Lysosomal integral membrane protein LGP85 (LIMP-2) is ubiquitinated at the N-terminal cytoplasmic domain.

LGP85/LIMP-2 is a type III transmembrane glycoprotein of lysosomes, which traverses the membrane twice with an N-terminal uncleaved signal sequence and C-terminal hydrophobic domain. In addition to functioning as a receptor for a lysosomal enzyme ß-glucocerebrosidase and for several enteroviruses, LGP85 plays a key role in the biogenesis and maintenance of endosomal/lysosomal compartments (ELCs). Our previous studies have demonstrated that overexpression of rat LGP85 into COS cells results in the enlarged ELCs, from where membrane trafficking is impaired. We show here that rat LGP85 is polyubiquitinated at the N-terminal short cytoplasmic domain that comprises of only three amino acid residues, alanine, arginine, and cysteine. Replacement of either arginine or cysteine with alanine within the N-terminal cytoplasmic domain did not influence the ubiquitination of LGP85, thereby indicating that ubiquitin (Ub) is conjugated to the α-NH2 group of the N-terminal alanine residue. Furthermore, we were able to define a domain necessary for ubiquitination in a region ranging from the amino acids 156 to 255 within the lumenal domain of LGP85. This is the first report showing that the integral lysosomal membrane protein LGP85 is ubiquitinated.

HM1.24/BST-2 is constitutively poly-ubiquitinated at the N-terminal amino acid in the cytoplasmic domain.

HM1.24 (also known as BST-2, CD317, and Tetherin) is a type II single-pass transmembrane glycoprotein, which traverses membranes using an N-terminal transmembrane helix and is anchored in membrane lipid rafts via a C-terminal glycosylphosphatidylinositol (GPI). HM1.24 plays a role in diverse cellular functions, including cell signaling, immune modulation, and malignancy. In addition, it also functions as an interferon-induced cellular antiviral restriction factor that inhibits the replication and release of diverse enveloped viruses, and which is counteracted by Vpu, an HIV-1 accessory protein. Vpu induces down-regulation and ubiquitin conjugation to the cytoplasmic domain of HM1.24. However, evidence for ubiquitination site(s) of HM1.24 remains controversial. We demonstrated that HM1.24 is constitutively poly-ubiquitinated at the N-terminal cytoplasmic domain, and that the mutation of all potential ubiquitination sites, including serine, threonine, cysteine, and lysine in the cytoplasmic domain of HM1.24, does not affect the ubiquitination of HM1.24. We further demonstrated that although a GPI anchor is necessary and sufficient for HM1.24 antiviral activities and virion-trapping, the deleted mutant of GPI does not influence the ubiquitination of HM1.24. These results suggest that the lipid raft localization of HM1.24 is not a prerequisite for the ubiquitination. Collectively, our findings demonstrate that the ubiquitination of HM1.24 occurs at the N-terminal amino acid in the cytoplasmic domain and indicate that the constitutive ubiquitination machinery of HM1.24 may differ from the Vpu-induced machinery.