The Single Subunit Transmembrane E3 Ligase Gene Related to Anergy in Lymphocytes (GRAIL) Captures and Then Ubiquitinates Transmembrane Proteins across the Cell Membrane

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 the of transmembrane for for the a E3 ligase a domain a membrane its domain an E3 ligase for of the tetraspanin 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 the of transmembrane for for the a E3 ligase a domain a membrane its domain an E3 ligase for of the tetraspanin The ubiquitin of a of proteins that the of The in of ubiquitin to a ubiquitin gene related to anergy in large to ubiquitin gene related to anergy in large to and E3 a of to the of ubiquitin to its The of specificity is a of protein the E3 ligase that the domains for of the ubiquitination of other of proteins the ubiquitin and RING finger proteins the and and proteins domains and protein the region in the for the RING finger E3 via domains to The for RING finger E3 and of the of E3 the of proteins ubiquitination that E3 ligase to protein E3 specificity the of is gene related to anergy in lymphocytes (GRAIL) is a type 1 transmembrane E3 ligase to that promoted T cell anergy GRAIL a of that T cell and and its activity an cytosolic RING finger domain GRAIL is to that and across the amino terminus of GRAIL contains a protease-associated (PA) domain, which to a in its is also that PA proteins a in degradation of the transmembrane in and of in the transmembrane E3 a of identified proteins. we the PA domain of GRAIL a to a luminal domain of transmembrane proteins ubiquitination of cytosolic residues the GRAIL RING finger. for proteins that the amino terminus of GRAIL identified the tetraspanin family member CD151, a transmembrane protein that and that GRAIL its PA domain to of both CD151 and and promoted cytosolic ubiquitination of the tetraspanin for proteasomal a ubiquitination in which a E3 ligase contains a domain for transmembrane proteins that is its ubiquitination across a Using membrane juxtaposition of and GRAIL transmembrane proteins for and and and and The and to and to and and amino terminus of GRAIL the the the a to the activity and identified for and tetraspanins for which a tetraspanins the in a tetraspanin protein the and in and using to the to and protein ubiquitination for to in for and to in for and for 1 protein the bound in proteins and to for using using and using the to the and 1 for to in in in and in in and using using an a and and in a and in in and for of for to both and the tetraspanin in for and of the GRAIL PA luminal portion of the type 1 transmembrane E3 ubiquitin ligase GRAIL contains a PA domain, which is in the of the and in the in we a in a two-hybrid the PA domain of the to of the in a two-hybrid the that of the for the the large of the transmembrane tetraspanin protein CD151 the CD151 and GRAIL in a of of proteins in of CD151 in of GRAIL and of also tetraspanin for the PA domain of GRAIL PA protein the CD151 to a tetraspanin is of and to the CD151 and the and GRAIL to CD151 and in a GRAIL and CD151 the GRAIL and CD151 and the of large tetraspanin for the of the the region of GRAIL that bound CD151, a of GRAIL CD151 in and for to As CD151 GRAIL in the domains and a the PA domain of CD151 to GRAIL GRAIL PA of the tetraspanin family is the of an domain. the for the tetraspanin a of tetraspanin family proteins of the family of we the PA domain of GRAIL an domain for other of the tetraspanin family in to of GRAIL in CD81 of an in an region of GRAIL the in a CD81 to CD151 an GRAIL amino terminus is for a GRAIL and a to The of the GRAIL and that the GRAIL PA domain bound to the luminal/extracellular region of the transmembrane tetraspanins and a in PA domain of GRAIL for GRAIL and CD81 and and for GRAIL and GRAIL and and CD81 and CD81 and in in and for and to and GRAIL PA for RING cytosolic RING finger of GRAIL to E3 ligase activity the family we tetraspanin the luminal PA domain of GRAIL in ubiquitination of of the cytosolic domains of transmembrane proteins. Using a ubiquitination in we the of the of a of both and to the to to proteins. the presence of a in the of CD81 and CD151 of proteins that cell for the ubiquitination in transmembrane tetraspanin ubiquitination both the of GRAIL to to the tetraspanin and its E3 ligase activity