Fig. 3

Selective degradation of non-encapsulated cargo. a Schematic of genetic construct showing a minimal C-terminal encapsulation signal (EncSig) fused to the photoactivatable fluorescent protein mEos4b (mEos4b-EncSig) to associate it to the inner surface of the nanocompartment. When mEos4b-EncSig is N-terminally fused to an FKBP12-derived destabilizing domain (DD), it is degraded by the proteasome unless it is sequestered into the encapsulin shell. This strategy thus selectively enriches cargo inside the lumen of the nanocompartment. b Cargo loading of mEos4b-EncSig into the nanocompartment composed of A^FLAG was demonstrated by co-immunoprecipitation (Co-IP) against the FLAG epitope followed by silver-stained SDS-PAGE (left panel). Corresponding analysis of whole cell lysate by BN-PAGE on a UV imager shows fluorescence of the native encapsulin band indicating the presence of the mEos4b-EncSig cargo. c Representative 2 × 2 table of epifluorescence microscopy images from HEK293T cells co-expressing DD-mEos4b-EncSig with or without A^FLAG (upper row) compared to coexpression of the cargo without destabilizing domain (mEos4b-EncSig, lower row). Scale bar represents 50 µm. d Corresponding quantification of the fluorescence intensities as exemplified in c. Co-expression of A^FLAG significantly protects degradation of DD-mEos4b-EncSig (p = 0.0286, Mann–Whitney test, n = 4 biological replicates, error bars represent mean ± SEM). DD-mEos4b-EncSig can also be stabilized by adding the small molecule Shield1 (0.5 mM, magenta label) to the cell culture medium. e Confocal microscopy images of HEK293T expressing DD-mEos4b-EncSig with or without A^FLAG. As a reference DD-mEos4b-EncSig was stabilized via the addition of 0.5 mM Shield1. Please note that the contrast of all images was linearly adjusted to the same extent optimizing for the condition shown on the left, which resulted in partial oversaturation of the condition shown on the right. Scale bar represents 20 µm