RESUMEN
Misfolded proteins in the endoplasmic reticulum (ER) activate IRE1α endoribonuclease in mammalian cells, which mediates XBP1 mRNA splicing to produce an active transcription factor. This promotes the expression of specific genes to alleviate ER stress, thereby attenuating IRE1α. Although sustained activation of IRE1α is linked to human diseases, it is not clear how IRE1α is attenuated during ER stress. Here, we identify that Sec63 is a subunit of the previously identified IRE1α/Sec61 translocon complex. We find that Sec63 recruits and activates BiP ATPase through its luminal J-domain to bind onto IRE1α. This leads to inhibition of higher-order oligomerization and attenuation of IRE1α RNase activity during prolonged ER stress. In Sec63-deficient cells, IRE1α remains activated for a long period of time despite the presence of excess BiP in the ER. Thus, our data suggest that the Sec61 translocon bridges IRE1α with Sec63/BiP to regulate the dynamics of IRE1α signaling in cells.
Asunto(s)
Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Endorribonucleasas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Canales de Translocación SEC/metabolismo , Respuesta de Proteína Desplegada , Proteína 1 de Unión a la X-Box/metabolismo , Chaperón BiP del Retículo Endoplásmico , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Células HEK293 , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Chaperonas Moleculares/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Empalme del ARN , Proteínas de Unión al ARN/genética , Canales de Translocación SEC/genética , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-Box/genéticaRESUMEN
The endoplasmic reticulum (ER) localized unfolded protein response (UPR) sensors, IRE1α, PERK, and ATF6α, are activated by the accumulation of misfolded proteins in the ER. It is unclear how the endogenous UPR sensors are regulated by both ER stress and the ER luminal chaperone BiP, which is a negative regulator of UPR sensors. Here we simultaneously examined the changes in the endogenous complexes of UPR sensors by blue native PAGE immunoblotting in unstressed and stressed cells. We found that all three UPR sensors exist as preformed complexes even in unstressed cells. While PERK complexes shift to large complexes, ATF6α complexes are reduced to smaller complexes on ER stress. In contrast, IRE1α complexes were not significantly increased in size on ER stress, unless IRE1α is overexpressed. Surprisingly, depletion of BiP had little impact on the endogenous complexes of UPR sensors. In addition, overexpression of BiP did not significantly affect UPR complexes, but suppressed ER stress mediated activation of IRE1α, ATF6α and, to a lesser extent, PERK. Furthermore, we captured the interaction between IRE1α and misfolded secretory proteins in cells, which suggests that the binding of unfolded proteins to preformed complexes of UPR sensors may be crucial for activation.
Asunto(s)
Factor de Transcripción Activador 6/metabolismo , Estrés del Retículo Endoplásmico , Endorribonucleasas/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , eIF-2 Quinasa/metabolismo , Chaperón BiP del Retículo Endoplásmico , Células HEK293 , Proteínas de Choque Térmico , Humanos , Mutación/genética , Unión Proteica , Pliegue de Proteína , Multimerización de Proteína , Respuesta de Proteína Desplegada , alfa 1-Antitripsina/metabolismoRESUMEN
IRE1α is an endoplasmic reticulum (ER) localized endonuclease activated by misfolded proteins in the ER. Previously, we demonstrated that IRE1α forms a complex with the Sec61 translocon, to which its substrate XBP1u mRNA is recruited for cleavage during ER stress (Plumb et al., 2015). Here, we probe IRE1α complexes in cells with blue native PAGE immunoblotting. We find that IRE1α forms a hetero-oligomeric complex with the Sec61 translocon that is activated upon ER stress with little change in the complex. In addition, IRE1α oligomerization, activation, and inactivation during ER stress are regulated by Sec61. Loss of the IRE1α-Sec61 translocon interaction as well as severe ER stress conditions causes IRE1α to form higher-order oligomers that exhibit continuous activation and extended cleavage of XBP1u mRNA. Thus, we propose that the Sec61-IRE1α complex defines the extent of IRE1α activity and may determine cell fate decisions during ER stress conditions.
Asunto(s)
Endorribonucleasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Canales de Translocación SEC/metabolismo , Transducción de Señal , Respuesta de Proteína Desplegada , Células HEK293 , Humanos , Immunoblotting , Unión Proteica , Multimerización de ProteínaRESUMEN
Upon endoplasmic reticulum (ER) stress, the transmembrane endoribonuclease Ire1α performs mRNA cleavage reactions to increase the ER folding capacity. It is unclear how the low abundant Ire1α efficiently finds and cleaves the majority of mRNAs at the ER membrane. Here, we reveal that Ire1α forms a complex with the Sec61 translocon to cleave its mRNA substrates. We show that Ire1α's key substrate, XBP1u mRNA, is recruited to the Ire1α-Sec61 translocon complex through its nascent chain, which contains a pseudo-transmembrane domain to utilize the signal recognition particle (SRP)-mediated pathway. Depletion of SRP, the SRP receptor or the Sec61 translocon in cells leads to reduced Ire1α-mediated splicing of XBP1u mRNA. Furthermore, mutations in Ire1α that disrupt the Ire1α-Sec61 complex causes reduced Ire1α-mediated cleavage of ER-targeted mRNAs. Thus, our data suggest that the Unfolded Protein Response is coupled with the co-translational protein translocation pathway to maintain protein homeostasis in the ER during stress conditions.