Fusion proteins containing the cytochrome b2 presequence are sorted to the mitochondrial intermembrane space independently of hsp60.
hsp60 is a chaperonin located in the mitochondrial matrix. It has been suggested that hsp60 participates in two processes: protein folding in the matrix, and the sorting of imported proteins to the intermembrane space. We analyzed hsp60 function by allowing isolated mitochondria to import two model precursor proteins and then measuring the binding of these proteins to the chaperonin. Of the methods that we tested for monitoring the association of imported proteins with hsp60, only co-immunoprecipitation with specific anti-hsp60 antibodies proved to be reliable. A chimeric matrix-targeted precursor, consisting of a mitochondrial presequence fused to a chloroplast-encoded protein, bound stably to hsp60 after import. In contrast, there was no detectable binding to hsp60 with a fusion protein that was targeted to the intermembrane space by the bipartite cytochrome b2 presequence. Analysis of a translocation intermediate demonstrated that the cytochrome b2 presequence arrests import through the inner membrane, with the result that the attached passenger protein is never exposed to hsp60.
Rospert
S
S
Biocenter, University of Basel, Switzerland.
Müller
S
S
Schatz
G
G
Glick
B S
BS
D001692:Biological Transport; D002451:Cell Compartmentation; D018833:Chaperonins; D004072:Digitonin / Q000494:pharmacology; D004734:Energy Metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D008928:Mitochondria / Q000378:metabolism*; D018272:Porins / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism
D006360:Heat-Shock Proteins; D018272:Porins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D013762:Tetrahydrofolate Dehydrogenase; D018833:Chaperonins; D004072:Digitonin