ubiquitination of CD81 and CD151 the GRAIL The of GRAIL ubiquitination a in the of tetraspanin GRAIL a of CD81 ubiquitination the PA and RING domains and in tetraspanin GRAIL CD151, and ubiquitin for to to for proteins. and and CD81 and in in and and of CD81 and GRAIL and for a of of for and the within the to lysine residues of proteins. of tetraspanins that the in the transmembrane domains and in the a lysine in the cytosolic amino terminus to the transmembrane region to the cytosolic RING finger of GRAIL for ubiquitin and a for tetraspanin lysine a for GRAIL-mediated of the lysines in CD81 and CD151 CD81 contains lysines only in the amino terminus and CD151 contains lysines in the amino terminus and lysine in of the other cytosolic domains GRAIL in ubiquitination lysine the lysine the of ubiquitination for both equally the ubiquitination of an protein of both lysines GRAIL-mediated a lysine for in the the of GRAIL to the the lysine the GRAIL-mediated ubiquitination the of lysines cytosolic lysines GRAIL of the lysines to GRAIL-mediated in GRAIL to a of the the the of the tetraspanin lysine residues only in the tetraspanin amino of the amino terminus of tetraspanins and of CD81 and CD151 lysine lysine GRAIL CD81 CD151, and ubiquitin in and and of CD81 and GRAIL and for a of of for and the the lysines of and for for GRAIL-mediated GRAIL only lysines in the amino terminus of the that GRAIL lysines in the amino the lysine of tetraspanins the for the GRAIL RING finger for lysine residues only within the domain of of contains lysine residues that other ubiquitin and in The of ubiquitin and the ubiquitin lysine in the to the of the substrate. of of lysines via ubiquitin lysine to proteasomal ubiquitin lysine the type of GRAIL CD151 GRAIL and ubiquitin ubiquitin for lysine lysine of ubiquitination of CD151, the CD151 that GRAIL tetraspanins using lysine The presence of ubiquitin in the presence of ubiquitin is ubiquitin and a to of lysine of using tetraspanins via ubiquitin Lys-48 for proteasomal GRAIL CD151, and ubiquitin in in and and CD81 CD151, and the for to in and and that tetraspanin to proteasomal and CD81 of both the and only the to the and The of both and ubiquitin both CD81 and CD151, of degradation that GRAIL tetraspanin via lysine and to degradation the of to in and of the and tetraspanins an membrane in to the cell surface and the of GRAIL and tetraspanins both GRAIL and CD81 and CD151 of of GRAIL and the tetraspanins only in of the of the tetraspanins and that of CD81 and CD151 that of GRAIL the GRAIL in down-regulation of cell surface GRAIL and CD81 to and and the GRAIL a in for CD81 CD151, and The of in the of the tetraspanin GRAIL a in for and The of in both the ubiquitination that GRAIL promoted tetraspanin the of GRAIL the of GRAIL-mediated ubiquitination tetraspanin of which a Using a for GRAIL in cell surface of CD81 and CD151 both in CD81 the in the of tetraspanin of the RING finger to cell surface tetraspanin that tetraspanin the luminal of the membrane cytosolic ubiquitination for cell surface down-regulation Using and for of transmembrane proteins the cell we down-regulation of cell surface GRAIL of the of the of GRAIL and domain CD81 GRAIL of RING finger E3 to the of RING finger E3 the family and the family in The type 1 transmembrane protein membrane proteins that residues in transmembrane domain, the and type transmembrane proteins that a of cell surface in the both of of E3 the transmembrane region the that the type 1 transmembrane E3 ligase GRAIL a for and transmembrane proteins. The presence of a PA domain of a luminal domain of a transmembrane ubiquitination of a cytosolic domain the RING finger of for proteins that bound to the GRAIL luminal PA domain identified the of the tetraspanin that an GRAIL PA domain for to only CD151 also to CD81 the of and that the PA domain a domain to transmembrane proteins of the of tetraspanins of for and transmembrane a tetraspanins the of the PA domain of GRAIL of the CD81 region of GRAIL and tetraspanins in promoted ubiquitination of transmembrane proteins in a of tetraspanins both a luminal the PA domain, and a RING finger for of cytosolic ubiquitination in GRAIL a in tetraspanin a that GRAIL the of GRAIL in down-regulation of cell surface tetraspanins of and to a protein down-regulation the