NcCyP41, a two domain neurospora crassa cyclophilin : characterization of its peptidyl-prolyl cis/trans isomerase activity ; isolation and functional analysis of two novel NcCyP41-binding proteins

NcCyP41 and its interactions were characterized. Its domains were cloned in different expression vectors and recombinant proteins purified from E. coli. Interaction of NcCyP41 and its domains with cellular proteins were then analysed by biochemical methods. <br> <br>PPIase activity of the entire protein and of its domains were tested in an in vitro assay. Catalytic efficiencies of NcCyP41 and its PPIase domain were nearly identical and inhibited by CsA. C-terminal TPR domain showed no PPIase activity. Thus, NcCyP41 has two distinct domains, the PPIase domain containing the full enzymatic activity. <br> <br>NcCyP41 and its domains were used in affinity experiments to identify binding proteins. Two abundant binding proteins were identified: Hsp80 and CyPBP37. A full-length cDNA for CyPBP37 was cloned and the protein purified. The characteristics of the interactions were then analysed. <br> <br>Hsp80 binds to the TPR domain of NcCyP41, while CyPBP37 binds to the PPIase domain. Binding of CyPBP37 to NcCyP41 is not sensitive to CsA, suggesting that CyPBP37 binds to the PPIase domain at a site different from the active site. <br> <br>CyPBP37 and NcCyP41 form two complexes with distinct electrophoretic mobility. Two dimensional gel electrophoresis indicates that CyPBP37 exists as different isoforms; mass spectrometry analysis has identified a potential ADP-ribosylation of CyPBP37. <br> <br>CyPBP37 is an abundant protein which is highly expressed in the logarithmic and the early stationary phase but only in thiamine free medium. The protein disappeared progressively from early to late stationary phase. In contrast to CyPBP37, Hsp80 and NcCyP41 expression is not regulated by thiamine. <br> <br>When NcCyP41 was expressed in a wild-type strain of S. cerevisiae, the growth at 37° C was severely reduced but only on a medium lacking thiamine, suggesting a similar type of interaction between NcCyP41 and a thiamine-regulated protein (probably Thi4p) in yeast.

Author dc:creator

• Faou, Pierre

Contributors dc:contributor

• Kleinig, Hans

Repository record source_url

https://freidok.uni-freiburg.de/data/371

OAI identifier oai:identifier

oai:freidok.uni-freiburg.de:371