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AAA proteins share a conserved active site for ATP hydrolysis and regulate many cellular processes. AAA proteins are oligomeric and often have multiple ATPase domains per monomer, which is suggestive of complex allosteric kinetics of ATP hydrolysis. Here, using wild-type Hsp104 in the hexameric state, we demonstrate that its two AAA modules (NBD1 and NBD2) have very different catalytic activities, but each displays cooperative kinetics of hydrolysis. Using mutations in the AAA sensor-1 motif of NBD1 and NBD2 that reduce the rate of ATP hydrolysis without affecting nucleotide binding, we also examine the consequences of keeping each site in the ATP-bound state. In vitro, reducing kcat at NBD2 significantly alters the steady-state kinetic behavior of NBD1. Thus, Hsp104 exhibits allosteric communication between the two sites in addition to homotypic cooperativity at both NBD1 and NBD2. In vivo, each sensor-1 mutation causes a loss-of-function phenotype in two assays of Hsp104 function (thermotolerance and yeast prion propagation), demonstrating the importance of ATP hydrolysis as distinct from ATP binding at each site for Hsp104 function. [ABSTRACT FROM AUTHOR]

The red algal chloroplast genome encodes an essential prokaryotic cell division gene, ftsH, which has never been found in the mitochondrial genome of any organism. To compare the conserved prokaryote-derived mechanism for mitochondrial division with that of chloroplasts, we cloned chloroplast- and nuclear-encoded ftsH genes from the primitive red alga Cyanidioschyzon merolae. The deduced amino-acid sequence of chloroplast ftsH (ftsHcp) consists of 603 amino acids and shows the highest similarity with algal-chloroplast and cyanobacterial FtsH. On the other hand, the nuclear-encoded ftsH (ftsH2) encodes a protein of 920 amino acids and has the highest similarity with two yeast mitochondrial FtsHs, Rca1p and Afg3p. Furthermore, the amino-terminal extension of FtsH2 appears to be an amphipathic α-helix, a characteristic mitochondrial targeting signal, suggesting that FtsH2 is a mitochondrial protein. Southern hybridization revealed that ftsH2 is a single gene located on chromosome III of the 17 C. merolae chromosomes. The level of expression of the 3.0 and 4.0 kb transcripts of this gene decreased in concert during the organelle division phase of a synchronized culture, indicating a cell-cycle-dependent manner of ftsH2 transcription, while northern hybridization did not detect ftsHcp transcripts. Nevertheless, reverse transcription-PCR and immunoblotting demonstrated for the first time that chloroplast-encoded ftsH is transcriptionally and translationally active. Overproduction of FtsHcp and FtsH2 in Escherichia coli disrupted cytokinesis and produced filamentous cells, but had no effect on the replication, segregation, or distribution of their nucleoids, as also occurs in ftsH-deficient E. coli. These observations suggest the possible involvement of both C. merolae FtsHs in organelle division. [ABSTRACT FROM AUTHOR]