Abstract
The small heat shock proteins (sHSPs) are molecular chaperones that share an alpha-crystallin domain but display a high diversity of sequence, expression, and localization. They are especially prominent in plants, populating most cellular compartments. In pea, mitochondrial HSP22 is induced by heat or oxidative stress in leaves but also strongly accumulates during seed development. The molecular function of HSP22 was addressed by studying the effect of temperature on its structural properties and chaperone effects using a recombinant or native protein. Overexpression of HSP22 significantly increased bacterial thermotolerance. The secondary structure of the recombinant protein was not affected by temperature in contrast with its quaternary structure. The purified protein formed large polydisperse oligomers that dissociated upon heating (42 °C) into smaller species (mainly monomers). The recombinant protein appeared thermosoluble but precipitated with thermosensitive proteins upon heat stress in assays either with single protein clients or within complex extracts. As shown by in vitro protection assays, HSP22 at high molar ratio could partly prevent the heat aggregation of rhodanese but not of malate dehydrogenase. HSP22 appears as a holdase that could possibly prevent the aggregation of some proteins while co-precipitating with others to facilitate their subsequent refolding by disaggregases or clearance by proteases.
Highlights
In all organisms, the homeostasis of proteins relies on several families of molecular chaperones that intervene in biogenesis, translocation, folding, and assembly of polypeptides to survey their structure and function in the crowded environment of cells [1]
There is mounting evidence that alpha-crystallin domain (ACD), N, and C-terminal regions contribute all together to small HSPs (sHSPs) structure and molecular chaperone activity [3,9]. sHSPs have been suggested to bind denatured proteins in an ATP-independent manner, limiting aggregation and allowing subsequent refolding in cooperation with other chaperones, such as HSP70 [1,10]. sHSPs were qualified as holdase chaperones by contrast to foldases that assist protein folding by ATP-dependent mechanisms [11]
We first examined whether the structure of the recombinant HSP22 (HSP22rec) could be affected by an increase of temperature from 20 ◦C to 42 ◦C, which corresponds to severe heat stress conditions for most plants
Summary
The homeostasis of proteins relies on several families of molecular chaperones that intervene in biogenesis, translocation, folding, and assembly of polypeptides to survey their structure and function in the crowded environment of cells [1]. An exception is the mitochondrial sHSP of the fruit fly Drosophila melanogaster, which accumulates during stress and aging [18] This sHSP was shown to protect mitochondria against oxidative damage and be a critical component of fly longevity [18,21,24,25]. Besides HSP22, pea seed mitochondria were found to accumulate in their matrix LEAM, a LEA protein shown to protect the inner membrane during desiccation [63,64]. The remarkable stress tolerance of pea seed mitochondria that still perform oxidative phosphorylation at -3.5 or 40 ◦C was correlated with the presence of HSP22 and LEAM [65] Both proteins were detected with similar abundance in the seeds of 91 different accessions of pea, which underlies their importance for the protection of mitochondria [66]. This strongly favors a role of HSP22 in the association with aggregated proteins, presumably to facilitate their refolding during stress recovery
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