CONICET | Buscador de Institutos y Recursos Humanos

Background Proteins may adopt diverse conformations during their folding in vivo, ranging from extended chains when they emerge from the ribosome to compact intermediates near the end of the folding process. A great variety of chaperones and folding assisting enzymes has evolved to deal with this diversity. Here we study the maturation of cruzipain (CZ), an abundant lysosomal protease of T. cruzi and the only endogenous substrate of UGGT identified so far. Our attention was focused on the conformations recognized in vivo by calreticulin (CRT), BiP and UDP-Glc:glycoprotein glucosyltransferase (UGGT). Methods CZ maturation in T. cruzi was followed by studying the formation of its disulfide bridges. To this end, CZ was labeled with [35S]-Met and chased for different times. Disulfide bond formation was assessed by a double alkylation procedure with iodoacetamide and AMS, followed by immunoprecipitation of CZ. A similar approach was employed to study the conformation of CZ associated with BiP or CRT. We compared wild type parasites with those lacking UGGT. Results In wild type parasites CZ associates sequentially with BiP and CRT during its folding pathway. Early and extended conformations were bound by BiP, while more advance and compact folding intermediates associated with CRT. The interaction between CZ and CRT was precluded by deleting the gene coding for UGGT. In this system, CZ associated with BiP displayed an extended conformation similar to that observed in wild type cells. Conclusions CZ association with CRT not only triggers its retention in the ER, but it is also necessary for its productive folding. In addition, we found that in vivo UGGT recognizes advanced folding intermediates of this endogenous substrate. These observations show that BiP and CRT activities are finely tuned to assist the entire folding pathway of CZ.