Abstract
Cellular proteostasis involves not only the expression of proteins in response to environmental needs, but also the timely repair or removal of damaged or unneeded proteins. AAA+ motor proteins are critically involved in these pathways. Here, we review the structure and function of AAA+ proteins ClpA, ClpB, and Hsp104. ClpB and Hsp104 rescue damaged proteins from toxic aggregates and do not partner with any protease. ClpA functions as the regulatory component of the ATP dependent protease complex ClpAP, and also remodels inactive RepA dimers into active monomers in the absence of the protease. Because ClpA functions both with and without a proteolytic component, it is an ideal system for developing strategies that address one of the major challenges in the study of protein remodeling machines: how do we observe a reaction in which the substrate protein does not undergo covalent modification? Here, we review experimental designs developed for the examination of polypeptide translocation catalyzed by the AAA+ motors in the absence of proteolytic degradation. We propose that transient state kinetic methods are essential for the examination of elementary kinetic mechanisms of these motor proteins. Furthermore, rigorous kinetic analysis must also account for the thermodynamic properties of these complicated systems that reside in a dynamic equilibrium of oligomeric states, including the biologically active hexamer.
Highlights
The central dogma of molecular biology tells us that proteins are constantly being produced by the cell upon exposure to environmental stresses, nutrients, and metabolites
The results clearly showed that ClpA drives translocation of a polypeptide chain into the proteolytic chamber of ClpP
With a method in hand that is sensitive to polypeptide translocation in the absence of proteolytic degradation the question that could be addressed is, does ClpAP translocate using the same mechanism as ClpA alone? A qualitative assessment of stopped-flow time courses had been reported previously that concluded ClpAP translocated faster than ClpA alone but rate constants were not reported (Kolygo et al, 2009)
Summary
The central dogma of molecular biology tells us that proteins are constantly being produced by the cell upon exposure to environmental stresses, nutrients, and metabolites. Certain AAA+ hexameric ring motors associate with each end of the barrel and couple the energy from ATP binding and hydrolysis to processive translocation of a polypeptide chain through the axial channel of the hexameric ring and into the proteolytic cavity of the protease. They do not covalently modify the substrate on which they operate This lack of proteolytic activity leads to a technical barrier in addressing the question of whether these enzymes pass a polypeptide chain through their axial channels fully or partially. This is, in part, because unfolding alone is not evidence for complete passage. These issues are discussed below, building on an overview of the structure of these proteins
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