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Inhibition of ER Translocation by Chaperone HSPA13 and Consequences for Proteostasis

Creative Commons 'BY' version 4.0 license

Import into the endoplasmic reticulum (ER) is a necessary process for the third of the eukaryotic proteome that participate in the secretory pathway. Proteins that take up residence in the ER or in a cellular membrane must successfully enter the Sec61 translocon in order to gain access to the ER lumenal complexes responsible for protein maturation.

Our studies identify HSPA13, a truncated ER-residing Hsp70 protein, as an inhibitor of ER import and protein processing. We demonstrate this effect by introducing a new peroxidase-based biotinylation assay for selectively labeling proteins on either side of the ER membrane. We utilize the secretory protein transthyretin (TTR) as a model substrate and show that TTR is inefficiently translocated. Furthermore, we discover that TTR that mistargets to the cytosol experiences slow degradation. We utilize ultracentrifugation to demonstrate a loss of protein solubility that is dependent on HSPA13.

Additionally, we explore HSPA13 function by identifying interactors using affinity purification followed by liquid chromatography coupled with tandem mass spectrometry. Among the discovered interactors were members of protein complexes that are involved with signal sequence cleavage and glycosylation of nascent protein entering the ER. We discover that mutations targeting HSPA13 ATPase activity increase HSPA13 regulatory functions, namely inhibition of ER protein translocation, maturation and secretion. We also associate the regulatory activity of HSPA13 to cytotoxicity, which is enhanced with HSPA13 ATPase mutants.

HSPA13 is conserved in metazoans and presumably provides an unidentified role in cell homeostasis. We employed the CRISPR/Cas9 gene editing system to develop HSPA13 knockout cells which were found to have impaired proteostasis which was observed when they were challenged with ER calcium disruption. Cells lacking HSPA13 also mishandled TTR, which provided additional evidence of compromised protein regulation.

In summary, this dissertation describes the effects of HSPA13 that diminish ER protein translocation and maturation, perhaps through direct interaction with the machinery involved with both processes. Additionally, overexpression or depletion of HSPA13 destabilizes proteostasis and confers deleterious effects.

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