Journal: Nature Communications

Article Title: Spatial proteomics reveals secretory pathway disturbances caused by neuropathy-associated TECPR2

doi: 10.1038/s41467-023-36553-6

Figure Lengend Snippet: a Scheme of RUSH (MAN2A1-SBP-GFP) coupling to APEX2-mediated biotinylation in proteinase K protected trafficking compartments. b RUSH conditions and proteomics workflow. c Homogenates from TECPR2 WT and MUT cells expressing RUSH-APEX2 were left untreated or incubated with proteinase K, RapiGest or both. d ER-to-Golgi trafficking and biotinylation of RUSH-APEX2. TECPR2 WT and MUT cells expressing RUSH-APEX2 were treated with biotin-phenol (BP) as indicated followed by fixation, immunostaining and analysis by confocal microscopy. Scale bars 10 µm. e Quantification of RUSH-APEX2 trafficking by rationing the increase of the Golgi-like GFP-positive area to the total GFP-positive area (normalized to 0 min BP, two-sided t-test analysis, n = 6 independent experiments, error bars represent mean ± SEM, p values = 0.03261, 0.00637 and <0.00001). f Volcano plots of protease-protected, biotinylated proteins altered upon expression of truncated TECPR2 at indicated trafficking conditions. Dark colors highlight class I and light colors class II proteins (two-sided t-test, n = 4 independent experiments). g Venn diagram of class I and class II proteins similarly altered in different trafficking conditions in RUSH-APEX2 expressing TECPR2 WT and MUT cells. h Total abundance of altered RUSH-APEX2 candidates (in percent) in the whole cell proteomic analysis of TECPR2 WT and MUT cells. Orange and brown depict same and opposing trend of changes in RUSH-APEX2 and whole cell proteomics; gray, not changed; white, not present. Selected examples are highlighted. i GO term enrichment of proteins similarly altered at least between 5 min and 30 min BP at 37 °C. Dot size correlates to number of proteins, dot color to term enrichment (p-value). GO BP, biological process; GO CC, cellular compartment; GO MF, molecular function; UP KW, uniport keyword; UP SEQ, uniport sequence feature. j TECPR2 WT and MUT cells were fixed and immunolabeled (left panel). Insets show area of magnification (right panel). Scale bars 10 µm. Quantification of trafficking features (normalized to WT, two-sided t-test analysis, n = 5 independent experiments for LGALS3BP ( p value = 0.02717) and n = 6 independent experiments for CLPTM1L, ERGIC1 and GOLIM4 ( p value = 0.00001, 0.00031 and 0.00003), error bars represent mean ± SEM). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used:ADAMTS1 (Abcam, #ab39194), APEX2 (IgG2A) (custom made by Regina Feederle, HZM München), ATP1A1 (Abcam, #ab7671), B4GALT3 (Proteintech, #11041-1-AP), BAG2 (Biomol, #A304-751A), beta-ACTIN (Sigma,#A1978), BIOTIN (Pierce, #31852), BiP (Cell Signaling, #3177), CALNEXIN (Abcam, #ab22595), Calreticulin (santa cruz, #sc-6467), CD63 (abcam, #ab59479), CLPTM1L (Sigma, #HPA014791), c-myc (Bethyl, #A190-104A), CRLF1 (Novus, #NBP1-85606), DTNBP1 (Bethyl, #A303-360A), EDIL3 (Abcam, #ab190692), ERGIC1 (Proteintech, #16108-1-AP), ERGIC53 (Santa Cruz, #sc398777), EXTL2 (Abcam, #ab168391), Flag M2 (Cell Signaling, #2368), GABARAP(Abcam,#109364), Giantin (Biolegend, #924302), GM130 (Abcam, #ab52649), GOLIM4 (Abcam, #ab28049), HA.11 Clone 16B2 (Covance/Biolegend, #MMS-101P/#901501), HS6ST2 (Abcam, # ab122220), LAMP1 (IF 1:100, Abcam/DSHB, #ab24170/H4A3), LAMP2 (IF 1:100, Abcam, #ab25631), LAMTOR1 (Cell Signaling, #8975S), LAMTOR2 (Cell Signaling, #8145); LAMTOR3 (Cell Signaling, #8168), LC3B (Cell Signaling, #2775S), LGALS3BP (Proteintech, #10281-1-AP), LRP1 (Abcam, #ab92544), M6PR (Abcam, #ab2733), MTOR (Cell Signaling, #2983), myc 9E10 (custom made by Regina Feederle, HZM), NCAM1 (WB: 1:500, IF 1:100, Merck, #AB5032), NEK9 (Abcam, #ab138488), NID1 (Invitrogen, #PA5-99666), NPC1 (WB: 1:500, Abcam, #ab134113), PCNA (Santa Cruz, #sc-7907), PLXDC2 (Novus, #NBP1-76858), PLXNA1 (R&D, #AF4309), PLXNA2 (Abcam, #ab39357), RAB5C (Sigma,#HPA003426), SEC12 (Novus, #NBP1-87056), SEC13 (Novus, #AF9055-100), SEC24C (Abcam, #ab122633), SEC24D (Cell Signaling, #14687), SEC31A (BD, #612351), SLC38A9 (Abcam, #ab81687), SPG20 (Proteintech, #13791-1-AP), TECPR2 (Christian Behrends, custom made), TNC (Abcam, #ab108930), TOLLIP (Abcam, #ab187198), TOMM40 (Abcam, #ab185543), TRAPPC11 (Sigma, #HPA045427), TRAPPC8 (Sigma, #HPA041107), TRAPPC9 (Proteintech, #16014-1-AP), TSG101 (Abcam, #ab30871), TUBULIN (Abcam, #ab7291), VAPA (Sigma, # HPA009174), VAPB (Sigma, # HPA013144), VCP (Bethyl, #A300-588A), VPS11 (Abcam, #ab125083).