GRAIL and tetraspanins in the of tetraspanin of tetraspanins GRAIL the of GRAIL tetraspanins in an GRAIL in T is the and the an in tetraspanin GRAIL to of ubiquitination E3 also lysine residues that in for ubiquitin of the lysine in the amino terminus of CD81 CD151 the of GRAIL to tetraspanin lysine in the amino of lysines in the amino terminus of CD151 the of GRAIL to CD151 even lysine in of the other cytosolic domains that GRAIL contains specificity for tetraspanin ubiquitination only the amino of the tetraspanins the for of ubiquitin to a the amino terminus of the tetraspanin is across a membrane and to the for CD81 that the amino terminus an which to the cytosolic RING finger of GRAIL the cytosolic GRAIL RING finger an an ubiquitin in the for ubiquitination of only the amino terminus of tetraspanin is an The of ubiquitination down-regulation of a GRAIL the specificity for tetraspanin ubiquitination GRAIL is a transmembrane E3 ligase that its its ubiquitination across a for via the luminal PA domain a to to cytosolic residues for ubiquitination via the RING finger. E3 GRAIL to other and transmembrane proteins. The ubiquitin of a of proteins that the of The in of ubiquitin to a ubiquitin gene related to anergy in large to ubiquitin gene related to anergy in large to and E3 a of to the of ubiquitin to its The of specificity is a of protein the E3 ligase that the domains for of the ubiquitination of other of proteins the ubiquitin E3 and RING finger proteins the and and proteins domains and protein the region in the for the RING finger E3 via domains to The for RING finger E3 and of the of E3 the of proteins ubiquitination that E3 ligase to protein E3 specificity the of is The gene related to anergy in lymphocytes (GRAIL) is a type 1 transmembrane E3 ligase to that promoted T cell anergy GRAIL a of that T cell and and its activity an cytosolic RING finger domain GRAIL is to that and across the amino terminus of GRAIL contains a protease-associated (PA) domain, which to a in its is also that PA proteins a in degradation of the transmembrane in and of in the transmembrane E3 a of identified proteins. we the PA domain of GRAIL a to a luminal domain of transmembrane proteins ubiquitination of cytosolic residues the GRAIL RING finger. for proteins that the amino terminus of GRAIL identified the tetraspanin family member CD151, a transmembrane protein that and that GRAIL its PA domain to of both CD151 and and promoted cytosolic ubiquitination of the tetraspanin for proteasomal a ubiquitination in which a E3 ligase contains a domain for transmembrane proteins that is its ubiquitination across a Using membrane juxtaposition of and GRAIL transmembrane proteins for and and and and The and to and to and and amino terminus of GRAIL the the the a to the activity and identified for and tetraspanins for which a tetraspanins the in a tetraspanin protein the and in and using to the to and protein ubiquitination for to in for and to in for and for 1 protein the bound in proteins and to for using using and using the to the and 1 for to in in in and in in and using using an a and and in a and in in and for of for to both and the tetraspanin in for and and and and and The and to and to and and amino terminus of GRAIL the the the a to the activity and identified for and tetraspanins for which a tetraspanins the in a tetraspanin protein the and in and using to the to and protein ubiquitination for to in for and to in for and for 1 protein the bound in proteins and to for using using and using the to the and 1 for to in in in and in in and using using an a and and in a and in in and for of for to both and the tetraspanin in for and of the GRAIL PA luminal portion of the type 1 transmembrane E3 ubiquitin ligase GRAIL contains a PA domain, which is in the of the and in the in we a in a two-hybrid the PA domain of the to of the in a two-hybrid the that of the for the the large of the transmembrane tetraspanin protein CD151 the CD151 and GRAIL in a of of proteins in of CD151 in of GRAIL and of also tetraspanin for the PA domain of GRAIL PA protein the CD151 to a tetraspanin is of and to the CD151 and the and GRAIL to CD151 and in a GRAIL and CD151 the GRAIL and CD151 and the of large tetraspanin for the of the the region of GRAIL that bound CD151, a of GRAIL CD151 in and for