Activation of an Endoribonuclease by Non-intein Protein Splicing.
The Chlamydomonas reinhardtii chloroplast-localized poly(A)-binding protein RB47 is predicted to contain a non-conserved linker (NCL) sequence flanked by highly conserved N- and C-terminal sequences, based on the corresponding cDNA. RB47 was purified from chloroplasts in association with an endoribonuclease activity; however, protein sequencing failed to detect the NCL. Furthermore, while recombinant RB47 including the NCL did not display endoribonuclease activity in vitro, versions lacking the NCL displayed strong activity. Both full-length and shorter forms of RB47 could be detected in chloroplasts, with conversion to the shorter form occurring in chloroplasts isolated from cells grown in the light. This conversion could be replicated in vitro in chloroplast extracts in a light-dependent manner, where epitope tags and protein sequencing showed that the NCL was excised from a full-length recombinant substrate, together with splicing of the flanking sequences. The requirement for endogenous factors and light differentiates this protein splicing from autocatalytic inteins, and may allow the chloroplast to regulate the activation of RB47 endoribonuclease activity. We speculate that this protein splicing activity arose to post-translationally repair proteins that had been inactivated by deleterious insertions or extensions.
From the Boyce Thompson Institute, Cornell University, Ithaca, New York 14853.
Stephen J
SJ
Campbell
From the Boyce Thompson Institute, Cornell University, Ithaca, New York 14853 ds28@cornell.edu.
David B
0000-0002-0653-6602
DB
Stern
D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D060365:Chloroplast Proteins / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D008027:Light; D019154:Protein Splicing / Q000502:physiology / Q000528:radiation effects*
D060365:Chloroplast Proteins; D004720:Endonucleases
chloroplast; endoribonuclease; intein; poly(A) binding protein; post-translational modification (PTM); protein splicing