Macromolecular Organization of the Rough Endoplasmic Reticulum at Homeostasis and Stress

Abstract

The endoplasmic reticulum (ER) is an organelle that exists as a patchwork offunctional membrane domains with unique protein components. The rough ER is onesuch domain characterized by associated ribosomal particles and is known as the site oftranslation for secretory and membrane proteins (SMPs). This has since been extendedto include the translation of the entire transcriptome and other diverse functionsincluding autophagosome assembly and miRNA silencing, however, little is knownabout the macromolecular organization of these complex biochemical processes. Usinga proximity labeling technique, BioID, four ribosome associated membrane proteins(RAMPs) were selected to create a rudimentary map of translation on the rough ER bycombining the BioID nonspecific biotin‐labeling with streptavidin pulldowns and massspectrometry. This revealed distinct environments surrounding each RAMP in additionto overlap that established spatial organization of these domains with each other. Of thefour selected, only two, LRRC59 and Sec61β of the Sec61 translocon, labeled ribosomesin vivo. Identification of the mRNAs associated with these labeled ribosomes revealedthat translation was spatially distinct between the two sites. This supports the existenceof complex sorting mechanisms within this domain that extend to sites of optimizedprotein synthesis for sub‐groups of mRNAs.Since organisms exist within changing environments and stressors, the next stepwas to determine if macromolecules associated with the ER during homeostasis changetheir localization during stress, namely the unfolded protein response (UPR).Combining DTT treatment for UPR stimulation with smFISH for ER chaperonestranscriptionally upregulated during this stress, it was determined that only specifictranscripts, e.g. GRP94, are localized to cytoplasmic granules during stress while others,e.g. BiP, are not. Combining these observations with the existing literature, the workinghypothesis is that transcripts, such as BiP, able to recruit ribosomes during times ofinefficient cap‐dependent translation, can escape stress granules while the nascenttranscript RBP environment, such as that on GRP94, is responsible for its recruitment tothese granules. Whether this observation is generalizable to all transcripts being madeupon UPR stimulation is the subject of further study.