to As CD151 GRAIL in the domains and a the PA domain of CD151 to GRAIL GRAIL PA of the tetraspanin family is the of an domain. the for the tetraspanin a of tetraspanin family proteins of the family of we the PA domain of GRAIL an domain for other of the tetraspanin family in to of GRAIL in CD81 of an in an region of GRAIL the in a CD81 to CD151 an GRAIL amino terminus is for a GRAIL and a to The of the GRAIL and that the GRAIL PA domain bound to the luminal/extracellular region of the transmembrane tetraspanins and a in GRAIL PA for RING cytosolic RING finger of GRAIL to E3 ligase activity the family we tetraspanin the luminal PA domain of GRAIL in ubiquitination of of the cytosolic domains of transmembrane proteins. Using a ubiquitination in we the of the of a of both and to the to to proteins. the presence of a in the of CD81 and CD151 of proteins that cell for the ubiquitination in transmembrane tetraspanin ubiquitination both the of GRAIL to to the tetraspanin and its E3 ligase activity ubiquitination of CD81 and CD151 the GRAIL The of GRAIL ubiquitination a in the of tetraspanin GRAIL a of CD81 ubiquitination the PA and RING domains and in tetraspanin GRAIL CD151, and ubiquitin for to to for proteins. and and CD81 and in in and and of CD81 and GRAIL and for a of of for and the within the to lysine residues of proteins. of tetraspanins that the in the transmembrane domains and in the a lysine in the cytosolic amino terminus to the transmembrane region to the cytosolic RING finger of GRAIL for ubiquitin and a for tetraspanin lysine a for GRAIL-mediated of the lysines in CD81 and CD151 CD81 contains lysines only in the amino terminus and CD151 contains lysines in the amino terminus and lysine in of the other cytosolic domains GRAIL in ubiquitination lysine the lysine the of ubiquitination for both equally the ubiquitination of an protein of both lysines GRAIL-mediated a lysine for in the the of GRAIL to the the lysine the GRAIL-mediated ubiquitination the of lysines cytosolic lysines GRAIL of the lysines to GRAIL-mediated in GRAIL to a of the the the of the tetraspanin lysine residues only in the tetraspanin amino of the amino terminus of tetraspanins and of CD81 and CD151 lysine lysine GRAIL CD81 CD151, and ubiquitin in and and of CD81 and GRAIL and for a of of for and the the lysines of and for for GRAIL-mediated GRAIL only lysines in the amino terminus of the that GRAIL lysines in the amino the lysine of tetraspanins the for the GRAIL RING finger for lysine residues only within the domain of of contains lysine residues that other ubiquitin and in The of ubiquitin and the ubiquitin lysine in the to the of the substrate. of of lysines via ubiquitin lysine to proteasomal ubiquitin lysine the type of GRAIL CD151 GRAIL and ubiquitin ubiquitin for lysine lysine of ubiquitination of CD151, the CD151 that GRAIL tetraspanins using lysine The presence of ubiquitin in the presence of ubiquitin is ubiquitin and a to of lysine of using tetraspanins via ubiquitin Lys-48 for proteasomal GRAIL CD151, and ubiquitin in in and and CD81 CD151, and the for to in and and that tetraspanin to proteasomal and CD81 of both the and only the to the and The of both and ubiquitin both CD81 and CD151, of degradation that GRAIL tetraspanin via lysine and to degradation the of to in and of the and tetraspanins an membrane in to the cell surface and the of GRAIL and tetraspanins both GRAIL and CD81 and CD151 of of GRAIL and the tetraspanins only in of the of the tetraspanins and that of CD81 and CD151 that of GRAIL the GRAIL in down-regulation of cell surface GRAIL and CD81 to and and the GRAIL a in for CD81 CD151, and The of in the of the tetraspanin GRAIL a in for and The of in both the ubiquitination that GRAIL promoted tetraspanin the of GRAIL the of GRAIL-mediated ubiquitination tetraspanin of which a Using a for GRAIL in cell surface of CD81 and CD151 both in CD81 the in the of tetraspanin of the RING finger to cell surface tetraspanin that tetraspanin the luminal of the membrane cytosolic ubiquitination for cell surface down-regulation Using and for of transmembrane proteins the cell we down-regulation of cell surface GRAIL of the of the of GRAIL and domain CD81 GRAIL of the GRAIL PA luminal portion of the type 1 transmembrane E3 ubiquitin ligase GRAIL contains a PA