In cells so called ribosomes synthesize proteins destined for various intra- and extracellular locations. Many of the newly synthesized proteins are chemically modified, for example by addition of sugars (glycosylation). Researchers from the Ludwig-Maximilians-Universität München (LMU), the Max Planck Institute for Biochemistry and prof. Friedrich Förster of Utrecht University, have now determined the three-dimensional structure of the macromolecular complex, which couples glycosylation to ribosomal protein synthesis and protein transport at the endoplasmic reticulum (ER) membrane. The ER is a tubular network of membranes that serves as a binding platform for the ribosomes. Their study provides the basis for a detailed understanding of this vital cellular process and has been published in the journal Science.
Science-publication about intracellular transport in 3D
Complex interplay between protein synthesis, transport and modification visualized
“The addition of sugars is a very fundamental mechanism in all higher organisms. It is essential for the successful functioning of a third of the proteins synthesized in the ribosomes”, Prof. Friedrich Förster of Utrecht University explains. “Thanks to the quantum leap in technologies such as Cryo-Electron Microscopy, we have now been able to visualize and model this mechanism.”
Biological membranes such as the endoplasmic reticulum (ER) membrane are essentially impermeable to polar (electrically charged) molecules such as proteins. However, certain specialized proteins can act as channels for the passage of other proteins. At the ER membrane such a ‘translocon’ allows proteins synthesized by ER-bound ribosomes to enter the interior of the ER or to be integrated into the ER membrane, respectively. As it passes across the membrane, the growing protein is modified at specific sites by the attachment of an ‘oligosaccharide’ made up of a chain of 14 sugar molecules, in a process known as glycosylation.
Delivery to the correct destination
This chemical label ensures that the protein is subsequently delivered to the correct destination. In addition, the label plays a vital role in enabling the nascent protein to fold into the appropriate shape required for its biological function. “Errors in glycosylation lead to the accumulation of improperly folded proteins, and these in turn activate cellular stress responses – often with fatal consequences for the cell,” says first author Katharina Braunger, a member of the Beckmann Lab in Munich.
Two forms that differ in function
The enzyme complex, which attaches the oligosaccharide while the nascent protein chain is fed through the translocon by the ribosome, is known as the OST (oligosaccharyltransferase) complex. In higher organisms the OST complex is present in two different compositions. Using a combination of cryo-electron microscopy and -tomography, the researchers have now obtained structural evidence that these two forms also differ in their function.
The image shows the three-dimensional structure of the OST-containing ribosome-translocon complex. Two of the OST subunits (yellow and magenta) link its catalytic subunit (red) to the protein-conducting channel (blue) and the ribosome (gray).
Source: K. Braunger, LMU
A- and B- variant
A-form OST interacts with both the active ribosome and the protein conducting channel to form a stable complex, and it modifies the growing protein during its production. In contrast, the other variant is unable to bind to the translocon. “The B variant is responsible for proof reading as well as the modification of sites that are not accessible to the A variant,” says Prof. Roland Beckmann of LUM.
Coupling protein transport and glycosylation
The new data has allowed the team to define the three-dimensional organization of the subunits in ribosome-bound OST complexes, and to develop a molecular model for their function. The model allows them to propose the basis for differences in specificity between the two variants, and explains how protein transport and glycosylation are coupled mechanistically.
Structural basis for coupling of protein transport and N-glycosylation at the mammalian endoplasmic reticulum
Katharina Braunger, Stefan Pfeffer, Shiteshu Shrimal, Reid Gilmore, Otto Berninghausen, Elisabet C. Mandon, Thomas Becker, Friedrich Förster, Roland Beckmann
Science, 8 March 2018