Figure 4.
ER receptor P180 is enriched in axons and regulates local translation
(A and B) Representative images of neurons expressing a fill and the ER-resident proteins GFP-Sec61β, P180-GFP, GFP-LRRC59, RPN1-GFP, or mNG-KTN1 (A), and respective quantification of polarity index (B).
(C) Scaled representation of proteins identified with mass spectrometry after pull-down of GFPbio or P180-GFPbio from adult rat brain extracts. The size and color of each dot reflect the number of PSMs or peptides identified as indicated in the legend.
(D) Western blot validation for ribosomal protein interactions with P180-GFPbio after streptavidin pull-down. Asterisk indicates that the presence of the coiled-coil domain in P180 causes protein instability, resulting in a banded pattern, as previously described.44 The double asterisk indicates an aspecific band at a different molecular weight than RpL24.
(E and F) Representative images of puromycilated peptides in distal axons from neurons expressing a fill plus control pSuper plasmid or shRNAs targeting P180 (E). Quantification in (F).
(G and H) Representative images of split APEX in distal axons for V5-AP-Sec61β and RpL10A-3xHA-EX, in the presence of control pSuper plasmid or shRNAs targeting P180 (G).Quantification in (H).
(I) Quantification of axonal ER-bound ribosomes, using split APEX assay, in neurons expressing a pSuper control shRNA or shRNAs against P180 stimulated with BSA (control) or NT-3 for 30 min.
(J) Western blot quantification of ribosomal proteins after GFPbio or P180-GFPbio pull-down with and without RNAseA/T1 treatment.
Individual data points each represent a neuron (B), (F), (H), and (I) or an independent experiment (J). Each color represents an independent experiment. Data are presented as mean values ± SEM in (F), (H), (I) or ± SD in (J). Boxplots show 25/75-percentiles, the median, and whiskers represent min to max in (B) ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 comparing conditions to control using Mann-Whitney tests (F), (H), and (J) or one-way ANOVA test (I). Scale bars represent 10 μm (A) and 5 μm (E) and (G).