Abstract
The ubiquitin E3 ligase gene related to anergy in lymphocytes (GRAIL) (Rnf128) is a type 1 transmembrane protein that induces T cell anergy through the ubiquitination activity of its cytosolic RING finger. GRAIL also contains an equally large luminal region consisting primarily of an uncharacterized protease-associated (PA) domain. Using two-hybrid technology to screen for proteins that bound the PA domain we identified CD151, a member of the tetraspanin family of membrane proteins. GRAIL bound to the luminal/extracellular portion of both CD151 and the related tetraspanin CD81 using its PA domain, which promoted ubiquitination of cytosolic lysine residues. GRAIL exhibited specificity for lysines only within the tetraspanin amino terminus even in the presence of other cytosolic lysine residues in the substrate. GRAIL-mediated ubiquitination promoted proteasomal degradation and cell surface down-regulation of tetraspanins via Lys-48 linkages. As a result, the juxtaposition of PA and RING finger domains across a lipid bilayer facilitates the capture of transmembrane substrates for subsequent ubiquitination. These findings identify for the first time a single subunit E3 ligase containing a substrate-binding domain spatially restricted by a membrane from its E2 recruitment domain as well as an E3 ligase for members of the tetraspanin family.
Highlights
E3 ligases are split into distinct families based on their E2 recruiting domains, including HECT, RING, and U box
The number of transmembrane, single subunit RING finger E3 ligases characterized to date remains exceedingly small compared with the total number of RING finger E3 ligases
The type 1 transmembrane protein Tul1 binds membrane proteins that contain inappropriately exposed polar residues in or near their transmembrane domain, whereas the K3 and MARCH families are type II transmembrane proteins that ubiquitinate a variety of cell surface markers important in the immune system
Summary
Materials and Antibodies—Ammonium chloride, Brij, CHAPS, chloroquine, Nonidet P-40, Triton. Cell lysates were split in half, and one set was cross-linked for 2 h on ice with 2 mM DSP followed by neutralization of free cross-linker with 50 mM Tris for 15 min on ice. SDS (0.1%) was added to both samples, and the tetraspanin was immunoprecipitated with anti-HA sive intracellular co-localization with GRAIL, the transferrin receptor [8], did not co-immunoprecipitate with transfected 3ϫHA-tagged. A, following transfection with either V5-tagged GRAIL or lacZ; 3ϫHA-tagged CD81, CD151, or vector alone; and FLAG-tagged ubiquitin (Ub), 293T cells were incubated with ALLN for 2 h prior to lysis to enrich for ubiquitinated proteins.
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