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. 2016 Dec 8;36(1):42–60. doi: 10.15252/embj.201695081

Figure EV3. TRIM16 recruits IL‐1β to LC3‐positive carrier membranes for secretion and motifs in TRIM16 and Sec22b.

Figure EV3

  • A, B
    IL‐1β levels in supernatants of HeLa cells reconstituted with flag‐pro‐IL‐1β and myc‐caspase‐1 and treated as indicated.
  • C
    CRISPR TRIM16 mutant A9 in HeLa cells (Chauhan et al, 2016).
  • D
    Processing of pro‐IL1β detected by immunoblotting of lysates from HeLa cells (wild type and TRIM16KO) reconstituted with pro‐IL‐1β and pro‐caspase‐1 by transfection and treated with LLOMe in the presence of autolysosomal/lysosomal degradation inhibitors E64D plus pepstatin. Ratios of mIL‐1β to tubulin levels are shown below the blots.
  • E
    Overlapping area (high content microscopy and processing) of GFP‐Sec22b and LC3B in HeLa cells fed or starved in EBSS.
  • F
    Alignments of the SNC1 domain of TRIM16, shown as they are annotated in the NCBI entry NP_006461, where TRIM16 SNC1 domain is compared with R‐SNAREs, including longin‐containing SNAREs, in NCBI CDD:227472 (http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=227472). Note that the SNC1 domain CDD: 227472 includes sequences from both longin and SNARE domains and that only the SNARE domains contain typical “0” ionic layers (the boxed R residues). Underlined residues, features of the SNARE motif in Saccharomyces cerevisiae (Sc) Snc1, with flanking layers maintaining hydrophobic interactions when this SNARE is in the four‐helix bundle SNARE fusion complex; the N‐terminal flanking parts encompass all layers of the SNARE motif of SNC1, whereas the C‐terminal part shown encompasses a half of C‐terminally positioned hydrophobic layers.
  • G
    Domain organization of TRIM16 and Sec22b. The relationship between the NCBI‐annotated domains (boxes) and predicted coiled‐coil regions of TRIM16 (overlined regions; as annotated in UniProtKB – O95361) are indicated. The relationships between NCBI‐annotated SNC1 motif (underlined region) and the NCBI‐annotated longin and SNARE domains of Sec22b are shown.
  • H, I
    LDH release controls corresponding to IL‐1β secretion complementation experiments of TRIM16KO cells with TRIM16 Sec22b‐interacting and its Sec22b‐nonbinding mutant shown in Fig 3M and N.
  • J
    Sub‐regions selected for cross‐correlation analysis of flag‐TRIM‐16 and GFP‐Sec22b super‐resolution data.
Data information: Means ± SEM; n ≥ 5, except for immunoblot quantifications and image analyses where n ≥ 3. *P < 0.05, † P ≥ 0.05 (t‐test for A, B, E; ANOVA for H, I).