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. 2009 Oct 20;106(42):17775-80.
doi: 10.1073/pnas.0910342106. Epub 2009 Oct 12.

In vitro reconstitution of ER-stress induced ATF6 transport in COPII vesicles

Adam J Schindler  1 , Randy Schekman
Affiliations

In vitro reconstitution of ER-stress induced ATF6 transport in COPII vesicles

Adam J Schindler et al. Proc Natl Acad Sci U S A. .

Abstract

The transcription factor ATF6 is held as a membrane precursor in the endoplasmic reticulum (ER), and is transported and proteolytically processed in the Golgi apparatus under conditions of unfolded protein response stress. We show that during stress, ATF6 forms an interaction with COPII, the protein complex required for vesicular traffic of cargo proteins from the ER. Using an in vitro budding reaction that recapitulates the ER-stress induced transport of ATF6, we show that no cytoplasmic proteins other than COPII are necessary for transport. ATF6 is retained in the ER by association with the chaperone BiP (GRP78). In the in vitro reaction, the ATF6-BiP complex disassembles when membranes are treated with reducing agent and ATP. A hybrid protein with the ATF6 cytoplasmic domain replaced by a constitutive sorting signal (Sec22b SNARE) retains stress-responsive transport in vivo and in vitro. These results suggest that unfolded proteins or an ER luminal -SH reactive bond controls BiP-ATF6 stability and access of ATF6 to the COPII budding machinery.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
ATF6 transports in vivo and in vitro in stably expressing cells. (A) CHO.K1 cells stably expressing 3×-FLAG-ATF6 were treated in 24-well culture dishes with toxins for indicated times. Full-length protein is 110 kDa, cleaved protein is 65 kDa. (B) Permeabilized CHO-ATF6 cells were incubated with GTP, ATP, and an ATP-regenerating system (ATPr), and 4 mg/mL rat liver cytosol for 1 h at room temperatur. Lane 1, 20% load of starting membranes; lanes 2 and 3, controls, in which either nucleotides or rat liver cytosol were excluded; lanes 4 and 5, duplicate incubations of the full budding reaction; lanes 6–17, full reactions with the addition of indicated toxins. Control proteins analyzed in parallel with ATF6 were ERGIC-53, a constitutively transported membrane lectin; Sec22b, a constitutively transported v-SNARE, and Ribophorin-I, a component of the oligosaccharyl transferase complex and resident ER protein. Proteins were analyzed by SDS/PAGE and immunoblotting. Quantification was performed by comparing the signal in vesicle lanes to the 20% starting membrane signal in lane 1. (C) Budding reaction in CHO.K1 cells with immunoblotting to endogenous ATF6. *, Nonspecific band.
Fig. 2.
Fig. 2.
In vitro budding reaction recapitulates ER-stress induced ATF6 transport. (A) Budding reaction in the presence of membrane permeable reducing agents DTT and BME, or membrane-impermeable reducing agent TCEP. (B) Budding reactions conducted in the presence of COPII inhibitors GTPγS and Sar1H79G, or controls GDP and Sar1, or in the presence of the COPI inhibitor Brefeldin A or the proteasome inhibitor ALLN. All lanes contained 2 mM DTT.
Fig. 3.
Fig. 3.
ATF6 forms a physical association with COPII proteins and transports without additional cytoplasmic proteins. (A) Diagram of the prebudding assay scheme to detect associations between ATF6 and COPII. (Left) GDP-restricted form of Sar1, Sar1T39N, is not able to recruit the Sec23/24 complex to membranes, and cargo proteins (such as ATF6) are not precipitated by glutathione pulldown to Sar1. (Right) GTP-restricted Sar1, Sar1H79G, recruits Sec23/24 and cargo proteins. ATF6 binding to COPII complexes is hypothesized to require DTT stimulation. Adapted from (25). (B) Prebudding assay in the presence of 10 μg of the Sec23/24 complex and 5 μg of GST-Sar1. (Left) Absence of 2 mM DTT pretreatment; (Right) After DTT pretreatment. Lanes 1 and 2, and 7 and 8 contain supernatants that did not bind to membranes in the reaction. These lanes demonstrate that Sec23/24 preferentially binds to Sar1·GTP, because unbound Sec23/24 is elevated in the presence of Sar1T39N and depleted in the presence of Sar1H79G. Lanes 3 and 4, and 9 and 10 contain proteins bound to membranes and solubilized by detergent. These lanes contain resident ER proteins and soluble proteins that adhered to membranes. Lane 5 and 6, and 11 and 12 are proteins extracted after glutathione pulldown of GST-Sar1 complexes. Cargo proteins ERGIC-53 and ATF6 are elevated in the Sar1H79G condition. ATF6 is present specifically after pretreatment with DTT (arrow). Bound fractions were 30% of the total; lysates were 1% of the total. (C) Quantification of four assays as in B. Binding efficiency was determined by subtracting the T39N signal from the H79G signal in the beads lanes, and calculating the bound amount as a percentage of total protein in lysates. n = 4; *, P < 0.05. (D) Budding reaction in HeLa-ATF6 cells in the presence of COPII and absence of cytosol. First four lanes represent the standard budding assay with 4 mg/mL cytosol. Remaining lanes had cytosol excluded and COPII added. Values for COPII indicate amounts each of Sar1, Sec23/24 complex, and Sec13/31 complex.
Fig. 4.
Fig. 4.
ATP and DTT act synergistically to dissociate BiP from ATF6. (A) Immunoprecipitation of FLAG-ATF6 in permeabilized CHO-ATF6 cells incubated for 30 min in the presence of 1 mM ATP or ATPγS, or 5 mM DTT. After incubation, cells were washed, lysed, and immunoprecipitated by α-FLAG followed by immunoblotting to FLAG and BiP. (B) Quantification of two replicates of assay as in A. BiP:ATF6 ratios were determined for individual reactions and normalized to the value for untreated cells. (C) Budding reaction to detect the presence of BiP in vesicles.
Fig. 5.
Fig. 5.
Pretreatment in culture overcomes the requirement for DTT in vitro. (A) CHO-ATF6 cells were pretreated in culture with 2 mM DTT for indicated times and washed to remove DTT. Budding reactions were conducted in the absence or presence of DTT. (B) Cells were pretreated as in A, permeabilized and salt washed with 1 M KoAc for 15 min to remove peripheral membrane proteins, followed by the budding reaction.
Fig. 6.
Fig. 6.
The ATF6 luminal domain controls its localization. (A) Cleavage assay of transiently transfected ATF6 constructs in HeLa cells, either untreated or treated with 2 mM DTT for 1 h. Red arrow indicates size of the cleaved N-terminal domain. (B) Schematic representation of chimeric proteins with the human Sec22b cytoplasmic domain (194 aa; yellow), ATF6 transmembrane domain (light blue), and varying ATF6 luminal domains (teal). Proteins contained an N-terminal FLAG tag. (C) Cleavage assay in HeLa cells transiently transfected with constructs from B. Treatment was for 1 h with 2 mM DTT. (D) Budding reactions on HeLa cells transiently transfected with Sec22b-ATF6 chimeras. (Upper) Sec22b-ATF6TM; (Lower) Sec22b-ATF6.
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References

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