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ATP-binding cassette (ABC) adenosine triphosphatases actively transport a wide variety of compounds across biological membranes. Here, the ABC protein Mdl was identified as an intracellular peptide transporter localized in the inner membrane of yeast mitochondria. Mdli was required for mitochondrial export of peptides with molecular masses of ~2100 to 600 daltons generated by proteolysis of inner-membrane proteins by the m-AAA protease in the mitochondrial matrix. Proteolysis by the i-AAA protease in the intermembrane space led to the release of similar-sized peptides independent of Mdli. Thus, two pathways of peptide efflux from mitochondria exist that may allow communication between mitochondria and their cellular environment.
Various adenosine 5'-triphosphate (ATP)-- dependent proteases within mitochondria constitute conserved, apparently ubiquitous protein families in eukaryotic cells. Two homologous proteolytic complexes that have been identified in the mitochondrial inner membrane-the m- and i-AAA protease-- expose their catalytic sites to the matrix and intermembrane space, respectively (1). They exert multiple functions that are essential for cell viability and mitochondrial biogenesis and constitute a quality-control system for the degradation of nonassembled inner-membrane proteins. Here we analyze the characteristics and the fate of proteolytic breakdown products of inner-membrane proteins, using the yeast Saccharomyces cerevisiae as a model system.
Mitochondrially encoded respiratory-- chain subunits, which have been synthesized in the presence of [^sup 35^S]methionine in isolated organelles, lack assembly partners encoded by the nucleus and are therefore rapidly degraded by the m-AAA protease (2, 3). Protein turnover can be quantified by determining the increase in radioactivity in the acid-soluble fraction after precipitation with trichloroacetic acid (TCA) (Fig. 1A). To examine whether these degradation products remain associated with mitochondria or are released from the organelle, we isolated mitochondria by centrifugation before the addition of TCA and determined radioactivity present in the supernatant fraction (Fig. 1A) (4). Degradation products were almost exclusively detected in the supernatant, indicating rapid release from mitochondria (Fig. 1A). The release was dependent on proteolysis by the m-AAA protease because it was abrogated in mitochondria that lacked m-AAA protease subunits Yta10 or Yta12 or that expressed proteolytically inactive variants of both subunits (Fig. 1B). Proteolytic products accumulated, however, at wild-type levels in the supernatant of (Delta)ymel mitochondria that lacked the i-AAA protease (Fig. 1B).
To characterize the released products, we performed...