight residues close to the N-terminus significantly i mproves import efficiency, whereas extension of this amphipathic struc ture, e.g. by replacing it with the homologous segment of Aky3p, a mit ochondrial matrix protein from yeast, leads to misdirection of the chi maera to the matrix compartment. This shows that the topogenic N-termi nal signal of Aky3p is dominant over the presumptive internal intermem brane space-targeting signal of Aky2p and argues that the sorting of w ild-type Aky2p to the intermembrane space is not due to the presence i n the protein of a specific sorting sequence for the intermembrane spa ce, but rather is the consequence of being imported but not being sort ed to the inner compartment. Some Aky2 mutant proteins are susceptible to proteolysis in the cytoplasm, indicating incorrect folding. They a re nevertheless efficiently rescued by uptake into mitochondria, sugge sting a negative correlation between folding velocity (or folding stab ility) and efficiency of import.