domain, which is in the of the and in the in we a in a two-hybrid the PA domain of the to of the in a two-hybrid the that of the for the the large of the transmembrane tetraspanin protein CD151 the CD151 and GRAIL in a of of proteins in of CD151 in of GRAIL and of also tetraspanin for the PA domain of GRAIL PA protein the CD151 to a tetraspanin is of and to the CD151 and the and GRAIL to CD151 and in a GRAIL and CD151 the GRAIL and CD151 and the of large tetraspanin for the of the the region of GRAIL that bound CD151, a of GRAIL CD151 in and for to As CD151 GRAIL in the domains and a the PA domain of CD151 to GRAIL The GRAIL PA of the tetraspanin family is the of an domain. the for the tetraspanin a of tetraspanin family proteins of the family of we the PA domain of GRAIL an domain for other of the tetraspanin family in to of GRAIL in CD81 of an in an region of GRAIL the in a CD81 to CD151 an GRAIL amino terminus is for a GRAIL and a to The of the GRAIL and that the GRAIL PA domain bound to the luminal/extracellular region of the transmembrane tetraspanins and a in The GRAIL PA for RING cytosolic RING finger of GRAIL to E3 ligase activity the family we tetraspanin the luminal PA domain of GRAIL in ubiquitination of of the cytosolic domains of transmembrane proteins. Using a ubiquitination in we the of the of a of both and to the to to proteins. the presence of a in the of CD81 and CD151 of proteins that cell for the ubiquitination in transmembrane tetraspanin ubiquitination both the of GRAIL to to the tetraspanin and its E3 ligase activity ubiquitination of CD81 and CD151 the GRAIL The of GRAIL ubiquitination a in the of tetraspanin GRAIL a of CD81 GRAIL within the to lysine residues of proteins. of tetraspanins that the in the transmembrane domains and in the a lysine in the cytosolic amino terminus to the transmembrane region to the cytosolic RING finger of GRAIL for ubiquitin and a for tetraspanin lysine a for GRAIL-mediated of the lysines in CD81 and CD151 CD81 contains lysines only in the amino terminus and CD151 contains lysines in the amino terminus and lysine in of the other cytosolic domains GRAIL in ubiquitination lysine the lysine the of ubiquitination for both equally the ubiquitination of an protein of both lysines GRAIL-mediated a lysine for in the the of GRAIL to the the lysine the GRAIL-mediated ubiquitination the of lysines cytosolic lysines GRAIL of the lysines to GRAIL-mediated in GRAIL to a of the the the of the tetraspanin the lysines of and for for GRAIL-mediated GRAIL only lysines in the amino terminus of the that GRAIL lysines in the amino the lysine of tetraspanins the for the GRAIL RING finger for lysine residues only within the domain of GRAIL of contains lysine residues that other ubiquitin and in The of ubiquitin and the ubiquitin lysine in the to the of the substrate. of of lysines via ubiquitin lysine to proteasomal ubiquitin lysine the type of GRAIL CD151 GRAIL and ubiquitin ubiquitin for lysine lysine of ubiquitination of CD151, the CD151 that GRAIL tetraspanins using lysine The presence of ubiquitin in the presence of ubiquitin is ubiquitin and a to of lysine of using that tetraspanin to proteasomal and CD81 of both the and only the to the and The of both and ubiquitin both CD81 and CD151, of degradation that GRAIL tetraspanin via lysine and to degradation the of to in and of the and tetraspanins an membrane in to the cell surface and the of GRAIL and tetraspanins both GRAIL and CD81 and CD151 of of GRAIL and the tetraspanins only in of the of the tetraspanins and that of CD81 and CD151 that of GRAIL the the ubiquitination that GRAIL promoted tetraspanin the of GRAIL the of GRAIL-mediated ubiquitination tetraspanin of which a Using a for GRAIL in cell surface of CD81 and CD151 both in CD81 the in the of tetraspanin of the RING finger to cell surface tetraspanin that tetraspanin the luminal of the membrane cytosolic ubiquitination for cell surface down-regulation Using and for of transmembrane proteins the cell we down-regulation of cell surface GRAIL of the of the of GRAIL and domain CD81 GRAIL of RING finger E3 to the of RING finger E3 the family and the family in The type 1 transmembrane protein membrane proteins that residues in transmembrane domain, the and type transmembrane proteins that a of cell surface in the both