Identifying insect cell proteins copurified with wild-type human UBR4/KCMF1/CALM1 complex and different domain deletions

The UBR4/KCMF1/CALM1 complex is an important general protein quality control enzyme which extends ubiquitin chains on pre-ubiquitinated proteins to target them for degradation. We observed by cryogenic electron microscopy that the complex purified recombinantly from insect cells is bound to a potential substrate which cannot be identified by experimental density. However, we could determine that the copurified protein is bound to a certain domain in the complex termed the ZZ-DZB domain. To identify the copurified insect cell proteins we performed LC-MS analysis of the purified wild-type complex and a complex with the ZZ-DZB domain deleted. We compared the two datasets to identify proteins which were highly enriched in the wildtype dataset but lost in the ZZ-DZB deletion dataset. To gain additional insight into the role of the domains of UBR4 in substrate recognition, we repeated this approach with two other domain deletions – the BS2 domain and the UBL domain. As the UBR4/KCMF1/CALM1 complex is expected to recognize proteins by the processing of their N-termini, we also performed digests with three different proteases to try and identify the native N-termini of the co-purified proteins.