Autophagy is the process of degradation of intracellular proteins through the 
lysosome. Members of the tripartite motif (TRIM) proteins have shown to directly 
recognize autophagic cargo and also to act as a hub for the phagophore 
nucleation complex. The TRIM proteins are classically characterized by the 
presence of an amino-terminal RING domain and a B-box domain followed by a 
coiled coil domain. Although regarded as ubiquitin E3 ligases, this activity has 
been shown only for a minor set of the 79 human TRIM proteins. Additionally, the 
role of each domain in the E3 ligase activity is unknown. We investigated the 
role of the SPRY and RING domains of the human TRIM49 protein in its E3 
ubiquitin ligase activity. Wild-type and mutant constructs of tagged TRIM49 were 
expressed in E. coli or mammalian cells, and the autoubiquitination activity of 
the purified protein was assessed. The purified TRIM49 showed no ubiquitin E3 
ligase activity in vitro. However, cells transfected with the wild-type or 
mutant protein showed increased levels of lower mass polyubiquitinated proteins 
and both proteins copurified with polyubiquitinated proteins. Taken together, 
these results indicate that the TRIM49 protein plays a role in autophagic 
protein degradation independently of an ubiquitin E3 ligase activity.