Post-translational N-Glycosylation of a Truncated Form of a Peptide Processing Enzyme

N-Glycosylation of membrane proteins is critical for their proper folding, co-assembly and subsequent matriculation through the secretory pathway. Here, we examine the kinetics of N-glycan addition to type I transmembrane KCNE1 K ؉ channel ␤-subunits, where point mutations that prevent N-glycosylation at one consensus site give rise to disorders of the cardiac rhythm and congenital deafness. We show that KCNE1 has two distinct N-glycosylation sites: a typical co-translational site and a consensus site ϳ20 residues away that unexpectedly acquires N-glycans after protein synthesis (post-translational). Mutations that ablate the co-translational site concomitantly reduce glycosylation at the post-translational site, resulting in unglycosylated KCNE1 subunits that cannot reach the cell surface with their cognate K ؉ channel. This long range inhibition is highly specific for post-translational N-glycosylation because mutagenic conversion of the KCNE1 post-translational site into a co-translational site restored both monoglycosylation and anterograde trafficking. These results directly explain how a single point mutation can prevent N-glycan attachment at multiple sites, providing a new biogenic mechanism for human disease.

Section: Discussionmentioning

confidence: 99%

Post-translational N-Glycosylation of Type I Transmembrane KCNE1 Peptides

Bas¹,

Gao²,

Lvov

et al. 2011

“…Moreover, the ensuing conformational change will allow for the post-translational N-glycan addition to new conformation-dependent glycosylation sequons. Although post-translational, as opposed to co-translational, N-glycosylation events appear very rare, at least one case involving peptidylglycine ␣-amidating monooxygenase has been rigorously documented (52). The folded tyrosinase protein will leave the ER in an apoenzymatic form and will bind the metal cofactor in the trans-Golgi network.…”

Section: Fig 8 a Cub Glycosylation-deficient Tyr Point Mutant Is Prmentioning

confidence: 99%

Conformation-dependent Post-translational Glycosylation of Tyrosinase

Olivares¹,

Solano

García‐Borrón

2003

Tyrosinase, the rate-limiting enzyme in mammalian melanogenesis, is a copper-containing transmembrane glycoprotein. Tyrosinase undergoes a complex posttranslational processing before reaching the melanosomal membrane. This processing involves N-glycosylation in several sites, including one located in the CuB copper binding site, movement from the endoplasmic reticulum (ER) to the Golgi, copper binding, and sorting to the melanosome. Aberrant processing is causally related to the depigmented phenotype of human melanomas. Moreover, some forms of albinism and several other pigmentary syndromes are considered ER retention diseases or trafficking defects. A critical step in tyrosinase maturation is the acquisition of an ER export-competent conformation recognized positively by the ER quality control system. However, the minimal structural requirements allowing exit from the ER to the Golgi have not yet been identified for tyrosinase or other melanosomal proteins. We addressed this question by analyzing the enzymatic activity and glycosylation pattern of mouse tyrosinase point mutants and chimeric constructs, where selected portions of tyrosinase were replaced by the homologous fragments of the highly similar tyrosinase-related protein 1. We show that a completely inactive tyrosinase point mutant lacking a critical histidine residue involved in copper binding is nevertheless able to exit from the ER and undergo further processing. Moreover, we demonstrate that tyrosinase displays at least two sites whose glycosylation is post-translational and most likely conformationdependent and that a highly specific interaction involving the CuB site is essential not only for correct glycosylation but also for exit from the ER and enzymatic activity.

“…This finding cannot be easily explained if one assumes that N-glycosylation is a strictly cotranslational process, in which growing nascent polypeptide chains are presented to the luminally oriented oligosaccharide transferase in an identical fashion, irrespective of the type of transmembrane or cytoplasmic domain of the mature polypeptide. Alternatively, N-glycosylation of the growing nascent chain may not occur in a strictly cotranslational manner (55), which is also suggested by the fact that the acylated tripeptide Asn-Tyr-Ser can be posttranslationally N-glycosylated (38,56). The extensive N-glycosylation of Asn 84 in II-T-T may therefore reflect their extended accessibility to the N-glycosylation machinery, possibly because of their inability to become incorporated into the oligomeric structure associated with the translocation apparatus in the rough endoplasmic reticulum (see also Ref.…”

Section: Fig 6 Ost48ss Is Retained In the Er When Coexpressed With mentioning

confidence: 99%

Retention of Subunits of the Oligosaccharyltransferase Complex in the Endoplasmic Reticulum

Fu¹,

Kreibich

2000

Membrane proteins of the endoplasmic reticulum (ER) may be localized to this organelle by mechanisms that involve retention, retrieval, or a combination of both. For luminal ER proteins, which contain a KDEL domain, and for type I transmembrane proteins carrying a dilysine motif, specific retrieval mechanisms have been identified. However, most ER membrane proteins do not contain easily identifiable retrieval motifs. ER localization information has been found in cytoplasmic, transmembrane, or luminal domains. In this study, we have identified ER localization domains within the three type I transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Together with DAD1, these membrane proteins form an oligomeric complex that has oligosaccharyltransferase (OST) activity. We have previously shown that ER retention information is independently contained within the transmembrane and the cytoplasmic domain of RII, and in the case of RI, a truncated form consisting of the luminal domain was retained in the ER. To determine whether other domains of RI carry additional retention information, we have generated chimeras by exchanging individual domains of the Tac antigen with the corresponding ones of RI. We demonstrate here that only the luminal domain of RI contains ER retention information. We also show that the dilysine motif in OST48 functions as an ER localization motif because OST48 in which the two lysine residues are replaced by serine (OST48ss) is no longer retained in the ER and is found instead also at the plasma membrane. OST48ss is, however, retained in the ER when coexpressed with RI, RII, or chimeras, which by themselves do not exit from the ER, indicating that they may form partial oligomeric complexes by interacting with the luminal domain of OST48. In the case of the Tac chimera containing only the luminal domain of RII, which by itself exits from the ER and is rapidly degraded, it is retained in the ER and becomes stabilized when coexpressed with OST48.