RESUMO
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.
Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Técnicas de Cultura de Células , Colágeno/metabolismo , Colágeno Tipo VII/metabolismo , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Sistemas de Translocação de Proteínas , Transporte Proteico , Fatores de Transcrição/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Mechanisms for exporting variably sized cargo from the endoplasmic reticulum (ER) using the same machinery remain poorly understood. COPII-coated vesicles, which transport secretory proteins from the ER to the Golgi apparatus, are typically 60-90 nm in diameter. However, collagen, which forms a trimeric structure that is too large to be accommodated by conventional transport vesicles, is also known to be secreted via a COPII-dependent process. In this paper, we show that Sec12, a guanine-nucleotide exchange factor for Sar1 guanosine triphosphatase, is concentrated at ER exit sites and that this concentration of Sec12 is specifically required for the secretion of collagen VII but not other proteins. Furthermore, Sec12 recruitment to ER exit sites is organized by its direct interaction with cTAGE5, a previously characterized collagen cargo receptor component, which functions together with TANGO1 at ER exit sites. These findings suggest that the export of large cargo requires high levels of guanosine triphosphate-bound Sar1 generated by Sec12 localized at ER exit sites.