Objective To verify that Rab guanine nucleotide exchange factor 1 (RabGEFl) is a novel substrate of linear ubiquitin.Methods Human RabGEFl gene was cloned into pEF6/Myc-His C vector. The interaction between RabGEFl and HOIP was verified by Co-IP experiment. The interaction domain between RabGEFl and HOIP was analyzed by GST-pulldown. The interaction and subcellular localization were verified by immunofluorescence. The linear ubiquitination of RabGEFl was detected by in vivo ubiquitination experiment. The NTA-His ubiquitination assay further confirmed that RabGEF1 can undergo linear ubiquitination modification. The ubiquitinated RabGEF1 proteinsamples were subjected to mass spectrometry to analyze the specific site modified by ubiquitin. The mutant plasmid of potential RabGEFl ubiquitination site was constructed according to the mass spectrometry results, and the ubiquitination site of RabGEFl was further verified with RabGEFl KR mutant plasmids in vivo ubiquitination experiment.Results RabGEFl interacts with HOIP via ZF-NEF domain. RabGEFl co-locates with HOIP in the cytoplasm. LUBAC mediated linear ubiquitination modification of RabGEFl depends on LUBAC enzyme activity, and the ubiquitination modification site of RabGEFl is K158.Conclusion RabGEFl is a novel substrate for linear ubiquitination modification, and the site of linear ubiquitination modification is K158.