Abstract
Previously we showed that membrane fusion is required for TANGO1-dependent export of procollagen VII from the endoplasmic reticulum (ER) (Nogueira, et al., 2014). Along with the t-SNARE Syntaxin 18, we now reveal the complete complement of SNAREs required in this process, t-SNAREs BNIP1 and USE1, and v-SNARE YKT6. TANGO1 recruits YKT6-containing ER Golgi Intermediate Compartment (ERGIC) membranes to procollagen VII-enriched patches on the ER. Moreover residues 1214-1396, that include the first coiled coil of TANGO1, specifically recruit ERGIC membranes even when targeted to mitochondria. TANGO1 is thus pivotal in concentrating procollagen VII in the lumen and recruiting ERGIC membranes on the cytoplasmic surface of the ER. Our data reveal that growth of a mega transport carrier for collagen export from the ER is not by acquisition of a larger patch of ER membrane, but instead by addition of ERGIC membranes to procollagen-enriched domains of the ER by a TANGO1-mediated process.
Highlights
Human cells express 28 collagens, most of which are secreted, constituting roughly 25% of our dry body weight (Kadler et al, 2007; Malhotra et al, 2015)
We present here the entire complement of t-SNAREs required for procollagen VII export from the endoplasmic reticulum (ER) - BNIP1, USE1 and the previously described Syntaxin 18; that ER Golgi Intermediate Compartment (ERGIC) membranes containing the v-SNARE YKT6 are required for fusion with the ER; and that the first coiled coil domain of TANGO1 is sufficient to recruit these membranes
Two other t-SNAREs reported to act in coordination with Syntaxin 18 (STX18) are BNIP1/Sec20 and USE1 (Belgareh-Touze et al, 2003; Burri et al, 2003; Cosson et al, 1997; Dilcher et al, 2003; Lewis and Pelham, 1996; Sweet and Pelham, 1992). siRNA oligos designed to knockdown t-SNAREs STX18, USE1 and BNIP1; or v-SNAREs YKT6, SEC22B and BET1 which function in transport between ER and the Golgi complex (Hong, 2005); and a scrambled oligo were transfected into RDEB/FB/C7 cells. 48 hr after transfection, cells were washed and re-plated in fresh medium containing ascorbate for 20 hr to promote procollagen export from the ER
Summary
Human cells express 28 collagens, most of which are secreted, constituting roughly 25% of our dry body weight (Kadler et al, 2007; Malhotra et al, 2015) Given their abundance and importance, an understanding of the mechanism by which collagens are sorted, packaged and exported across the secretory pathway, is of fundamental importance. The second coiled coil domain of TANGO1 binds a protein called cTAGE5 and the C-terminal proline rich domains (PRD’s) of TANGO1 and cTAGE5 interact with the COPII coat proteins Sec23A and Sec24C (Saito et al, 2009; Saito et al, 2011) Together, these proteins nucleate a complex that drives procollagen VII export from the ER (Malhotra et al, 2015; Miller and Schekman, 2013).
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