Molecular Chaperones and Proteases
In the age of post-genomics, understanding how protein structure and function is regulated throughout the cell will be of crucial importance. It is known that molecular chaperones and ATP-dependent proteases play a central role in such regulatory networks, and that their influence over protein biogenesis and stability occurs in all living organisms. Chaperones are intricately involved in protein maturation, promoting activities such as protein synthesis, translocation, folding and assembly into multimeric structures. Chaperones stabilise partially unfolded polypeptides until conditions are favourable for correct folding or protein association, thereby reducing the probability of premature folding and formation of functionally inert structures. This function is particularly vital during stress, when the potential for protein misfolding, denaturation and aggregation greatly increases.
Of equal importance to cell homeostasis are ATP-dependent proteases. By efficiently degrading selected proteins, proteases help modulate key metabolic enzymes and regulatory proteins, and also remove aberrant polypeptides arising from translational errors and post-synthetic damage. Again, the activity of these proteases is vital during stress due to the dramatic rise in abnormal and thus potentially toxic polypeptides. Although the study of molecular chaperones and proteases is now one of the most exciting and developing fields of research today, our understanding of such systems in photosynthetic organisms remains rudimentary. This is especially true for cyanobacteria and in chloroplasts of higher plants. The protein environment within both cell locales is complex and dynamic, with many processes potentially requiring the action of one or more chaperones and proteases. The discovery of a new family of proteins has provided exciting clues towards identifying a novel chaperone/proteolytic system in photosynthetic organisms that may play a vital role in regulating the structure and function of many important proteins.
The Clp/Hsp100 Protein Family
The Clp/Hsp100 proteins comprise a new family of molecular chaperones, with at least one present in all eubacteria and eukaryotes studied to date. The family can be divided into two broad groups, which are separated further into different types based on specific sequence signatures. Proteins in the first group are large (85 to 105 kD), and contain two distinct ATP-binding domains. They are subdivided into five types, ClpA-E. The second group has only two members, ClpX and ClpY, which differ from those in the first group by having only one ATP-binding domain. Being the newest chaperone family, little is still known about the role of Clp/Hsp100 proteins in different organisms, or their specific modes of chaperone action. Despite this, many genetic studies have shown that different Clp/Hsp100 proteins have an important, and in many cases, essential role. An added dimension to the importance of Clp/Hsp100 proteins is their association with a proteolytic subunit, ClpP. This complex forms the ATP-dependent Clp protease, in which the chaperone subunit is essential for activating the proteolytic activity of ClpP (ClpP alone is proteolytically inactive). In steps requiring ATP, the Hsp100 partner selectively binds the target protein and presents it to ClpP in an unfolded state, ready for degradation. Because of their involvement in both chaperone and proteolytic activities, certain Clp/Hsp100 proteins are thought to play a vital regulatory role in determining the fate of many proteins.
Our group studies the various Clp proteins in two model photosynthetic organisms: the unicellular cyanobacterium Synechococcus sp. PCC , and the higher plant Arabidopsis thaliana. We have now identified eight and eighteen distinct clp genes in Synechococcus and Arabidopsis, respectively, coding for by far many more different Clp proteins than present in non-photosynthetic organisms. In Synechococcus, we have so far shown that certain Clp proteins are essential for either photosynthetic growth or survival to various stresses, including extremes of temperature, high light intensity and supplementary UV-B. In both model organisms, we are continuing to characterise the specific functions of each Clp protein, using a combination of molecular, genetic, biochemical and physiological approaches. We hope to eventually elucidate the chaperone or proteolytic nature of each Clp protein, their role in protein biogenesis and targeted proteolysis, and the native proteins and pathways they affect in cyanobacteria and plant chloroplasts.
Selected publications:
Clarke, A.K., Gustafsson, P. & Lidholm, J. () Identification and expression of the chloroplast clpP gene in the conifer Pinus contorta. Plant Mol. Biol. 26, 851-862.
Clarke, A.K. & Eriksson, M-J. () The cyanobacterium Synechococcus sp. PCC possesses a close homologue to the chloroplast ClpC protein of higher plants. Plant Mol. Biol. 31, 721-730.
Eriksson, M-J. & Clarke, A.K. () The heat shock protein ClpB mediates the development of thermotolerance in the cyanobacterium Synechococcus sp. strain PCC . J. Bacteriol. 178, .
Porankiewicz, J. & Clarke, A.K. () Induction of the heat shock protein ClpB affects cold acclimation in the cyanobacterium Synechococcus sp. strain PCC . J. Bacteriol. 179, .
Clarke, A.K., Schelin, J. & Porankiewicz, J. () Inactivation of the clpP1 gene for the proteolytic subunit of the Clp protease in the cyanobacterium Synechococcus impairs growth and light acclimation. Plant Mol. Biol. 37, 791-801.
Porankiewicz, J., Schelin, J. & Clarke, A.K. () The ATP-dependent Clp protease is essential for cold and UV-B acclimation in the cyanobacterium Synechococcus. Mol. Microbiol. 29, 275-284.
Porankiewicz, J., Wang, J. & Clarke, A.K. () New insights into the ATP-dependent Clp protease: E. coli and beyond. Mol. Microbiol. 32, 449-458.
Clarke, A.K. () ATP-dependent Clp proteases in photosynthetic organisms: A cut above the rest! Annal. Bot. 83, 593-599.
