Directionality of polypeptide transfer in the mitochondrial pathway of chaperone-mediated protein folding.

Protein folding in mitochondria depends on the functional cooperation of the Hsp70 and Hsp60 chaperone systems, at least for a subset of mitochondrial polypeptides. As suggested previously, Hsp70 and Hsp60 act sequentially. However, recent proposals that the chaperonin Hsp60 functions by releasing substrate protein in an unfolded state would predict a lateral partitioning of folding intermediates between chaperone systems. Firefly luciferase, carrying a mitochondrial targeting signal, was used as a model protein to analyze the degree of coupling and the directionality of substrate transfer between the Hsp70 and Hsp60 chaperones. In vitro, Hsp60 binds unfolded luciferase with high affinity but is unable to promote its folding, whereas the Hsp70 system assists the folding of luciferase efficiently. Upon import into yeast mitochondria, luciferase interacted first with Hsp70. Surprisingly, most of the protein subsequently accumulated in a complex with Hsp60 and never reached the native state. Import into mitochondria that lack a functional Hsp60 did not result in increased folding, but in the aggregation of luciferase. Thus, in intact organelles the two chaperone systems do not function independently in de novo folding of aggregation-sensitive proteins but rather act in an ordered pathway with substrate transfer predominantly in the direction from Hsp70 to Hsp60.

Conservation of chromosomal arrangement among three strains of the genetically unstable archaeon Halobacterium salinarium.

Phenotypic variants of Halobacterium salinarium NRC-1 arise at a frequency of 10(-2). These result from transpositions of halobacterial insertion sequences and rearrangements mediated by halobacterial insertion sequences. We have tested the hypothesis that such mutations are confined to only a portion of the genome by comparing the chromosomal restriction map of H. salinarium NRC-1 and that of the derivative S9, which was made in 1969. The two chromosomes were mapped by using two-dimensional pulsed-field gel electrophoresis and the restriction enzymes AflII, AseI, and DraI. A comparison of the two deduced maps showed a domain of about 210 kbp to be subject to many rearrangements, including an inversion in S9 relative to NRC-1. However, the rest of the chromosome was conserved among NRC-1, S9, and an independent Halobacterium isolate, GRB, previously mapped by St. Jean et al. (A. St. Jean, B. A. Trieselmann, and R. L. Charlebois, Nucleic Acids Res. 22:1476-1483, 1994). This concurs with data from eubacteria suggesting strong selective forces maintaining gene order even in the face of rearrangement events occurring at a high frequency.