Figure 5. Secretion of tandem α-syn oligomers and α-syn fused with thermostable helix-bundle protein.

(A) Schematic diagrams of tandem α-syn oligomers. α-syn protomers (yellow) were linked head to tail by flexible linker (green) to mimic increased size of α-syn oligomers. (B) Secretion of tandem α-syn oligomers in medium. Secretion assay was performed with media from HEK293T cells transfected with indicated tandem α-syn oligomers. Tandem α-syn oligomers are more sensitively detected by immunoblot which were exposed for shorter time compared with WT α-syn. (C) Expression of tandem α-syn oligomers in HEK293T cells. *a non-specific band. (D) Schematic diagrams of N-terminal fused and C-terminal fused thermostable three helix-bundle (3H-) α-syn. 3H shown as three blue dashes, α-syn shown as orange circle. (E) Secretion of 3H-α-syn and α-syn-3H in medium. Secretion assay was performed with media from HEK293T cells transfected with indicated 3H-fused α-syn constructs. *a non-specific band. (F) Expression of 3H-α-syn and α-syn-3H in HEK293T cells. *a non-specific band.

Figure 5—figure supplement 1. Medium fractionation of secreted tandem α-syn oligomers.

Medium fractionation of secreted tandem α-syn oligomers. P-DNAJC5 was depleted in the supernatant after centrifugation at 100,000 (100k)×g. However, no significant decrease of tandem α-syn oligomers in the supernatant was observed.

Figure 5—figure supplement 2. Thermostable three helix bundle blocked pOTC-mediated mitochondrial import of GFP.

(A) Schematic diagrams of constructs used in mitochondrial import assay. GFP, GFP alone. pOTC-GFP, GFP with leader peptide from ornithine ranscarbamylase (OTC) fused at amino-terminus. pOTC-3H-GFP, the thermostable three helix bundle was inserted between the leader peptide pOTC and GFP. (B) Immunoblot analysis of proteins in crude mitochondria fraction. Whole-cell lysate (W) was prepared by mixing homogenized cells with equal volume of 2.3 M sucrose buffer and centrifugation at 1200×g to remove large debris. Soluble fraction (S) and particulate fraction (P) were separated by centrifuging W fraction at 7000×g for 10 min. Mitochondria were enriched in P fraction. Tom20, mitochondrial marker. Tubulin, soluble marker. (C) The mitochondrial localization efficiency was calculated by quantifying the protein amount in P fraction divided by the amount in S fraction. (D) Cartoon depicting relative localization of proteins inside or outside of mitochondria. Tom20, a mitochondrial outer membrane protein. Citrate synthase (CS), a mitochondrial matrix protein. The import of pOTC-3H-GFP is blocked by 3H. (E) Proteinase K protection assay of crude mitochondria. SDS (0.5%) was added in addition to 1% TX-100 to sensitize protease treatment of well-folded protein, for example, CS. The protease accessibility of pOTC-3H-GFP was revealed by the production of GFP fragment and disappearance of the full-length (FL) band. (F) Quantification of percentage of protection of proteins in (E).