Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jul 1;27(13):2008-13.
doi: 10.1091/mbc.E16-03-0180. Epub 2016 May 11.

Dual function of cTAGE5 in collagen export from the endoplasmic reticulum

Tomoya Tanabe  1 , Miharu Maeda  1 , Kota Saito  2 , Toshiaki Katada  1
Affiliations

Dual function of cTAGE5 in collagen export from the endoplasmic reticulum

Tomoya Tanabe et al. Mol Biol Cell. .

Abstract

Two independent functions of cTAGE5 have been reported in collagen VII export from the endoplasmic reticulum (ER). cTAGE5 not only forms a cargo receptor complex with TANGO1, but it also acts as a scaffold to recruit Sec12, a guanine-nucleotide exchange factor for Sar1 GTPase, to ER exit sites. However, the relationship between the two functions remains unclear. Here we isolated point mutants of cTAGE5 that lost Sec12-binding ability but retained binding to TANGO1. Although expression of the mutant alone could not rescue the defects in collagen VII secretion mediated by cTAGE5 knockdown, coexpression with Sar1, but not with the GTPase-deficient mutant, recovered secretion. The expression of Sar1 alone failed to rescue collagen secretion in cTAGE5-depleted cells. Taken together, these results suggest that two functionally irreplaceable and molecularly separable modules in cTAGE5 are both required for collagen VII export from the ER. The recruitment of Sec12 by cTAGE5 contributes to efficient activation of Sar1 in the vicinity of ER exit sites. In addition, the GTPase cycle of Sar1 appears to be responsible for collagen VII exit from the ER.

