Retrograde Transport by Clathrin-Coated Vesicles is Involved in Intracellular Transport of PrPSc in Persistently Prion-Infected Cells

Takeshi Yamasaki,Rie Hasebe,Motohiro Horiuchi,Akio Suzuki

doi:10.1038/s41598-018-30775-1

Takeshi Yamasaki, Rie Hasebe + Show 2 more

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https://doi.org/10.1038/s41598-018-30775-1

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Journal: Scientific Reports	Publication Date: Aug 16, 2018
Citations: 6	License type: open-access

Affiliation: Hokkaido University

Abstract

Intracellular dynamics of an abnormal isoform of prion protein (PrPSc) are tightly associated with prion propagation. However, the machineries involved in the intracellular trafficking of PrPSc are not fully understood. Our previous study suggested that PrPSc in persistently prion-infected cells dynamically circulates between endocytic-recycling compartments (ERCs) and peripheral regions of the cells. To investigate these machineries, we focused on retrograde transport from endosomes to the trans-Golgi network, which is one of the pathways involved in recycling of molecules. PrPSc was co-localized with components of clathrin-coated vesicles (CCVs) as well as those of the retromer complex, which are known as machineries for retrograde transport. Fractionation of intracellular compartments by density gradient centrifugation showed the presence of PrPSc and the components of CCVs in the same fractions. Furthermore, PrPSc was detected in CCVs isolated from intracellular compartments of prion-infected cells. Knockdown of clathrin interactor 1, which is one of the clathrin adaptor proteins involved in retrograde transport, did not change the amount of PrPSc, but it altered the distribution of PrPSc from ERCs to peripheral regions, including late endosomes/lysosomes. These data demonstrated that some PrPSc is transported from endosomes to ERCs by CCVs, which might be involved in the recycling of PrPSc.

Highlights

The intracellular dynamics of PrPSc in cells persistently infected with prions have been analyzed in order to investigate the mechanisms of PrPSc formation
In a subclone of Neuro2a cells persistently infected with 22 L prion (ScN2a-3-22L cells), PrPSc at the perinuclear region was partially co-localized with the components of clathrin-coated vesicles (CCVs)—clathrin heavy chain (CHC), Ap1g1, and clathrin interactor 1 (Clint1)—as well as the components of the retromer complex: Snx[1], vacuolar protein sorting (Vps)[26], Vps[29], and Vps[35]
Ratios of PrPSc signals co-localized with the signals of CHC, Clint[1], Ap1g1, Snx[1], Vps[26], Vps[29], Vps[35] and Rab[9] relative to the total PrPSc signals were 13%, 8%, 29%, 14%, 9%, 14%, 11%, and 13%, respectively, which confirmed the partial co-localization of PrPSc with these component molecules

Summary

Introduction

The intracellular dynamics of PrPSc in cells persistently infected with prions have been analyzed in order to investigate the mechanisms of PrPSc formation. Recent studies showed that some part of the internalized PrPSc is sorted by the retromer complex within early or late endosomes[17,19] It is not clear whether the destination of PrPSc transported by the retromer complex is to either the retrograde pathway for recycling or the endolysosomal pathway for degradation. We showed that PrPSc in persistently prion-infected cells shared the endocytic pathway with exogenously introduced STxB and CTxB that passed through ERCs during their retrograde transport from early endosomes to the TGN13. These facts raised the hypothesis that PrPSc is transported to ERCs by a certain cellular machinery for the retrograde transport from endosomes to the TGN in a similar manner to cargo proteins such as STxB and CTxB. We found that PrPSc is present within clathrin-coated vesicles (CCVs) in the cells, and at least part of the PrPSc is transported from peripheral endosomal compartments to ERCs by CCVs

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