Split-ubiquitin two-hybrid assay to analyze protein-protein interactions at the endosome: application to Saccharomyces cerevisiae Bro1 interacting with ESCRT complexes, the Doa4 ubiquitin hydrolase, and the Rsp5 ubiquitin ligase.

ABSTRACT: Targeting of membrane proteins into the lysosomal/vacuolar lumen for degradation requires their prior sorting into multivesicular bodies (MVB). The MVB sorting pathway depends on ESCRT-0, -I, -II, and -III protein complexes functioning on the endosomal membrane and on additional factors, such as Bro1/Alix and the ubiquitin ligase Rsp5/Nedd4. We used the split-ubiquitin two-hybrid assay to analyze the interaction partners of yeast Bro1 at its natural cellular location. We show that Bro1 interacts with ESCRT-I and -III components, including Vps23, the Saccharomyces cerevisiae homologue of human Tsg101. These interactions do not require the C-terminal proline-rich domain (PRD) of Bro1. Rather, this PRD interacts with the Doa4 deubiquitinating enzyme to recruit it to the endosome. This interac

SUBMITTER: Nikko E 

PROVIDER: S-EPMC1951119 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature