Macromolecular Organization of the Rough Endoplasmic Reticulum at Homeostasis and Stress

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2019

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Abstract

The endoplasmic reticulum (ER) is an organelle that exists as a patchwork of

functional membrane domains with unique protein components. The rough ER is one

such domain characterized by associated ribosomal particles and is known as the site of

translation for secretory and membrane proteins (SMPs). This has since been extended

to include the translation of the entire transcriptome and other diverse functions

including autophagosome assembly and miRNA silencing, however, little is known

about the macromolecular organization of these complex biochemical processes. Using

a proximity labeling technique, BioID, four ribosome associated membrane proteins

(RAMPs) were selected to create a rudimentary map of translation on the rough ER by

combining the BioID nonspecific biotin‐labeling with streptavidin pulldowns and mass

spectrometry. This revealed distinct environments surrounding each RAMP in addition

to overlap that established spatial organization of these domains with each other. Of the

four selected, only two, LRRC59 and Sec61β of the Sec61 translocon, labeled ribosomes

in vivo. Identification of the mRNAs associated with these labeled ribosomes revealed

that translation was spatially distinct between the two sites. This supports the existence

of complex sorting mechanisms within this domain that extend to sites of optimized

protein synthesis for sub‐groups of mRNAs.

Since organisms exist within changing environments and stressors, the next step

was to determine if macromolecules associated with the ER during homeostasis change

their localization during stress, namely the unfolded protein response (UPR).

Combining DTT treatment for UPR stimulation with smFISH for ER chaperones

transcriptionally upregulated during this stress, it was determined that only specific

transcripts, 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 working

hypothesis is that transcripts, such as BiP, able to recruit ribosomes during times of

inefficient cap‐dependent translation, can escape stress granules while the nascent

transcript RBP environment, such as that on GRP94, is responsible for its recruitment to

these granules. Whether this observation is generalizable to all transcripts being made

upon UPR stimulation is the subject of further study.

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Hoffman, Alyson Marie (2019). Macromolecular Organization of the Rough Endoplasmic Reticulum at Homeostasis and Stress. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/19798.

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