![Generation and validation ofa photoactivatable proNRG3 cleavage reporter (LA-NRG3). (A) Schematic overview of proNRG3-LOV2/Jα fusion proteins tested to derive a configuration with photoactivatable BACE1 processing properties. Fusion constructs varied with regards to the LOV2/Jα insertion site and number of deleted residues in proNRG3 (carboxyl-terminal to L362 [no deletion], P358 [ΔTDHL], S353 [ΔILSDP], or P349 [ΔKTDS]), as well as the extent of carboxyl-terminal deletions of the LOV2 Jα helix, ranging from 0 in LA144 to 8 in LA136 (superscript numbers indicate the overall length of the LOV2/Jα moiety). All fusion protein are based on NGFP-proNRG3-mCherryC (shown above; BACE1 site indicated). LOV2/Jα–encoding residues are shown in blue. Superscript numbers denote amino acid positions relative to their native proteins (proNRG3 and A. sativa phototropin 1, respectively). (B) Effect of LOV2/Jα insertion on NRG3 accumulation at the PM in transfected HEK293 cells. GFP fluorescence intensities at TGN and PM were measured under dark conditions and used to derive TGN over PM ratios illustrating the extent of retention in the TGN. The LA143-NRG3∆ILSDP variant (bold) showed the highest ratio indicative of maximum retention. As controls, TGN retention of proNRG3 lacking the LOV2/Jα domain after 24 h of BACE1 inhibition by BACE-IV (1 µM) is included. Data are plotted as signal intensity ratios and represent the mean ± SEM from three independent experiments (n = 7 cells). (C) Representative images showing subcellular NRG3 NTF/CTF distribution in HEK293 cells transfected with LA144-NRG3362, LA143-NRG3362, LA143-NRG3∆TDHL, and LA143-NRG3∆ILSDP under dark conditions. Note the very low NTF signal for LA143-NRG3∆ILSDP in the PM. (D) Quantitative analysis of experiment shown in C. TGN retention values are plotted as ratios of integrated green or red fluorescence pixel intensities in the TGN over the PM and support the notion that LA143-NRG3∆ILSDP accumulates in the TGN as an unprocessed protein. Data represent the mean ± SEM from three independent experiments (n = 8 cells). Scale bar: 10 μm. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (one-way ANOVA).](https://cdn.rupress.org/rup/content_public/journal/jcb/221/7/10.1083_jcb.202110167/4/jcb_202110167_figs3.png?Expires=1768786142&Signature=pt-bWGpGMQUFkHUAU3yQ577Xi91v7WT60FYVaccYUrtZJIYbtB0Rle8yGRnWfVtlqaQUkmPMz~XAbj5NXL9qJvBTh9Yg-Nxtt8piMhvLMFqhtVlnYlhdhVYgkMXel2uMaIVxeWZVy-zYTWqmMN6nsQns8DkGOKGLU0wvDg1u1LAjWReCRfwW~tjyGx8CcZG2MXP4f~d~hehOjExLBuyYrWht7TPjOxb~1zL~-ZiVGjRWxt3rFZ-CuDviGuM13l4ZBWO9TLq6NcAc63iN4XT32fORHRKgmKpvI4c57Mn1nId-Ilece31EgfQvdaGFGepO~7JX7yMx45ex~NWYTy3MHA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Generation and validation of a photoactivatable proNRG3 cleavage reporter (LA-NRG3). (A) Schematic overview of proNRG3-LOV2/Jα fusion proteins tested to derive a configuration with photoactivatable BACE1 processing properties. Fusion constructs varied with regards to the LOV2/Jα insertion site and number of deleted residues in proNRG3 (carboxyl-terminal to L362 [no deletion], P358 [ΔTDHL], S353 [ΔILSDP], or P349 [ΔKTDS]), as well as the extent of carboxyl-terminal deletions of the LOV2 Jα helix, ranging from 0 in LA144 to 8 in LA136 (superscript numbers indicate the overall length of the LOV2/Jα moiety). All fusion protein are based on NGFP-proNRG3-mCherryC (shown above; BACE1 site indicated). LOV2/Jα–encoding residues are shown in blue. Superscript numbers denote amino acid positions relative to their native proteins (proNRG3 and A. sativa phototropin 1, respectively). (B) Effect of LOV2/Jα insertion on NRG3 accumulation at the PM in transfected HEK293 cells. GFP fluorescence intensities at TGN and PM were measured under dark conditions and used to derive TGN over PM ratios illustrating the extent of retention in the TGN. The LA143-NRG3∆ILSDP variant (bold) showed the highest ratio indicative of maximum retention. As controls, TGN retention of proNRG3 lacking the LOV2/Jα domain after 24 h of BACE1 inhibition by BACE-IV (1 µM) is included. Data are plotted as signal intensity ratios and represent the mean ± SEM from three independent experiments (n = 7 cells). (C) Representative images showing subcellular NRG3 NTF/CTF distribution in HEK293 cells transfected with LA144-NRG3362, LA143-NRG3362, LA143-NRG3∆TDHL, and LA143-NRG3∆ILSDP under dark conditions. Note the very low NTF signal for LA143-NRG3∆ILSDP in the PM. (D) Quantitative analysis of experiment shown in C. TGN retention values are plotted as ratios of integrated green or red fluorescence pixel intensities in the TGN over the PM and support the notion that LA143-NRG3∆ILSDP accumulates in the TGN as an unprocessed protein. Data represent the mean ± SEM from three independent experiments (n = 8 cells). Scale bar: 10 μm. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (one-way ANOVA).