Abstract
AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP hydrolysis in the AAA-domains is mediated by a non-catalytic N-terminal domain. The exact mechanisms that transmit the signal from the N-domain and coordinate the individual AAA-domains in the hexameric complex are still the topic of intensive research. Here, we present the characterization of a novel mutant variant of the eukaryotic AAA-ATPase Drg1 that shows dysregulation of ATPase activity and altered interaction with Rlp24, its substrate in ribosome biogenesis. This defective regulation is the consequence of amino acid exchanges at the interface between the regulatory N-domain and the adjacent D1 AAA-domain. The effects caused by these mutations strongly resemble those of pathological mutations of the AAA-ATPase p97 which cause the hereditary proteinopathy IBMPFD (inclusion body myopathy associated with Paget’s disease of the bone and frontotemporal dementia). Our results therefore suggest well conserved mechanisms of regulation between structurally, but not functionally related members of the AAA-family.
Highlights
The N-domain of p97 mediates the direct physical interactions with the majority of co-factors and adaptor proteins that recruit the enzyme to the target pathways[7,10,17]
We present novel mutations in the N-domain of Drg[1] that cause a disturbed regulation of ATPase activity and altered nucleotide-dependent interaction with the substrate protein Rlp[24]
The growth defect caused by the drg[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21] allele was enhanced in combination with the EQ2 exchange, but not when combined with the EQ1 mutation. This is obvious on the SD-ura plate lacking copper where the CUP1 promoter shows only low activity, but strong growth inhibition is observed for the allele containing the two drg[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21] exchanges (T100I/leucine 108 for proline (L108P)) in combination with the EQ2 exchange (Fig. 1d)
Summary
The N-domain of p97 mediates the direct physical interactions with the majority of co-factors and adaptor proteins that recruit the enzyme to the target pathways[7,10,17]. We present novel mutations in the N-domain of Drg[1] that cause a disturbed regulation of ATPase activity and altered nucleotide-dependent interaction with the substrate protein Rlp[24].
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