Morphologically Discrete ER Subdomains Support the Synthesis of Different Types of Protein

Abstract

The endoplasmic reticulum (ER) has a complex morphology comprised of stacked sheets, tubules, and three-way junctions, which together function as a platform for protein synthesis of the membrane and secretory proteins. It is widely believed that specific ER subdomains are spatially organized for protein synthesis activities, but precisely where these domains occur in relation to the ER's overall architecture is unclear due to lack of functional-spatial resolution. Here, we use single-molecule tracking of ribosomes and mRNAs in combination with simultaneous imaging of ER to assess sites of membrane protein synthesis in the ER. We found that ribosomes were widely distributed throughout the different ER morphologies. However, synthesis of membrane proteins (including type I, II and multi-spanning) and an ER luminal protein occurred primarily at the three-way junctions. Unlike mRNAs coding for membrane proteins, mRNA coding for the translocon-independent, tail-anchored protein Sec61β was found primarily on ER tubules and was actively translated on the ER. Surprisingly, mRNA coding for the cytosolic protein β-actin was also primarily associated with ER tubules. These results support the idea that discrete ER subdomains exist to support specific types of protein synthesis activities, with ER morphological features playing an important role in this organization