Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum

Abstract

Protein synthesis, transport, and N-glycosylation are coupled at the mammalian endoplasmic reticulum by complex formation of a ribosome, the Sec61 protein-conducting channel, and oligosaccharyltransferase (OST). Here we used different cryo-electron microscopy approaches to determine structures of native and solubilized ribosome-Sec61-OST complexes. A molecular model for the catalytic OST subunit STT3A (staurosporine and temperature sensitive 3A) revealed how it is integrated into the OST and how STT3-paralog specificity for translocon-associated OST is achieved. The OST subunit DC2 was placed at the interface between Sec61 and STT3A, where it acts as a versatile module for recruitment of STT3A-containing OST to the ribosome-Sec61 complex. This detailed structural view on the molecular architecture of the cotranslational machinery for N-glycosylation provides the basis for a mechanistic understanding of glycoprotein biogenesis at the endoplasmic reticulum.

Fig. 1.. RTCs harbor exclusively STT3A complexes.

(A) Schematic representation and membrane topology of OST subunits for the STT3A (red frame) and STT3B complexes (green frame, yeast names in parentheses). Shared subunits are depicted in pink. OST subcomplexes are indicated for the STT3A complex. (B) Microsomes from wild type or mutant HEK293 cells were analyzed by immunoblotting using rabbit polyclonal antibodies. The arrowhead in the STT3B blot designates a nonspecific background band. (C)-(E) Ribosome-bound translocon populations observed for microsomes from wild type HEK293 (C), STT3B(−/−) (D) and STT3A(−/−) (E) cell lines after in silico sorting. The absolute number and ratio of subtomograms contributing to each class are given. All densities were filtered to 30 Å resolution.

Fig. 2.. Localization of STT3A, RPN1 and DC2 in ribosome-bound OST.

(A) Cryo-EM structure of the active solubilized RTC. Ribosome and P-Site tRNA are shown before, the membrane region including Sec61, TRAP and OST after focused refinement (Fig. S2) low-pass filtered to 4 Å. (B) Zoom on the translocon region omitting TRAP as depicted in (A) (upper panel) or rotated by 90° (lower panel). (C, D) Fitted homology model for mammalian STT3A. Density for phosphate groups in the catalytic center is green. (E) Close up view of the cytosolic RPN1 four-helix bundle binding to the ribosome. (F) Zoom on the Sec61-OST interface with a fitted model for the DC2 TMs.

Fig. 3.. Translocon dynamics and scheme for cotranslational N-glycosylation.

(A) Models for Sec61 and OST were fitted into the RTC densities with laterally closed (left: programmed, central: non-programmed) and opened Sec61 (right). (B) Trajectories of Cα atoms connecting the observed conformational states with color-coded length. (C) Schematic representation of the RTC with an interpolated example path for a nascent secretory protein. The STT3A catalytic site and a signal peptide (SP) or TM in the Sec61 lateral gate are separated by ~6.5 nm. (D) Molecular basis for STT3 paralog specificity in the RTC. The DC2 and RPN1 subunits tie the STT3A complex into the RTC (upper panel). The lack of DC2 and potential interference of the STT3B-specififc cytosolic domain with ribosome binding exclude STT3B complexes from the RTC.