Abstract
Folding assistance is a fundamental requirement of certain proteins, and it may be subjected to physicochemical constraints in case of organisms adapted to polar temperatures. Limited information is available about protein folding in the polar environment. Folding of tubulin provides one of the few studied cases. Here, we report a pilot folding analysis of a divergent beta-tubulin isotype, named EFBT3, from the Antarctic psychrophilic ciliate Euplotes focardii. To attain its native monomeric structure, beta-tubulin needs the assistance of the eukaryotic class II chaperonin CCT and cofactor A (CofA). The in vitro folding reaction of EFBT3 with CCT and CofA purified from rabbit did not generate any folded product. In contrast, the reaction performed with the rabbit reticulocyte lysate, that contains all the chaperones required for efficient tubulin folding, was productive, suggesting that additional factors besides purified CCT and CofA are required for EFBT3 to attain its monomeric structure. We also demonstrated that the rare Cys281 of EFBT3 is critical for the folding reaction. Model predictions indicate that EFBT3 binds to CofA differently from yeast beta-tubulin, suggesting a diverse folding mechanism that may be correlated with microtubule cold adaptation.
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Abbreviations
- RRL:
-
Rabbit reticulocyte lysate
- CCT:
-
Cytosolic chaperonin containing TCP-1
- TCP-1:
-
T-complex polypeptide 1
- TriC:
-
TCP-1 ring complex
- CofA:
-
Cofactor A
- CofB:
-
Cofactor B
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Acknowledgments
This research was supported by grants from the Italian Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) (PRIN 2008) to CM and from the Italian Programma Nazionale di Ricerche in Antartide (PNRA) to CM, and PEA2009 to SP and DS. FC acknowledges support from the Italian Ministry of Education and Research through the Flagship (PB05) InterOmics HIRMA (RBAP11YS7 K) and the European IMOMICSprojects.
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300_2013_1390_MOESM1_ESM.tif
In vitro folding reactions of labeled denatured EFBT3 in rabbit reticulocyte lysate (RRL) at 30 and 4 °C (lanes 1 and 3, respectively), and in presence of CCT purified from RRL at 30 and 4 °C (lanes 2 and 5, respectively) and from N. coriiceps testis, at 4 °C (lane 4), in the presence of Cof A (lanes 2, 4, and 5). In all lanes, the upper arrow indicates the location of CCT/EFBT3 complex. The lower arrow indicates CofA/EFBT3 complex (only in lane 1). (TIFF 7069 kb)
300_2013_1390_MOESM2_ESM.tif
Primary structure alignment of beta-tubulin from a variety of living organisms (the Acc. Nos are reported in the figure) obtained from ClustalW server, Secondary structures are based on the 1JFF mode: The nucleotide binding site is colored in red, the intermediate domain is in blue, and the C-terminal domain in green (Nogales et al. 2008). The EFBT3 sequence is shown in red. The unique Cys-281 of EFBT3, in the M-loop, is boxed and indicated by the arrow. In violet are the beta-tubulin isotypes that have been used as probes in folding reactions. (TIFF 747 kb)
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Pucciarelli, S., Chiappori, F., Sparvoli, D. et al. Tubulin folding: the special case of a beta-tubulin isotype from the Antarctic psychrophilic ciliate Euplotes focardii . Polar Biol 36, 1833–1838 (2013). https://doi.org/10.1007/s00300-013-1390-9
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DOI: https://doi.org/10.1007/s00300-013-1390-9