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. 2007 Jun 1;129(5):943–955. doi: 10.1016/j.cell.2007.03.046

Figure 4.

Figure 4

Retrotranslocation Dependence on Lumenal Components

(A) RRMs containing Δgpαf-BOF and 2 mM ATP were reconstituted with total lumenal proteins (black), no lumenal proteins (orange, n = 5), BiP (cyan, n = 4), or PDI (red, n = 7). Each RRM was then incubated with cptcyto at 30°C. For comparison, cptRRMs incubated in the absence of all cytosolic proteins are shown (green). Net αBOF-dependent spectral changes are shown after subtraction of –cyto background (cf. Figure 2C).

(B) Δgpαf-BOF (0.2 μM in buffer A) was titrated at 4°C with oxidized PDI (red, n = 6), reduced PDI (blue, n = 7), or purified BiP (black, n = 3). PDI was reduced or oxidized by 30 min incubation at 30°C in 5 mM DTT or 5 mM GSSG, respectively; BiP was incubated with 2 mM ATP for 10 min at 30°C.

(C) RRMs containing Δgpαf-BOF and 2 mM ATP were reconstituted with total lumenal proteins (black), no cytosolic proteins (green), 10 μM oxidized PDI + 5 mM GSSG (red, n = 3), or 10 μM reduced PDI + 5 mM DTT (orange, n = 3). Data were analyzed as in (A).