LAPTM5 is a lysosomal transmembrane protein expressed in immune cells. UIM is usually retained in the Golgi as is usually LAPTM5 expressed in cells in which Nedd4 or GGA3 is usually knocked-down with RNAi. However ubiquitination-impaired LAPTM5 can still traffic to the lysosome suggesting that Nedd4 binding to LAPTM5 not LAPTM5 ubiquitination is required for targeting. Interestingly Nedd4 is also able to ubiquitinate CSF2RA GGA3. These results demonstrate a novel mechanism by which the ubiquitin-ligase Nedd4 via interactions with GGA3 and cargo (LAPTM5) regulates cargo trafficking to the lysosome without requiring cargo ubiquitination. Introduction Ubiquitin especially monoubiquitin has been a well-documented sorting transmission for both endocytosed plasma membrane (PM) proteins and intracellular resident proteins trafficking from your TGN to endosomes or lysosomes (Hicke 2001 This has been extensively studied in yeast where ubiquitination serves an important sorting transmission to determine protein targeting to either the lumen or the limiting membrane of the vacuole (lysosome; Helliwell et al. 2001 Katzmann et al. 2001 Soetens et al. 2001 Blondel et al. 2004 The role of ubiquitin in sorting proteins from your DL-cycloserine Golgi to endosomes/lysosomes in mammalian cells is not as obvious. Either blocking ubiquitin modification or adding a single ubiquitin to cargo proteins diverts them away from their normal cellular destination. For example blocking ubiquitination of the vacuolar carboxypeptidase S or adding a ubiquitin to the hydrolase dipeptidylaminopeptidase B in yeast diverts these proteins to DL-cycloserine the limiting membrane or lumen of the vacuole respectively (Katzmann et DL-cycloserine al. 2001 2002 Similarly the ubiquitination status of the yeast Space1 and Fur4 permeases determines their fate during sorting at the Golgi (Blondel et al. 2004 DL-cycloserine Helliwell et al. 2001 Soetens et al. 2001 In both yeast and mammalian cells most of the ubiquitinated proteins that reach the vacuole/lysosome interior are either PM proteins which then undergo degradation (Levkowitz et al. 1998 Hicke 2001 Rocca et al. 2001 or hydrolytic enzymes activated inside the vacuolar lumen (Odorizzi et al. 1998 If proteins are not ubiquitinated they are either recycled back to the PM (Levkowitz et al. 1998 or delivered to the limiting membrane of multivesicular body (MVBs) or the vacuole/lysosome (Katzmann et al. 2001 Therefore mono- or multimonoubiquitination plays a critical role in targeting cargo proteins to their proper cellular destination (Terrell et al. 1998 Shih et al. 2000 Mosesson et al. 2003 hence deciding their fate (degradation vs. recycling). The monoubiquitin sorting signal can be acknowledged and transmitted by several proteins involved in both the endocytic and biosynthetic pathways through direct physical conversation. These proteins often possess ubiquitin-binding motifs/domains (“ubiquitin receptors”) such as ubiquitin-interacting motif (UIM; Hofmann and Falquet 2001 GAT (GGA and Tom homologue) UBA UEV VHS and CUE (Hicke et al. 2005 The UIMs in epsin and eps15/eps15R play an important role in internalization and the early stages of endocytosis of PM proteins such as the EGFR (Oldham et al. 2002 Polo et al. 2002 whereas the UIMs of Vps27/Hrs and Hse1/STAM are associated with sorting function at the early and late DL-cycloserine endosome (Bilodeau et al. 2002 Raiborg et al. 2002 Shih et al. 2002 Interestingly several UIM-containing endocytic proteins (e.g. epsin eps15 and Hrs) are themselves ubiquitinated outside of the UIM sequence itself (Katz et al. 2002 Polo et al. 2002 Miller et al. 2004 At least in some DL-cycloserine cases this ubiquitination appears to involve Nedd4 family members. Nedd4 is usually a ubiquitin ligase comprised of a C2 domain name 3 WW domains that bind PY motifs (L/PPxY) on target proteins and a ubiquitin ligase Hect domain name (Staub and Rotin 2006 However it is not yet obvious how Nedd4 proteins are recruited to these endocytic proteins which do not possess PY motifs. Moreover the precise role of ubiquitination of these UIM-containing endocytic proteins in cargo sorting and transport is not known. The Golgi-localized γ-ear-containing ADP ribosylation factor-binding proteins (GGAs) are primarily associated with the TGN and.