Sequential action of mitochondrial chaperones in protein import into the matrix.

Abstract

Translocation and folding of proteins imported into mitochondria are mediated by two matrix‐localized chaperones, mhsp70 and hsp60. In order to investigate whether these chaperones act sequentially or in parallel, we studied their interaction with newly imported precursor proteins in isolated yeast mitochondria by coimmunoprecipitation. All precursors bound transiently to mhsp70. Release from mhsp70 required hydrolysis of ATP and did not immediately generate a tightly folded protein. For example, after imported mouse dihydrofolate reductase (a soluble monomeric enzyme) had been released from mhsp70, folding to a protease resistant conformation occurred only after a lag and was much slower than the release. Under standard import conditions, no significant association of DHFR with hsp60 could be detected. Similarly, newly imported hsp60 subunit was released from mhsp70 as an incompletely folded, unassembled intermediate which accumulated at low temperature and assembled to hsp60 14‐mer at higher temperature in an ATP‐dependent manner. Mas2p (the larger subunit of the MAS‐encoded processing protease) first bound to mhsp70, then to hsp60, and only then assembled with its partner subunit, Mas1p. We propose that ATP‐dependent release from mhsp70 is insufficient to cause folding of imported proteins and that assembly of hsp60 and Mas2p requires sequential, ATP‐dependent interactions with mhsp70 and hsp60.

Mitochondrial protein import: Mechanisms, components and energetics

“…Mitochondria harboring a temperature-sensitive Hsp60 allele accumulated unfolded proteins in the matrix [39]. Thus, the two mitochondrial heat-shock proteins, Hsp70 and Hsp60, appear to act in a sequential process: Hsp70 mediates the translocation of precursor proteins across the membranes, Hsp60 promotes folding to the native conformation and assembly into oligomeric complexes [40]. As expected, mitochondrial Hsp60 interacts with its cognate Hspl0 which was recently identified [41,42].…”

Import of cytochrome b2 to the mitochondrial intermembrane space: The tightly folded heme‐binding domain makes import dependent upon matrix ATP

“…The presence of ATP in the matrix appears to generate a pulling force that acts on the presequence. A logical candidate for an ATP-dependent "import motor" is mitochondrial hsp70 (mhsp70; Kang et al, 1990;Scherer et al, 1990;Manning-Krieg et al, 1991). It is conceivable that the heme-binding domain remains completely folded during translocation, as the mitochondrial import machinery can accommodate structures other than extended polypeptide chains (Vestweber & Schatz, 1988b).…”

Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding

“…Heat shock proteins of the Hsp70 and Hsp60 family have been shown to play essential roles as molecular chaperones in the translocation of proteins into mitochondria and in the subsequent folding and assembly of the newly imported precursor proteins within the organelle (Cheng et al, 1989;Ostermann et al, 1989;Scherer et al, 1990;Kang et al, 1990;Manning-Krieg et al, 1991;Craig et al, 1993;Ellis, 1993;Neupert and Pfanner, 1993). Cytosolic members of the Hsp70 class are required to maintain proteins in a transport-competent conformation during translocation across membranes (Chirico et al, 1968;Deshaies et al, 1988).…”