Both the NH2 and COOH regions of Morgue protein exhibit association with polyubiquitin.
A. Schematic representation of MBP (Maltose binding protein) and 6xHis-tagged full length Morgue (MBP:Morgue:6xHis) as well as either the Morgue NH2 (MBP-MorgueN:6xHis) or COOH (MBP:MorgueC:6xHis) region. Note that MorgueN contains the zinc finger and F box while MorgueC contains the UEV domain. B. Western blot analysis of in vitro immunoprecipitation assays using ubiquitin (anti-Ub) or MBP (anti-MBP) antisera and native or tagged Morgue proteins. Note presence of ubiquitin immunoreactive bands at approximate sizes of ubiquitin dimers and tetramers that associate with tagged Morgues but not native Morgue protein. Also, MBP immunoreactive bands are observed for each tagged Morgue but not native Morgue. The distinct sizes of these bands correspond to distinct sizes of tagged Morgue proteins. C. Semi-quantitative analysis of Morgue association with ubiquitin. The tagged MorgueN protein exhibits slightly stronger association to polyubiquitin than the tagged MorgueC protein. Neither exhibits the full level of association seen for full length Morgue. Minimal association is observed between MBP and polyubiqutin.