Methods for NAD-Dependent Ubiquitination Catalyzed by Legionella pneumophila Effector Proteins

Ubiquitination is one of the most important posttranslational modifications in eukaryotic cells where it regulates the activity, cellular localization, and half-life of proteins. Ubiquitination thus affects many essential cellular processes, including vesicle trafficking, cell cycle, DNA repair, immune response, and protein homeostasis. The ubiquitin system is exclusive to eukaryotes; however, pathogenic bacteria have developed effective strategies to influence the host ubiquitin system for their own benefit. Legionella pneumophila is the causative agent of Legionnaires' disease, a severe form of pneumonia. This bacterium utilizes a type IV secretion system to translocate more than 300 effector proteins into host cells. These virulence factors modulate a wide spectrum of host processes to support its intracellular survival and replication. Hijacking of host ubiquitin system is an important theme in Legionella virulence, and a number of L. pneumophila effector proteins have been shown to possess ubiquitin ligase or deubiquitinase activity. Among these, members of the SidE family effector proteins (SidEs) catalyze ubiquitination of several ER-associated Rab small GTPases by a mechanism that bypasses the requirement of ATP and the E1, E2 enzymes. Here, we summarize the experimental details of Rab small GTPases ubiquitination catalyzed by SdeA, a member of the SidE family.