of of E3 the transmembrane region the that the type 1 transmembrane E3 ligase GRAIL a for and transmembrane proteins. The presence of a PA domain of a luminal domain of a transmembrane ubiquitination of a cytosolic domain the RING finger of for proteins that bound to the GRAIL luminal PA domain identified the of the tetraspanin that an GRAIL PA domain for to only CD151 also to CD81 the of and that the PA domain a domain to transmembrane proteins of the of tetraspanins of for and transmembrane a tetraspanins the of the PA domain of GRAIL of the CD81 region of GRAIL and tetraspanins in promoted ubiquitination of transmembrane proteins in a of tetraspanins both a luminal the PA domain, and a RING finger for of cytosolic ubiquitination in GRAIL a in tetraspanin a that GRAIL the of GRAIL in down-regulation of cell surface tetraspanins of and to a protein down-regulation the GRAIL and tetraspanins in the of tetraspanin of tetraspanins GRAIL the of GRAIL tetraspanins in an GRAIL in T is the and the an in tetraspanin GRAIL to of ubiquitination E3 also lysine residues that in for ubiquitin of the lysine in the amino terminus of CD81 CD151 the of GRAIL to tetraspanin lysine in the amino of lysines in the amino terminus of CD151 the of GRAIL to CD151 even lysine in of the other cytosolic domains that GRAIL contains specificity for tetraspanin ubiquitination only the amino of the tetraspanins the for of ubiquitin to a the amino terminus of the tetraspanin is across a membrane and to the for CD81 that the amino terminus an which to the cytosolic RING finger of GRAIL the cytosolic GRAIL RING finger an an ubiquitin in the for ubiquitination of only the amino terminus of tetraspanin is an The of ubiquitination down-regulation of a GRAIL the specificity for tetraspanin ubiquitination GRAIL is a transmembrane E3 ligase that its its ubiquitination across a for via the luminal PA domain a to to cytosolic residues for ubiquitination via the RING finger. E3 GRAIL to other and transmembrane proteins. The of RING finger E3 to the of RING finger E3 the family and the family in The type 1 transmembrane protein membrane proteins that residues in transmembrane domain, the and type transmembrane proteins that a of cell surface in the both of of E3 the transmembrane region the that the type 1 transmembrane E3 ligase GRAIL a for and transmembrane proteins. The presence of a PA domain of a luminal domain of a transmembrane ubiquitination of a cytosolic domain the RING finger of for proteins that bound to the GRAIL luminal PA domain identified the of the tetraspanin that an GRAIL PA domain for to only CD151 also to CD81 the of and that the PA domain a domain to transmembrane proteins of the of tetraspanins of for and transmembrane a tetraspanins the of the PA domain of GRAIL of the CD81 region of GRAIL and tetraspanins in promoted ubiquitination of transmembrane proteins in a of tetraspanins both a luminal the PA domain, and a RING finger for of cytosolic ubiquitination in GRAIL a in tetraspanin a that GRAIL the of GRAIL in down-regulation of cell surface tetraspanins of and to a protein down-regulation the GRAIL and tetraspanins in the of tetraspanin of tetraspanins GRAIL the of GRAIL tetraspanins in an GRAIL in T is the and the an in tetraspanin GRAIL to of ubiquitination E3 also lysine residues that in for ubiquitin of the lysine in the amino terminus of CD81 CD151 the of GRAIL to tetraspanin lysine in the amino of lysines in the amino terminus of CD151 the of GRAIL to CD151 even lysine in of the other cytosolic domains that GRAIL contains specificity for tetraspanin ubiquitination only the amino of the tetraspanins the for of ubiquitin to a the amino terminus of the tetraspanin is across a membrane and to the for CD81 that the amino terminus an which to the cytosolic RING finger of GRAIL the cytosolic GRAIL RING finger an an ubiquitin in the for ubiquitination of only the amino terminus of tetraspanin is an The of ubiquitination down-regulation of a GRAIL the specificity for tetraspanin ubiquitination GRAIL is a transmembrane E3 ligase that its its ubiquitination across a for via the luminal PA domain a to to cytosolic residues for ubiquitination via the RING finger. E3 GRAIL to other and transmembrane proteins. to for and the of the for and also and for and for of the to and for