PubMed Disclaimer

Figures

FIGURE 1:
FIGURE 1:
Construction of cTAGE5 point mutants lacking Sec12-binding ability. (A) 293T cells were transfected with FLAG tag only, FLAG-tagged cTAGE5 coil1 (aa 61–300), cTAGE5 coil2 (aa 301–650), cTAGE5 (aa 61–221), or cTAGE5 (aa 115–300) with HA-tagged Sec12 (aa 1–386). Cell lysates were immunoprecipitated with anti-FLAG antibody and eluted with the FLAG peptide. Eluates and cell lysates were analyzed by SDS–PAGE followed by Western blotting with FLAG or HA antibodies. (B) Alignment of human cTAGE5 (aa 61–115) with corresponding regions from other species. Identical amino acids are shaded in black, and similar amino acids are shaded in gray. (C) 293T cells were transfected with FLAG tag only, FLAG-tagged cTAGE5 wild type, cTAGE5 (aa 61–300) T1, or cTAGE5 (aa 61–115) T1 with HA-tagged Sec12 or TANGO1. (D) 293T cells were transfected with FLAG tag only, FLAG-tagged cTAGE5 wild type, cTAGE5 R61A S65A, S68A R69A, Y71A, E75A K76A, K89A L93A, S97A, or L112A with HA-tagged Sec12. (E) 293T cells were transfected with FLAG tag only, FLAG-tagged cTAGE5 wild type, E75A K76A, K89A L93A, or S97A with HA-tagged TANGO1. (F) 293T cells were transfected with FLAG tag only, FLAG-tagged cTAGE5 wild type, K89A L93A, or K89A with HA-tagged Sec12 or TANGO1. (C–F) Cell lysates were immunoprecipitated with anti-FLAG antibody and eluted with the FLAG peptide. Eluates and cell lysates were analyzed by SDS–PAGE followed by Western blotting with FLAG or HA antibodies.
FIGURE 2:
FIGURE 2:
cTAGE5 mutants lacking Sec12-binding ability fail to recruit Sec12 to the ER exit sites. (A) HSC-1 cells were transfected with cTAGE5 siRNA and cultured for 24 h. cTAGE5-FLAG wild-type and mutant constructs were transfected, and the cells were cultured for another 24 h, fixed, and stained with Sec12 (clone 6B3), FLAG, and Sec16 antibodies. Scale bar, 10 μm. Typically, >90% of the cells showed efficient reduction of cTAGE5 expression by siRNA transfection (cells with asterisks). Less than 10% of the cells were transfected with FLAG-tagged constructs. Cells with asterisks indicate that the cells were efficiently depleted for cTAGE5, but FLAG-tagged constructs were not transfected. (B) Quantification of Sec12 immunofluorescence intensity at ER exit sites in A. A.U., arbitrary units. Fifty ER exit sites from 10 cells, n = 50 (analysis of variance). Error bars represent mean ± SEM; **p < 0.001 compared with wild-type expression; n.s., p > 0.05 compared with wild-type expression. The data shown are from a single representative experiment out of three repeats.
FIGURE 3:
FIGURE 3:
Sar1 coexpression with cTAGE5 mutant recovers collagen VII secretion from the ER. HSC-1 cells were treated with control or cTAGE5 siRNA and cultured for 24 h. For cTAGE5 siRNA-treated cells, cTAGE5-FLAG wild type or mutants (A) or cTAGE5-FLAG constructs together with HA-Sar1a constructs (B) were transfected and further cultured for 24 h. The cells were fixed and stained with collagen VII and FLAG (A) or collagen VII, FLAG, and HA antibodies (B). Collagen VII immunofluorescence signal per cell (A.U., arbitrary units) were quantified in each cell category as described later. The cells positively stained with FLAG or HA antibodies were categorized as the constructs expressed, and the surrounding unstained cells were categorized as nontransfected counterparts. Within each well, cells transfected with constructs are labeled as +, and nontransfected cells are labeled as –. Analysis of variance. Error bars represent mean ± SEM; **p < 0.001; *p < 0.05; n.s., p > 0.05. The data shown are from a single representative experiments out of three repeats. (A) Cells treated with control siRNA (n = 78); cells treated with cTAGE5 siRNA and wild type– (n = 140); wild type+ (n = 49); 60-300aaT1– (n = 111); 60-300aaT1+ (n = 49); S68A R69A– (n = 131); S68A R69A+ (n = 50); E75A K76A– (n = 114); E75A K76A+ (n = 48); and K89A– (n = 167); K89A+ (n = 51). (B) Cells treated with control siRNA (n = 75); cells treated with cTAGE5 siRNA and HA-Sar1aWT– (n = 62); HA-Sar1aWT+ (n = 12); HA-Sar1aH79G– (n = 135); HA-Sar1aH79G+ (n = 37); E75AK76A–, Sar1aWT– (n = 358); E75AK76A+, Sar1aWT– (n = 74); E75AK76A+, Sar1aWT+ (n = 54); E75AK76A–, Sar1aH79G– (n = 272); E75AK76A+, Sar1aH79G– (n = 67); and E75AK76A+, Sar1aH79G+ (n = 54).
FIGURE 4:
FIGURE 4:
Schematic representation of collagen receptor complex under conditions used in this study. In wild-type cells, cTAGE5/TANGO1/Sec12 acts as a cargo receptor for collagen VII and also produces activated Sar1 in the vicinity of ER exit sites (scheme 1). cTAGE5 knockdown renders cargo receptor incompetent and leads to Sec12 dispersion, which inhibits the production of activated Sar1 around ER exit sites. Therefore collagen VII secretion is impaired (scheme 2). The expression of cTAGE5 mutants in cTAGE5-depleted cells rescues the formation of cargo receptor complex, but production of activated Sar1 remains inefficient. Collagen VII cannot be secreted (scheme 3). The expression of only Sar1 is not efficient for the formation of cargo–receptor complex, thereby inhibiting collagen VII secretion (scheme 4). The coexpression of Sar1 and cTAGE5 mutants rescues the formation of the cargo–receptor complex and the amount of activated Sar1 around ER exit sites. Thus collagen VII secretion can be rescued (scheme 5).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bharucha N, Liu Y, Papanikou E, McMahon C, Esaki M, Jeffrey PD, Hughson FM, Glick BS. Sec16 influences transitional ER sites by regulating rather than organizing COPII. Mol Biol Cell. 2013;24:3406–3419. - PMC - PubMed
    1. Farhan H, Weiss M, Tani K, Kaufman RJ, Hauri HP. Adaptation of endoplasmic reticulum exit sites to acute and chronic increases in cargo load. EMBO J. 2008;27:2043–2054. - PMC - PubMed
    1. Ishikawa Y, Boudko S, Bachinger HP. Ziploc-ing the structure: triple helix formation is coordinated by rough endoplasmic reticulum resident PPIases. Biochim Biophys Acta. 2015;1850:1983–1993. - PubMed
    1. Jin L, Pahuja KB, Wickliffe KE, Gorur A, Baumgartel C, Schekman R, Rape M. Ubiquitin-dependent regulation of COPII coat size and function. Nature. 2012;482:495–500. - PMC - PubMed
    1. Kung LF, Pagant S, Futai E, D’Arcangelo JG, Buchanan R, Dittmar JC, Reid RJ, Rothstein R, Hamamoto S, Snapp EL, et al. Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat. EMBO J. 2012;31:1014–1027. - PMC - PubMed

MeSH terms

LinkOut - more resources