Techniques: Expressing, Incubation, Immunostaining, Confocal Microscopy, Sequencing, Immunolabeling

Journal: Nature Communications

Article Title: Spatial proteomics reveals secretory pathway disturbances caused by neuropathy-associated TECPR2

doi: 10.1038/s41467-023-36553-6

Figure Lengend Snippet: a Scheme of RUSH (MAN2A1-SBP-GFP) coupling to APEX2-mediated biotinylation in proteinase K protected trafficking compartments. b RUSH conditions and proteomics workflow. c Homogenates from TECPR2 WT and MUT cells expressing RUSH-APEX2 were left untreated or incubated with proteinase K, RapiGest or both. d ER-to-Golgi trafficking and biotinylation of RUSH-APEX2. TECPR2 WT and MUT cells expressing RUSH-APEX2 were treated with biotin-phenol (BP) as indicated followed by fixation, immunostaining and analysis by confocal microscopy. Scale bars 10 µm. e Quantification of RUSH-APEX2 trafficking by rationing the increase of the Golgi-like GFP-positive area to the total GFP-positive area (normalized to 0 min BP, two-sided t-test analysis, n = 6 independent experiments, error bars represent mean ± SEM, p values = 0.03261, 0.00637 and <0.00001). f Volcano plots of protease-protected, biotinylated proteins altered upon expression of truncated TECPR2 at indicated trafficking conditions. Dark colors highlight class I and light colors class II proteins (two-sided t-test, n = 4 independent experiments). g Venn diagram of class I and class II proteins similarly altered in different trafficking conditions in RUSH-APEX2 expressing TECPR2 WT and MUT cells. h Total abundance of altered RUSH-APEX2 candidates (in percent) in the whole cell proteomic analysis of TECPR2 WT and MUT cells. Orange and brown depict same and opposing trend of changes in RUSH-APEX2 and whole cell proteomics; gray, not changed; white, not present. Selected examples are highlighted. i GO term enrichment of proteins similarly altered at least between 5 min and 30 min BP at 37 °C. Dot size correlates to number of proteins, dot color to term enrichment (p-value). GO BP, biological process; GO CC, cellular compartment; GO MF, molecular function; UP KW, uniport keyword; UP SEQ, uniport sequence feature. j TECPR2 WT and MUT cells were fixed and immunolabeled (left panel). Insets show area of magnification (right panel). Scale bars 10 µm. Quantification of trafficking features (normalized to WT, two-sided t-test analysis, n = 5 independent experiments for LGALS3BP ( p value = 0.02717) and n = 6 independent experiments for CLPTM1L, ERGIC1 and GOLIM4 ( p value = 0.00001, 0.00031 and 0.00003), error bars represent mean ± SEM). