TANGO1 recruits ERGIC membranes to the endoplasmic reticulum for procollagen export
- The Barcelona Institute of Science and Technology, Spain
- Universitat Pompeu Fabra, Spain
- Institució Catalana de Recerca i Estudis Avançats, Spain
Figures
Figure 1 with 1 supplement
V- and t-SNAREs required for procollagen VII export from the ER RDEB/FB/C7 cells were transfected with siRNAs directed against BNIP1, STX18, USE1, BET1, SEC22B, YKT6 or a scrambled siRNA.
(A) Collagen VII secretion was measured by western blotting and probing for collagen VII in RDEB/FB/C7 cell lysates and supernatants collected for 20 hr in the presence of ascorbic acid. In three …
Figure 1—figure supplement 1
SNAREs Syntaxin 18, BNIP1, USE1 and YKT6 are required for procollagen VII export from the ER in Het-1A cells.
(A) Het-1A cells transfected with siRNA as described in the methods section were incubated with ascorbate for 20h, fixed with methanol and immunostained with for collagen VII, HSP47 and TGN46. …
Figure 2 with 1 supplement
TANGO1-dependent recruitment of ERGIC membranes to ER-retained patches of procollagen VII.
(A) Procollagen VII was accumulated in the ER of RDEB/FB/C7 cells that were either incubated at 15°C to accumulate secretory cargo in the ER and then released at 37°C for five minutes (control) or …
Figure 2—figure supplement 1
TANGO1 domains.
A schematic representation of human TANGO1 domain organization and its known interactors. SS, signal sequence; SH3, Src homology 3-like domain that is required for binding procollagen VII (Saito et …
Figure 3
Expression of TEER recruits YKT6-containing ERGIC membranes to the ER.
(A) A scheme depicting the coding regions of the two constructs - TEER-Myc (residues 1177–1396 of TANGO1, tagged with Myc) and CC-TMCC3-Myc (residues 282–437 of TMCC3, tagged with Myc). HeLa cells …
Figure 4
The sequence and topology of mitochondrially targeted TEER (Mit-TEER) and CC-TMCC3 (Mit-CC-TMCC3).
(A) A scheme depicting the coding regions of the two constructs for mitochondrial targeting - Mit-TEER-Myc (residues 1187–1396 of TANGO1, including 11 amino acids of its transmembrane domain, tagged …
Figure 5
Recruitment of YKT6-containing ERGIC membranes to TEER expressed at the mitochondria (Mit-TEER).
HeLa cells were seeded on coverslips and transfected with Mit-TEER-Myc or Mit-CC-TMCC3-Myc for 48 hr. For visualisation, cells were stained with DAPI (blue) and immunostained for Myc (green), and …
Figure 6 with 2 supplements
TEER recruits ERGIC membranes, but not Golgi membranes or COPII components HeLa cells were seeded on coverslips and 48 hr after transfection with Mit-TEER-Myc they were fixed and permeabilised.
(A) For visualisation, cells were stained with DAPI (blue) and immunostained for Myc (green). The red channel shows immunostaining for ERGIC-53; YKT6 (after cytosol washout); early and late Golgi …
Figure 6—figure supplement 1
Generation of TANGO1-deficient HeLa by CRISPR/Cas9.
(A) Immunoblot of the parental HeLa cell line and two Tango1 CRISPR knock-out HeLa clones. Protein lysates were separated on a 6% SDS-PAGE and transferred onto a nitrocellulose membrane and probed …
Figure 6—figure supplement 2
A model for the generation of procollagen VII containing mega-carriers.
(A) A pool of ERGIC membranes fuse to the ER exit site enriched in procollagen VII. This fusion is mediated by the t-SNARES BNIP1, Syntaxin18 and USE1; and the v-SNARE YKT6. The TEER domain of …
