Date of Award

Fall 10-1-2021

Document Type


Degree Name

Doctor of Philosophy (PhD)


Cell Biology

First Advisor

Mariappan, Malaiyalam


Membrane proteins contain hydrophobic transmembrane domains (TMDs) which reside in the nonpolar section of cellular membranes. The synthesis of the majority of membrane proteins in eukaryotes is routed through the co-translational insertion pathway. As they are translating nascent membrane proteins, ribosomes are delivered to the endoplasmic reticulum (ER) where the new membrane protein is co-translationally inserted directly into the membrane as it emerges from the ribosome exit tunnel. Thus, newly translated hydrophobic TMDs are never released into the aqueous cytosol. However, exceptions to the co-translational insertion route exist. Certain membrane proteins are released into the cytosol and must be captured by chaperones and targeted to a membrane for post-translational insertion. One class of proteins that require post-translational membrane insertion is tail-anchored (TA) membrane proteins. As their name suggests, TA proteins contain a single TMD at their carboxy (C) terminus. When the TMD of a nascent TA protein emerges from the ribosome exit tunnel, it cannot be routed for co-translational insertion because translation has already terminated. Thus, TA proteins containing hydrophobic TMDs are released into the aqueous cytosol. Such exposed hydrophobicity can trigger nonspecific protein-protein interactions or protein aggregation, harming healthy cellular function. To address the dangers posed by hydrophobic TMDs in the cytosol, the TRC/GET chaperone pathways capture, shield, and post-translationally insert TA proteins. The mammalian TRC (transmembrane recognition complex) pathway and the homologous yeast GET (guided entry of tail-anchored proteins) pathway are conserved in all eukaryotes. Although the chaperones and mechanisms that drive nascent TA protein insertion have been elucidated, how cells recognize and handle exposed hydrophobicity remains less clear, particularly when comparing misfolded proteins and nascent membrane proteins. Misfolded proteins are targeted due to their exposed hydrophobicity by quality control machinery. Since both nascent TA proteins and misfolded proteins contain regions of exposed hydrophobic amino acids, how do cells accurately distinguish between TA proteins intended for insertion and misfolded proteins intended for degradation? My research published in the Journal of Cell Biology (DOI: 10.1083/jcb.202004086) shows that a portion of nascent TA proteins are ubiquitinated in the cytosol. Typically, ubiquitination routes a protein for proteasomal degradation. However, these ubiquitinated TA proteins are properly inserted into the ER and are deubiquitinated by the homologous deubiquitinases USP20 and USP33. Therefore, despite both containing exposed hydrophobicity and being ubiquitinated, TA proteins and misfolded proteins are distinguished from each other within cells as evidenced by their different fates. To follow up the novel finding of ubiquitin on nascent TA proteins, I ask what function this ubiquitination may serve. Does the presence of ubiquitination impact how TA proteins behave or how they are handled? It appears that nascent TA proteins are well folded regardless of ubiquitination status. I have found that disrupting the AAA+ ATPase P97 lowers the insertion efficiency of ubiquitinated TA proteins, but not lysine-free (and thus ubiquitin-free) TA proteins. This may indicate a currently undiscovered role for P97 in TA protein production. In addition to exposed hydrophobicity and ubiquitination, TA proteins and misfolded proteins also share machinery such as BAG6. BAG6 acts as a scaffold to bring TA protein insertion factors together. BAG6 also routes misfolded proteins for degradation by recruiting the E3 ligase RNF126 to add ubiquitin to misfolded proteins. Properly balanced protein synthesis and degradation is referred to as protein homeostasis. Certain environmental conditions or human diseases can disrupt protein homeostasis leading to the accumulation of misfolded proteins. Therefore, I examined if the accumulation of misfolded proteins could block TA protein synthesis by occupying their shared chaperones. Indeed, I observed slower insertion of TA proteins into the ER when misfolded proteins accumulate. Furthermore, the shared chaperones shift in binding kinetics and can localize to protein aggregates during stress. Understanding how misfolded proteins interfere with TA protein synthesis would shed light on the dangers faced by cells during protein folding diseases and how cells distinguish between misfolded and nascent proteins under normal conditions. Lastly, a link between the O-linked glycosylation and TA protein synthesis. Previously, BAG6 was found to interact with O-linked N-acetylglucosamine transferase (OGT). OGT adds O-linked glycosylation to proteins. I found that the BAG6-OGT interaction is sensitive to protein homeostasis stress. Perhaps O-linked glycosylation assists relevant chaperones in determining the fate of bound substrates i.e., sending TA proteins to the ER and misfolded proteins to the proteasome.