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used:ADAMTS1 (Abcam, #ab39194), APEX2 (IgG2A) (custom made by Regina Feederle, HZM München), ATP1A1 (Abcam, #ab7671), B4GALT3 (Proteintech, #11041-1-AP), BAG2 (Biomol, #A304-751A), beta-ACTIN (Sigma,#A1978), BIOTIN (Pierce, #31852), BiP (Cell Signaling, #3177), CALNEXIN (Abcam, #ab22595), Calreticulin (santa cruz, #sc-6467), CD63 (abcam, #ab59479), CLPTM1L (Sigma, #HPA014791), c-myc (Bethyl, #A190-104A), CRLF1 (Novus, #NBP1-85606), DTNBP1 (Bethyl, #A303-360A), EDIL3 (Abcam, #ab190692), ERGIC1 (Proteintech, #16108-1-AP), ERGIC53 (Santa Cruz, #sc398777), EXTL2 (Abcam, #ab168391), Flag M2 (Cell Signaling, #2368), GABARAP(Abcam,#109364), Giantin (Biolegend, #924302), GM130 (Abcam, #ab52649), GOLIM4 (Abcam, #ab28049), HA.11 Clone 16B2 (Covance/Biolegend, #MMS-101P/#901501), HS6ST2 (Abcam, # ab122220), LAMP1 (IF 1:100, Abcam/DSHB, #ab24170/H4A3), LAMP2 (IF 1:100, Abcam, #ab25631), LAMTOR1 (Cell Signaling, #8975S), LAMTOR2 (Cell Signaling, #8145); LAMTOR3 (Cell Signaling, #8168), LC3B (Cell Signaling, #2775S), LGALS3BP (Proteintech, #10281-1-AP), LRP1 (Abcam, #ab92544), M6PR (Abcam, #ab2733), MTOR (Cell Signaling, #2983), myc 9E10 (custom made by Regina Feederle, HZM), NCAM1 (WB: 1:500, IF 1:100, Merck, #AB5032), NEK9 (Abcam, #ab138488), NID1 (Invitrogen, #PA5-99666), NPC1 (WB: 1:500, Abcam, #ab134113), PCNA (Santa Cruz, #sc-7907), PLXDC2 (Novus, #NBP1-76858), PLXNA1 (R&D, #AF4309), PLXNA2 (Abcam, #ab39357), RAB5C (Sigma,#HPA003426), SEC12 (Novus, #NBP1-87056), SEC13 (Novus, #AF9055-100), SEC24C (Abcam, #ab122633), SEC24D (Cell Signaling, #14687), SEC31A (BD, #612351), SLC38A9 (Abcam, #ab81687), SPG20 (Proteintech, #13791-1-AP), TECPR2 (Christian Behrends, custom made), TNC (Abcam, #ab108930), TOLLIP (Abcam, #ab187198), TOMM40 (Abcam, #ab185543), TRAPPC11 (Sigma, #HPA045427), TRAPPC8 (Sigma, #HPA041107), TRAPPC9 (Proteintech, #16014-1-AP), TSG101 (Abcam, #ab30871), TUBULIN (Abcam, #ab7291), VAPA (Sigma, # HPA009174), VAPB (Sigma, # HPA013144), VCP (Bethyl, #A300-588A), VPS11 (Abcam, #ab125083).

Techniques: Expressing, Incubation, Immunostaining, Confocal Microscopy, Sequencing, Immunolabeling