Fig 2: POTEE was essential for NMT1-mediated N-myristoylation. (A) RPL7A, HBB and POTE family were identified specifically and commonly interacted with NDP and NUP. Three NDP (including LXN, RPL29 and FAU) and three NUP (including AHSG, TF and ALB) were immuno-precipitated by their corresponding antibodies, respectively. The immuno-precipitates were then subjected into MS and the data (two biological replicates) were further evaluated by software STRING. (B) NMT1 stimulated global N-myristoylation depended on POTEE. Global N-myristoylation were measured by CuAAC in control cells, Bel-7402 and Bel-7404 cells with or without POTEE knocked down or overexpressed, as indicated. (C) POTEE was essential for N-myristoylation of both NDP and NUP. N-myristoylation of NDP and NUP was measured by CuAAC and WB in the immuno-precipitates that were immuno-precipitated by the corresponding antibodies, as indicated, in Bel-7402 and Bel-7404 cells. (D) POTEE was critical for the interaction between NMT1 and NDP/NUP. Endogenous NMT1 was immuno-precipitated by anti-NMT1 antibodies in Bel-7402 and Bel-7404 cells overexpressing exogenous WT (with functional NDP/NUP motif) or Mutant (without functional NDP/NUP motif) NDP/NUP-HA. The cells were also transfected with or without increasing concentration of POTEE expressing plasmids. Images of WB are representative ones of 3 independent experiments.

Fig 3: NMT1 regulated RPL7A and HBB to control HIST1H4H. (A) The NDP/NUP motifs were critical for RPL7A, HBB and HIST1H4H binding. NDP/NUP with (WT) or without (Mut) functional NDP/NUP motifs were immuno-precipitated by anti-HA antibodies in Bel-7402 cells, and co-immuno-precipitations of RPL7A, HBB and HIST1H4H were measured by indicated antibodies, as indicated. (B) Ubiquitination of NDP/NUP by HIST1H4H was NDP/NUP motif-dependent Ubiquitination of NDP/NUP was measured by firstly immuno-precipitation of NDP/NUP by anti-HA antibodies, followed by WB using anti-Ub antibodies in Bel-7402 cells expressing NDP/NUP with (WT) or without (Mut) NDP/NUP motifs in the absence or presence of HIST1H4H. (C) NMT1 oppositely controlled HIST1H4H binding to NDP/NUP. HIST1H4H was immuno-precipitated by anti-HIST1H4H antibodies in control cells, Bel-7402 cells expressing increasing concentration of NMT1 (left), and Bel-7402 cells with NMT1 knocked down with or without simultaneous overexpression of NMT1 (right). The co-immuno-precipitation of NDP and NUP was measured by WB using specific antibodies against NDP or NUP, as indicated. (D) The interaction between HIST1H4H and NDP/NUP was alerted when NMT1 was knocked out. The interaction between HIST1H4H and NDP/NUP in the liver of mice, as indicated, was measured using co-IP by firstly immuno-precipitation of HIST1H4H using anti-HIST1H4H antibodies followed by WB using anti-NDP/NUP antibodies, as indicated. (E) RPL7A and HBB are required for HIST1H4H binding to NDP/NUP. HIST1H4H was immuno-precipitated by anti-HIST1H4H antibodies in control cells, and Bel-7402 cells with RPL7A/HBB knocked down or ectopic expressed, as indicated. The co-immuno-precipitations of NDP and NUP were measured by antibodies, as indicated. (F) NMT1 is essential for RPL7A/HBB binding to NDP/NUP. RPL7A/HBB were immuno-precipitated in control cells, Bel-7402 cells with NMT1 knocked down or ectopic expressed, as indicated, and co-immuno-precipitations of NDP and NUP were measured by antibodies, as indicated. (G) POTEE was essential for NMT1-meidated RPL7A/HBB binding to NDP/NUP. RPL7A/HBB were immuno-precipitated by anti-RPL7A/HBB antibodies, and co-immuno-precipitation of NDP/NUP were measured by indicated antibodies in control cells, Bel-7402 cells with or without POTEE knocked down or ectopic expressed in the absence or presence of NMT1 overexpression. Images of WB are representative ones of 3 independent experiments.

Fig 4: pSILAC-SP3 Reveals 75 Nascent Proteins Regulated by the Sema3A-p-eIF2α Pathway(A) Schematic of the pSILAC-SP3 methodology applied to somaless retinal axons.(B) Subset of NSPs regulated in response to Sema3A by p-eIF2α. Only significant NSP changes are shown (blue, downregulation; red, upregulation; p < 0.10).(C) KEGG pathway analysis (red, upregulated pathway; blue, downregulated pathway; cutoff ≥ 2 proteins per pathway).(D) Network-based cluster analysis of the enriched NSP changes induced by Sema3A-p-eIF2α signaling and their associated functional classes (blue nodes, downregulated NSPs; red nodes, upregulated NSPs; light blue edges, interactions known from databases; purple edges, interactions experimentally determined; green stars, NSPs belonging to the “response to stress” category; false discovery rate [FDR] < 0.05).(E and F) IF representative images (E) and quantification (F) for Gsn, Actb, and RpL7a in growth cones pre-incubated with ISRIB for 30 min and co-stimulated with Sema3A for 15 min (one-way ANOVA with Bonferroni’s multiple comparisons test).(G) Correlation analysis of pSILAC- and qIF-derived detection of protein changes (r = 0.92).Error bars indicate SEM. Scale bar, 5 μm. See also Figure S2 and Table S1.

Fig 5: RPL7A and HBB regulated NDP and NUP via HIST1H4H. (A) HIST1H4H was predicted to co-interact with RPL7A and HBB. STRING was performed to reveal the interactions among HIST1H4H, RPL7A and HBB. The domain within HIST1H4H was aligned using Blastp, and the data were extracted from Uniprot. (B) The PPVxxAxxxxV motif was predicated to be similar between RPL7A and HBB, and the bioinformatics was conducted using the CDD database and Blastp. (C) RPL7A and HBB interacted with HIST1H4H. WT or mutant HIST1H4H (without functional pfam02291), as indicated, was co-expressed with WT RPL7A and HBB, respectively in Bel-7402 cells. The exogenous HIST1H4H-HA was immuno-precipitated by anti-HA antibodies, and co-immuno-precipitation of exogenous RPL7A-FLAG and HBB-FLAG was visualized by WB using anti-FLAG antibodies. (D) RPL7A and HBB oppositely controlled ubiquitination of NDP and NUP that mediated by HIST1H4H. Bel-7402 cells were co-overexpressed with WT or mutant HIST1H4H (without functional pfam02291) and WT or mutant RPL7A/HBB (without functional PPVxxAxxxxV motif), as indicated. Ubiquitination of NDP/NUP was firstly immuno-precipitation of NDP/NUP by the corresponding antibodies, and then visualized by anti-Ub antibodies using WB. (E) RPL7A and HBB regulate NDP and NUP expression via HIST1H4H. The protein expression of NDP and NUP were measured by WB in Bel-7402 cells with or without HIST1H4H knocked down or overexpressed, in the presence or absence of RPL7A (upper) or HBB (lower) overexpression. Images of WB are representative ones of 3 independent experiments.