Abstract
We have used misfolded prion protein (PrP*) as a model to investigate how mammalian cells recognize and degrade misfolded GPI-anchored proteins. While most misfolded membrane proteins are degraded by proteasomes, misfolded GPI-anchored proteins are primarily degraded in lysosomes. Quantitative flow cytometry analysis showed that at least 85% of PrP* molecules transiently access the plasma membrane en route to lysosomes. Unexpectedly, time-resolved quantitative proteomics revealed a remarkably invariant PrP* interactome during its trafficking from the endoplasmic reticulum (ER) to lysosomes. Hence, PrP* arrives at the plasma membrane in complex with ER-derived chaperones and cargo receptors. These interaction partners were critical for rapid endocytosis because a GPI-anchored protein induced to misfold at the cell surface was not recognized effectively for degradation. Thus, resident ER factors have post-ER itineraries that not only shield misfolded GPI-anchored proteins during their trafficking, but also provide a quality control cue at the cell surface for endocytic routing to lysosomes.
Highlights
Maintenance of a correctly folded proteome is critical for cellular and organismal homeostasis
To perform quantitative analysis of misfolded GPI-anchored protein degradation, we first generated and characterized a stable doxycycline-inducible HEK293T cell line expressing GFP-tagged prion protein (PrP)* (GFP-PrP*) integrated into a single defined locus in the genome. This mutant of PrP contains a Cys to Ala change at position 179, thereby preventing the formation of a critical disulfide bond required for PrP folding (Satpute-Krishnan et al, 2014)
A matched cell line expressing wild type GFP-PrP from the same locus served as a control in these studies
Summary
Maintenance of a correctly folded proteome is critical for cellular and organismal homeostasis. In the specific case of PrP, surface-exposed misfolded forms may facilitate uptake of prions into cells (Fehlinger et al, 2017) From these combined studies in yeast and mammalian cells, it is thought that both folded and misfolded GPI-anchored proteins engage TMED family export receptors at the ER and traffic to the Golgi. We used quantitative flow cytometry and proteomic analyses to show that the majority of PrP* traffics via the cell surface to lysosomes in a complex with resident ER chaperones and cargo receptors This suggests that minor populations of abundant factors long thought to be restricted to the early secretory pathway have functional excursions to the cell surface during quality control of GPIanchored proteins
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