In vivo zippering of inner and outer mitochondrial membranes by a stable translocation intermediate.
It was previously assumed that the import of cytoplasmically synthesized precursor proteins into mitochondria occurs through a single structure spanning both outer and inner membranes at contact sites. Based on recent findings, however, the two membranes appear to contain independent translocation elements that reversibly cooperate during protein import. This feature makes it difficult to generate a means of isolating a fully integrated and functional translocation complex. To study these independent translocases in vitro and in vivo, we have constructed a chimeric protein consisting of an N-terminal authentic mitochondrial precursor (delta1-pyrroline-5-carboxylate dehydrogenase) linked, through glutathione S-transferase, to IgG binding domains derived from staphylococcal protein A. This construct becomes trapped en route to the matrix, spanning both outer and inner membranes in such a way that the entire signal-less delta1-pyrroline-5-carboxylate dehydrogenase moiety reaches the matrix, while only the folded protein A domain remains outside. During in vivo import of this precursor, outer and inner membranes of yeast mitochondria become progressively "zippered" together, forming long stretches of close contact. Using this novel intermediate, the outer and inner mitochondrial membrane channels, which normally interact only transiently, can be tightly joined (both in vitro and in vivo), forming a stable association. This suggests a method for isolating the functional translocation complex as a single entity.
Schülke
N
N
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6085, USA.
Sepuri
N B
NB
Pain
D
D
D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism
D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D005982:Glutathione Transferase