Figure 7. Properties of the pre-SufI-IAC^biotin/avidin complex.

(A) Effect of avidin on the transport of pre-SufI-IAC^biotin. Proteins were detected by Western blotting using avidin-HRP. When present, biotin (16 μM) was in large excess over neutravidin (1.6 μM), and neutravidin was in large excess over the precursor protein (40 nM, 1.4 pmol). Thus, the binding interactions were saturated. [IMV] = 5 (A₂₈₀) (B) Competition between pre-SufI-IAC^biotin and pre-SufI-IAC^atto in the presence of avidin. The transport efficiency of pre-SufI-IAC^atto (40 nM, 1.4 pmol) was determined in the absence (black) and presence (red) of 25 μM neutravidin and the indicated molar equivalents of pre-SufI-IAC^biotin (n = 3). Gels were visualized by the Atto565 fluorescence emission. [NADH] = 4 mM; [IMV] = 5 (A₂₈₀). (C) Membrane binding efficiency of pre-SufI-IAC^biotin in the presence of avidin. This anti-SufI immunoblot shows the binding efficiency of pre-SufI-IAC and pre-SufI-IAC^biotin to ΔTat and pTat IMVs in the presence and absence of 25 μM neutravidin, as indicated. In lanes 10–15, the boxes indicate which reagent was added first. In lanes 10–12, neutravidin was preincubated with pre-SufI-IAC^biotin for 10 min prior to addition of IMVs (A₂₈₀ = 2). In lanes 13–15, IMVs were preincubated with pre-SufI-IAC^biotin for 10 min prior to addition of neutravidin. The lower plot corresponds to integrated values from the lanes below in the gel (averaged over 3 identical experiments). The upper plot corresponds to the amount of translocon-bound precursor, calculated for each set of experiments by subtracting the ΔTat IMV values from the pTat IMV values. (D) Effect of precursor addition order on the transport efficiency of pre-SufI-IAC^atto. For lanes 5–9, the reactions contained pre-SufI-IAC^atto, 25 μM neutravidin, and pTat* IMVs (A₂₈₀ = 5). For lanes 7–9, the reactions contained pre-SufI-IAC^biotin. The boxes indicate which precursor (90 nM, 3.1 pmol each) was added first to the reaction solution. In lane 7, both precursors were added simultaneously, and 4 mM NADH was added 5 min later. In lane 8, pre-SufI-IAC^biotin was added 5 min prior to the simultaneous addition of pre-SufI-IAC^atto and NADH. Lane 9 was obtained similarly to lane 8, but the precursors were reversed. This gel was visualized by the Atto565 fluorescence emission.