N-linked glycosylation and homeostasis of the endoplasmic reticulum

Abstract

As a major site of protein biosynthesis, homeostasis of the endoplasmic reticulum is critical for cell viability. Asparagine linked glycosylation of newly synthesized proteins by the oligosaccharyltransferase plays a central role in ER homeostasis due to the use of protein-linked oligosaccharides as recognition and timing markers for glycoprotein quality control pathways that discriminate between correctly folded proteins and terminally malfolded proteins destined for ER associated degradation. Recent findings indicate how the oligosaccharyltransferase achieves efficient and accurate glycosylation of the diverse proteins that enter the endoplasmic reticulum. In metazoan organisms two distinct OST complexes cooperate to maximize the glycosylation of nascent proteins. The STT3B complex glycosylates acceptor sites that have been skipped by the translocation channel associated STT3A complex.

Figure 1

Asparagine linked glycosylation allows glycoprotein entry into the CNX/CRT cycle. (a–d) GN₂M₉G₁ dependent binding of unfolded proteins to CNX and CRT. For simplicity, the interaction of ERp57 and CypB with CNX and CRT is only depicted in diagrams c and d. (e, f) Gls2 and UGGT promote cyclic binding and release of glycoproteins form CNX (d) and CRT(c). (g,h) ERMan1 cleavage of the B-branch mannose residue reduces the reglucosylation of the glycan by UGGT. (i, j) Folded glycoproteins that exit the CNX/CRT cycle are trimmed by ERMan1 to yield protein linked GN₂M_8–9. (k) Secretory cargo proteins can be recognized by the lectins ERGIC53, VIP36 and VIPL. Non-glycosylated glycoproteins (l) do not enter the CNX/CRT cycle and are prone to misfolding (m). (n) The diagram shows the symbol code for saccharide residues in GN₂M₉G₃, and indicates the location of the A, B and C branches. (o) The folding status of glycoproteins in diagrams a–m. The gray arrow in diagram h indicates that the pathway shown is less favored relative to that shown with black arrows.

Figure 2

Trimmed N-linked glycans promote ERAD of terminally misfolded proteins. The glycans on unfolded glycoproteins that exit the CNX/CRT cycle (a) are trimmed by ERMan1 (b). Successive processing by α1–2 mannosidase activity of the EDEMs yields protein-linked glycans on misfolded proteins with 5–7 mannose residues (c–e) that are recognized by the ERAD lectins OS-9 and XTP3-B (f) for targeting to the HRD1-Sel1 complex (g) for subsequent retrotranslocation of the misfolded protein into the cytosol. Accumulation of hypoglycosylated misfolded glycoproteins (h, red asterisks indicate skipped sequons) is one cause of ER stress that is sensed by the UPR sensors (i). For simplicity, only a single stress sensor (Ire1, PERK or ATF-6) is shown. The gray arrow in diagram f indicate that the pathway shown is less favored relative to those shown with black arrows.

Figure 3

Cooperation between the STT3A and STT3B complexes maximizes protein glycosylation efficiency. (a, b) Subunit composition of the STT3A and STT3B complexes. Ribophorin 1, ribophorin 2 and TMEM258 are abbreviated as Rb1, Rb2 and Ost5, respectively. (c) Cotranslational translocation of a nascent glycoprotein through the Sec61 protein translocation channel allows cotranslational glycosylation of acceptor sequons by the STT3A complex. The malectin-OST association is only shown in this diagram for simplicity. (d) Cotranslational glycosylation of cysteine rich proteins before disulfide bond formation. (e) Examples of acceptor sequons that have been skipped by the STT3A complex including extreme N and C-terminal sites, closely spaced NXS sites, cysteine proximal sites (NCS) and a poorly understood class of non-optimal sequons (designated as NXS). (f, g) Posttranslocational glycosylation of extreme C-terminal sequons (red asterisks) does not occur by an N to C-terminal scanning mechanism but is instead determined by the relative affinity of the STT3B active site for NXT and NXS sequons. (h–j) Posttranslocational glycosylation of cysteine proximal sequons by the STT3B complex can be facilitated by reversible formation of a mixed disulfide between MagT1 (or TUSC3) and the unfolded glycoprotein. Red asterisks in diagrams e–h indicate skipped acceptor sites.