JCB_201702159 3625..3638

Asparagine-linked glycosylation is an essential protein modification reaction critical for the folding, trafficking, and function of many proteins that enter the secretory pathway. In most eukaryotes, the oligosaccharyltransferase (OST) complex is composed of a catalytic subunit (an STT3 protein) plus as many as eight accessory subunits. Plant and metazoan organisms encode two STT3 proteins (STT3A and STT3B) that are assembled with a set of six shared OST subunits (ribophorins I and 2, OST48, DAD1, OST4, and TMEM258). An oxidoreductase, either MagT1 or TUSC3, is assembled with STT3B as a complex-specific accessory subunit (Cherepanova et al., 2014; Cherepanova and Gilmore, 2016). DC2 (OSTC gene) and KCP2, two small membrane proteins, were initially detected as novel OST subunits in translocation channel–associated OST complexes (Shibatani et al., 2005). STT3A complexes interact with the protein translocation channel (Shibatani et al., 2005; Ruiz-Canada et al., 2009) and mediate co-translational N-glycosylation when the acceptor site (NXT/S/C, sequon) is positioned ∼65–75 residues from the peptidyltransferase site on the ribosome (Whitley et al., 1996; Nilsson et al., 2003). The STT3A and STT3B complexes have complementary roles in N-linked glycosylation. Depletion of STT3A by siRNA treatment or knockout of STT3A by CRI SPR/Cas9 gene editing causes hypoglycosylation of proteins, resulting in induction of the unfolded protein response (UPR) pathway (Ruiz-Canada et al., 2009; Cherepanova and Gilmore, 2016). The STT3B complex can mediate posttranslocational glycosylation of acceptor sites that have been skipped by the STT3A complex, including sequons that are located in the last 65 residues of the protein (Shrimal et al., 2013b). Co-translational glycosylation by the STT3A complex is responsible for the modification of the majority of the sequons in the glycoproteome; hence, depletion of STT3B or disruption of the STT3B gene causes little or no induction of the UPR pathway (Ruiz-Canada et al., 2009; Cherepanova and Gilmore, 2016). A co-translational scanning mechanism enhances glycosylation efficiency because acceptor sites in folded polypeptides do not have access to the catalytic site in STT3 proteins, as revealed by the structures of the eubacterial and archaebacterial STT3 homologues PglB and AglB (Lizak et al., 2011; Matsumoto et al., 2013). The mechanism responsible for localization of the STT3A complex adjacent to the protein translocation channel has not been addressed at either a biochemical or structural level. The shared OST subunits are unlikely to contribute to localization, as their presence in both complexes would result in competition between STT3A and STT3B for the Sec61-proximal location. One hypothesis would be that the STT3 proteins, which are only 59% identical in sequence, contain segments that either promote for STT3A, or prevent for STT3B, a stable interaction with the protein translocation channel. Alternatively, the DC2 or KCP2 proteins might mediate the interaction with the Sec61 In metazoan organisms, the STT3A isoform of the oligosaccharyltransferase is localized adjacent to the protein translocation channel to catalyze co-translational N-linked glycosylation of proteins in the endoplasmic reticulum. The mechanism responsible for the interaction between the STT3A complex and the translocation channel has not been addressed. Using genetically modified human cells that are deficient in DC2 or KCP2 proteins, we show that loss of DC2 causes a defect in co-translational N-glycosylation of proteins that mimics an STT3A−/− phenotype. Biochemical analysis showed that DC2 and KCP2 are responsible for mediating the interaction between the protein translocation channel and the STT3A complex. Importantly, DC2and KCP2-deficient STT3A complexes are stable and enzymatically active. Deletion mutagenesis revealed that a conserved motif in the C-terminal tail of DC2 is critical for assembly into the STT3A complex, whereas the lumenal loop and the N-terminal cytoplasmic segment are necessary for the functional interaction between the STT3A and Sec61 complexes. DC2 and KCP2 mediate the interaction between the oligosaccharyltransferase and the ER translocon